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<!doctype html> <html data-n-head-ssr lang="en" data-n-head="%7B%22lang%22:%7B%22ssr%22:%22en%22%7D%7D"> <head > <link data-n-head="ssr" rel="icon" type="image/png" sizes="16x16" href="https://brand.frontiersin.org/m/ed3f9ce840a03d7/favicon_16-tenantFavicon-Frontiers.png"> <link data-n-head="ssr" rel="icon" type="image/png" sizes="32x32" href="https://brand.frontiersin.org/m/ed3f9ce840a03d7/favicon_32-tenantFavicon-Frontiers.png"> <link data-n-head="ssr" rel="apple-touch-icon" type="image/png" sizes="180x180" href="https://brand.frontiersin.org/m/ed3f9ce840a03d7/favicon_180-tenantFavicon-Frontiers.png"> <title>Frontiers | Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation</title><meta data-n-head="ssr" charset="utf-8"><meta data-n-head="ssr" name="viewport" content="width=device-width, initial-scale=1"><meta data-n-head="ssr" data-hid="charset" charset="utf-8"><meta data-n-head="ssr" data-hid="mobile-web-app-capable" name="mobile-web-app-capable" content="yes"><meta data-n-head="ssr" data-hid="apple-mobile-web-app-title" name="apple-mobile-web-app-title" content="Frontiers | Articles"><meta data-n-head="ssr" data-hid="theme-color" name="theme-color" content="#0C4DED"><meta data-n-head="ssr" data-hid="description" property="description" name="description" content="Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose e..."><meta data-n-head="ssr" data-hid="og:title" property="og:title" name="title" content="Frontiers | Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation"><meta data-n-head="ssr" data-hid="og:description" property="og:description" name="description" content="Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose e..."><meta data-n-head="ssr" data-hid="keywords" name="keywords" content="acidophiles,Acidophilic bacteria,acid mine drainage,Biodiversity,extremozymes,esterase,Metagenomics,Plastic"><meta data-n-head="ssr" data-hid="og:site_name" property="og:site_name" name="site_name" content="Frontiers"><meta data-n-head="ssr" data-hid="og:image" property="og:image" name="image" content="https://images-provider.frontiersin.org/api/ipx/w=1200&f=png/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg"><meta data-n-head="ssr" data-hid="og:type" property="og:type" name="type" content="article"><meta data-n-head="ssr" data-hid="og:url" property="og:url" name="url" content="https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.868839/full"><meta data-n-head="ssr" data-hid="twitter:card" name="twitter:card" content="summary_large_image"><meta data-n-head="ssr" data-hid="citation_volume" name="citation_volume" content="13"><meta data-n-head="ssr" data-hid="citation_journal_title" name="citation_journal_title" content="Frontiers in Microbiology"><meta data-n-head="ssr" data-hid="citation_publisher" name="citation_publisher" content="Frontiers"><meta data-n-head="ssr" data-hid="citation_journal_abbrev" name="citation_journal_abbrev" content="Front. Microbiol."><meta data-n-head="ssr" data-hid="citation_issn" name="citation_issn" content="1664-302X"><meta data-n-head="ssr" data-hid="citation_doi" name="citation_doi" content="10.3389/fmicb.2022.868839"><meta data-n-head="ssr" data-hid="citation_firstpage" name="citation_firstpage" content="868839"><meta data-n-head="ssr" data-hid="citation_language" name="citation_language" content="English"><meta data-n-head="ssr" data-hid="citation_title" name="citation_title" content="Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation"><meta data-n-head="ssr" data-hid="citation_keywords" name="citation_keywords" content="acidophiles; Acidophilic bacteria; acid mine drainage; Biodiversity; extremozymes; esterase; Metagenomics; Plastic"><meta data-n-head="ssr" data-hid="citation_abstract" name="citation_abstract" content="<p>Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and <italic>in silico</italic> metagenomic approaches, we retrieved 16 esterases from the α/β-hydrolase fold superfamily with the closest match from uncultured acidophilic <italic>Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium</italic>, and <italic>Ferrimicrobium), Acidiphilium</italic>, and other <italic>Proteobacteria</italic> inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in <italic>Escherichia coli</italic>. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.</p>"><meta data-n-head="ssr" data-hid="citation_pdf_url" name="citation_pdf_url" content="https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.868839/pdf"><meta data-n-head="ssr" data-hid="citation_online_date" name="citation_online_date" content="2022/03/14"><meta data-n-head="ssr" data-hid="citation_publication_date" name="citation_publication_date" content="2022/05/19"><meta data-n-head="ssr" data-hid="citation_author_0" name="citation_author" content="Vidal, Paula"><meta data-n-head="ssr" data-hid="citation_author_institution_0" name="citation_author_institution" content="Institute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Spain"><meta data-n-head="ssr" data-hid="citation_author_1" name="citation_author" content="Martínez-Martínez, Mónica"><meta data-n-head="ssr" data-hid="citation_author_institution_1" name="citation_author_institution" content="Institute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Spain"><meta data-n-head="ssr" data-hid="citation_author_2" name="citation_author" content="Fernandez-Lopez, Laura"><meta data-n-head="ssr" data-hid="citation_author_institution_2" name="citation_author_institution" content="Institute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Spain"><meta data-n-head="ssr" data-hid="citation_author_3" name="citation_author" content="Roda, Sergi"><meta data-n-head="ssr" data-hid="citation_author_institution_3" name="citation_author_institution" content="Department of Life Sciences, Barcelona Supercomputing Center, Spain"><meta data-n-head="ssr" data-hid="citation_author_4" name="citation_author" content="Méndez-García, Celia"><meta data-n-head="ssr" data-hid="citation_author_institution_4" name="citation_author_institution" content="Área de Microbiología, Departamento Biología Funcional e Instituto de Biotecnología de Asturias, Universidad de Oviedo, Spain"><meta data-n-head="ssr" data-hid="citation_author_5" name="citation_author" content="Golyshina, Olga V."><meta data-n-head="ssr" data-hid="citation_author_institution_5" name="citation_author_institution" content="Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, United Kingdom"><meta data-n-head="ssr" data-hid="citation_author_6" name="citation_author" content="Guallar, Víctor"><meta data-n-head="ssr" data-hid="citation_author_institution_6" name="citation_author_institution" content="Department of Life Sciences, Barcelona Supercomputing Center, Spain"><meta data-n-head="ssr" data-hid="citation_author_7" name="citation_author" content="Peláez, Ana I."><meta data-n-head="ssr" data-hid="citation_author_institution_7" name="citation_author_institution" content="Área de Microbiología, Departamento Biología Funcional e Instituto de Biotecnología de Asturias, Universidad de Oviedo, Spain"><meta data-n-head="ssr" data-hid="citation_author_8" name="citation_author" content="Ferrer, Manuel"><meta data-n-head="ssr" data-hid="citation_author_institution_8" name="citation_author_institution" content="Institute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Spain"><meta data-n-head="ssr" data-hid="dc.identifier" name="dc.identifier" content="doi:10.3389/fmicb.2022.868839"><link data-n-head="ssr" rel="manifest" href="/article-pages/_nuxt/manifest.c499fc0a.json" data-hid="manifest"><link data-n-head="ssr" rel="canonical" href="https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.868839/full"><script data-n-head="ssr" data-hid="newrelic-browser-script" type="text/javascript">window.NREUM||(NREUM={});NREUM.info = {"agent":"","beacon":"bam.nr-data.net","errorBeacon":"bam.nr-data.net","licenseKey":"598a124f17","applicationID":"588603994","agentToken":null,"applicationTime":2.513994,"transactionName":"MQcDMkECCkNSW0YMWghNIgldDQFTRxd1IGFJTQ==","queueTime":0,"ttGuid":"250f259fe006a72e"}; (window.NREUM||(NREUM={})).init={privacy:{cookies_enabled:true},ajax:{deny_list:["bam.nr-data.net"]},distributed_tracing:{enabled:true}};(window.NREUM||(NREUM={})).loader_config={agentID:"594400880",accountID:"230385",trustKey:"230385",xpid:"VgUHUl5WGwYIXFdSBAgOUg==",licenseKey:"598a124f17",applicationID:"588603994"};;/*! 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class="Ibar__userArea"></div></div></div></nav> <div class="ArticlePage"><div><div class="Layout Layout--withAside Layout--withIbarMix ArticleDetails"> <main class="Layout__main"> <div class="ArticleDetails__main"><div class="ArticleLayoutHeader"><div class="ArticleLayoutHeader__info"><p class="ArticleLayoutHeader__info__title"> ORIGINAL RESEARCH article </p> <p class="ArticleLayoutHeader__info__journalDate"><span>Front. Microbiol.</span> <span>, 19 May 2022</span></p> <p class="ArticleLayoutHeader__info__journalDate"> Sec. Evolutionary and Genomic Microbiology </p> <p class="ArticleLayoutHeader__info__doiVolume"><span> Volume 13 - 2022 | </span> <a href="https://doi.org/10.3389/fmicb.2022.868839" class="ArticleLayoutHeader__info__doi"> https://doi.org/10.3389/fmicb.2022.868839 </a></p> </div>  <p class="ArticleLayoutHeader__isPartOfRT"><span class="ArticleLayoutHeader__isPartOfRT__label">This article is part of the Research Topic</span> <span class="ArticleLayoutHeader__isPartOfRT__title">Functional Metagenomics for Enzyme Discovery</span> <span class="Link__wrapper"><a aria-label="View all 6 articles" href="https://www.frontiersin.org/research-topics/20327/functional-metagenomics-for-enzyme-discovery/articles" target="_self" data-event="customLink-link-a_viewAll6Articles" class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right"><span>View all 6 articles</span></a></span></p></div> <div class="ArticleDetails__main__content"><div class="ArticleDetails__main__content__main ArticleDetails__main__content__main--fullArticle"><div class="JournalAbstract"><div class="JournalAbstract__titleWrapper"><h1>Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation</h1> </div> </div> <div class="JournalFullText"><div class="JournalAbstract"> <a id="h1" name="h1"></a> <div class="authors"><span class="author-wrapper notranslate"> <img class="pr5" src="https://loop.frontiersin.org/cdn/images/profile/default_32.jpg" alt="\r\nPaula Vidal" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';">Paula Vidal<sup>1</sup></span><span class="author-wrapper notranslate"><img class="pr5" src="https://loop.frontiersin.org/cdn/images/profile/default_32.jpg" alt="Mnica Martínez-Martínez&#x;" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';">Mónica Martínez-Martínez<sup>1†</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/1685724" class="user-id-1685724"><img class="pr5" src="https://loop.frontiersin.org/images/profile/1685724/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Laura Fernandez-Lopez">Laura Fernandez-Lopez</a><sup>1</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/1685246" class="user-id-1685246"><img class="pr5" src="https://loop.frontiersin.org/images/profile/1685246/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Sergi Roda">Sergi Roda</a><sup>2</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/217882" class="user-id-217882"><img class="pr5" src="https://loop.frontiersin.org/images/profile/217882/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Celia Mndez-García">Celia Méndez-García</a><sup>3</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/224799" class="user-id-224799"><img class="pr5" src="https://loop.frontiersin.org/images/profile/224799/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Olga V. Golyshina">Olga V. Golyshina</a><sup>4</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/517366" class="user-id-517366"><img class="pr5" src="https://loop.frontiersin.org/images/profile/517366/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Víctor Guallar,">Víctor Guallar</a><sup>2,5</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/93877" class="user-id-93877"><img class="pr5" src="https://loop.frontiersin.org/images/profile/93877/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Ana I. Pelez">Ana I. Peláez</a><sup>3</sup></span><span class="author-wrapper notranslate"><a href="https://loop.frontiersin.org/people/236735" class="user-id-236735"><img class="pr5" src="https://loop.frontiersin.org/images/profile/236735/74" onerror="this.onerror=null;this.src='https://loop.frontiersin.org/cdn/images/profile/default_32.jpg';" alt="Manuel Ferrer*\r\n">Manuel Ferrer</a><sup>1*</sup> </span></div> <ul class="notes"> <li><span><sup>1</sup></span>Institute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Madrid, Spain</li> <li><span><sup>2</sup></span>Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain</li> <li><span><sup>3</sup></span>Área de Microbiología, Departamento Biología Funcional e Instituto de Biotecnología de Asturias, Universidad de Oviedo, Oviedo, Spain</li> <li><span><sup>4</sup></span>Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, United Kingdom</li> <li><span><sup>5</sup></span>Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain</li> </ul> <p class="mb0">Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and <i>in silico</i> metagenomic approaches, we retrieved 16 esterases from the α/β-hydrolase fold superfamily with the closest match from uncultured acidophilic <i>Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium</i>, and <i>Ferrimicrobium), Acidiphilium</i>, and other <i>Proteobacteria</i> inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in <i>Escherichia coli</i>. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.</p> <div class="clear"></div> </div> <div class="JournalFullText"> <a id="h2" name="h2"></a><h2>Introduction</h2> <p class="mb15">In biotechnology, there is high interest in finding enzymes with new or improved properties (<a href="#B55">Pellis et al., 2017</a>; <a href="#B23">Ferrer et al., 2019</a>). This interest is especially increased in relation to enzymes from extremophiles, which are microorganisms that have evolved to thrive in extreme environments (<a href="#B9">Baweja et al., 2016</a>), as they can efficiently operate under multiple conditions requested by industry. One example application is the eco-friendly bioconversion of cellulosic biomass by extremozymes, which produces green products and has less substrate loss (<a href="#B64">Thapa et al., 2020</a>). Furthermore, plastic disposal is one of the major problems currently faced by the environment, as enormous quantities of synthetic plastics are non-degradable. Researchers are constantly exploring new ways to degrade plastics, and one of these ways involves using enzymes from microorganisms or microbial communities, including some that inhabit extreme environments (<a href="#B53">Nchedo Ariole and George-West, 2020</a>).</p> <p class="mb15">Acid mine drainage (AMD) systems deserve special attention as a source of extremozymes. AMDs are extremely acidic runoff formations that originate from the microbial oxidation of pyrite and other sulfide minerals, which results in the production of sulfuric acid and metal-rich solutions (<a href="#B51">Méndez-García et al., 2015</a>; <a href="#B43">Johnson and Quatrini, 2020</a>). AMD systems are common in our planet, although only a limited number of them have been microbiologically characterized (<a href="#B51">Méndez-García et al., 2015</a>; <a href="#B43">Johnson and Quatrini, 2020</a>). Although it has recently been demonstrated that some of these AMD formations, such as the Los Rueldos mercury mine in northwestern Spain (<a href="#B50">Méndez-García et al., 2014</a>), appear to be populated by a great diversity of prokaryotes, the majority of them are inhabited by a restricted set of acidophilic bacteria and archaea (<a href="#B20">Dopson et al., 2004</a>; <a href="#B30">Golyshina, 2011</a>; <a href="#B51">Méndez-García et al., 2015</a>; <a href="#B14">Chen et al., 2016</a>; <a href="#B43">Johnson and Quatrini, 2020</a>), whose variety and abundance depend on geochemical constraints (<a href="#B50">Méndez-García et al., 2014</a>, <a href="#B51">2015</a>; <a href="#B39">Huang et al., 2016</a>).</p> <p class="mb15">Major bacterial lineages detected in AMD systems include the phyla <i>Proteobacteria</i> (<i>Acidithiobacillus, Acidiphilium</i>, <i>Acidocella</i>, <i>Acidicaldus</i>, <i>Acidomonas</i>, <i>Acidisphaera</i>, “<i>Ferrovum</i>,” <i>Acidibacter</i>, and <i>Metallibacterium</i> spp.), <i>Nitrospirae</i> (<i>Leptospirillum</i> spp. such as <i>Leptospirillum ferrooxidans</i>, <i>Leptospirillum ferriphilum</i>, and “<i>Leptospirillum ferrodiazotrophum</i>”), <i>Actinobacteria</i>, <i>Firmicutes</i> (<i>Sulfobacillus</i> spp., and <i>Alicyclobacillus</i> spp.), <i>Acidobacteria</i>, <i>Saccharibacteria</i> (TM7) and other candidate phyla radiation (CPR) organisms. Archaea include the phyla <i>Euryarchaeota</i> (<i>Ferroplasma</i> spp. such as <i>Ferroplasma acidiphilum</i> and “<i>Ferroplasma acidarmanus</i>,” <i>Acidiplasma cupricumulans</i>, and <i>Cuniculiplasma divulgatum</i>), <i>Thaumarchaeota</i>, and the Candidate divisions “<i>Micrarchaeota</i>” and “<i>Parvarchaeota</i>” (<a href="#B33">Golyshina et al., 2000</a>, <a href="#B34">2009</a>, <a href="#B32">2016</a>; <a href="#B20">Dopson et al., 2004</a>; <a href="#B6">Baker et al., 2006</a>, <a href="#B5">2010</a>; <a href="#B30">Golyshina, 2011</a>; <a href="#B14">Chen et al., 2016</a>, <a href="#B15">2018</a>; <a href="#B28">Gavrilov et al., 2019</a>; <a href="#B45">Korzhenkov et al., 2019</a>). These microorganisms are expected to be reservoirs of enzymes selected to resist acidic harsh conditions (at least regarding extracellular products) (<a href="#B63">Sharma et al., 2012</a>), some of which might be of biotechnological relevance (<a href="#B35">Gomes et al., 2003</a>; <a href="#B1">Adrio and Demain, 2014</a>).</p> <p class="mb15">In this category, esterases and lipases from the α/β-hydrolase fold superfamily are appropriate biocatalysts for use in a modern circular bioeconomy because of their abundance (at least one per genome; <a href="#B21">Ferrer et al., 2015</a>); the extensive knowledge that has been accumulated after the analysis of the biochemical features, sequences, and structures of more than 280,638 such proteins (<a href="#B8">Bauer et al., 2020</a>); their ease of identification (multiple available screening methods; <a href="#B60">Reyes-Duarte et al., 2012</a>); and their outstanding properties in terms of stability, reactivity, and scalability, which make them third-choice tools for the functionalization and modification of low-reactivity hydrocarbon-like blocks, oils, and fats (<a href="#B16">Daiha et al., 2015</a>). Genomics and metagenomics can potentially make accessible an enormous reserve of such important biocatalysts in organisms or microbial communities inhabiting any environment, including AMD systems. However, only 239 of the 280,638 sequences available at the Lipase Engineering (LED) Database (<a href="#B8">Bauer et al., 2020</a>) have been retrieved from cultured microorganisms (listed above) and uncultured microorganisms that are inhabitants of AMD systems, including <i>Alicyclobacillus</i> spp., 118 in total; <i>Sulfobacillus</i> spp., 53; <i>Acidobacteria</i>, 34; <i>Acidithiobacillus</i>, 13; <i>Leptospirillum</i>, 9; “<i>Ferrovum</i>,” 5; <i>Acidocella</i>, 3; and <i>Ferroplasma</i>, <i>Aciditrix</i>, <i>Acidiphilium</i>, and <i>Metallibacterium</i>, with 1 each. Among these biocatalysts, only a low-pH optimum carboxylesterase from <i>F. acidiphilum</i> (<a href="#B54">Ohara et al., 2014</a>) has been characterized. This limits the assessment of the biotechnological potential of acidophiles living in AMD systems, at least regarding esterases and lipases. The minimal enzyme-level information that is known about these systems is restricted to two endo-acting amylases with no similarity to any known protein and two genes conferring metal and acid resistance from the microbial community inhabiting the AMD systems of the Carnoulès (lead–zinc) mine in France (<a href="#B18">Delavat et al., 2012</a>) and the Tinto River in southwestern Spain (<a href="#B18">Delavat et al., 2012</a>; <a href="#B36">Guazzaroni et al., 2013</a>), respectively.</p> <p class="mb15">To fill this knowledge gap, we initiated a metagenomic investigation to isolate carboxylesterases from a recently discovered and microbiologically characterized AMD formation, namely, the Los Rueldos mercury mine in northwestern Spain (<a href="#B50">Méndez-García et al., 2014</a>). By applying homology searches in metagenomic sequences and naïve screening in clone libraries with enzyme substrates, we discovered a number of such enzymes whose characteristics are reported herein. Both function- and DNA sequence-based metagenomic methods are complementary, with each having advantages and disadvantages. Bioinformatics methods allow a rapid process of enzyme searching. However, in prokaryotic genomes, >30% of genes remain annotated as “hypothetical, conserved hypothetical or with general prediction,” and large numbers of genes may have non-specific annotations (such as putative hydrolases). The analysis of biochemical functions is likely to provide a superior approach to avoid this limitation, especially when screening novel enzymes. However, only a few hundred specific enzymatic assays exist, with a limited number of them applied in a high-throughput manner for the naïve screening of metagenomics libraries.</p> <p class="mb0">Although the <i>in vivo</i> roles and expression levels of the genes encoding the hydrolases presented in this study are unknown, their sequences and results of biochemical analyses shed new light on the enzymology of the microbial inhabitants of the Los Rueldos AMD formation, which have been neglected in enzyme prospecting.</p> <a id="h3" name="h3"></a><h2>Materials and Methods</h2> <h3 class="pt0">General Experimental Procedures</h3> <p class="mb0">The source and brand of each of the esters (purity ≥99%) used in this study was Merck Life Science S.L.U., Madrid, Spain. The oligonucleotides used for DNA amplification were synthesized by Sigma Genosys, Ltd. (Pampisford, Cambridgeshire, United Kingdom). The <i>Escherichia coli</i> EPI-300-T1R strain used for pCCFOS1 fosmid library construction and screening was from Epicentre Biotechnologies (Madison, WI, United States). The <i>E. coli</i> strain GigaSingles used for gene cloning and <i>E. coli</i> strain BL21 (DE3) used for gene expression were from Novagen (Darmstadt, Germany).</p> <h3>Sampling Site and Sample Collection</h3> <p class="mb0">The Los Rueldos gallery is located along the northwestern slope of the Morgao Valley (2 km northeast of the town of Mieres and 20 km southeast of Oviedo, which is the capital city of Asturias in northwestern Spain; 43°15′47″N, 5°46′9″W). It is a 70 m-long gallery with 10–30 cm depths in the shallower areas and 40–70 cm depths in the deeper sections (<a href="#B50">Méndez-García et al., 2014</a>). Microorganisms are developed along the AMD system (pH ∼2), forming a bedded acidic biofilm with uppermost oxic (B1A) and lowermost anoxic (B1B) strata. The DNA samples from B1A and B1B (see below) samples collected and used in this study were the same as those in the previous work (<a href="#B50">Méndez-García et al., 2014</a>). Briefly, samples were collected in sterile 50 ml tubes at two sampling sites determined by the presence of each different macroscopic microbial growth morphology (B1A: up to 2 cm deep; B1B: from 2 to 15 cm deep) and kept on ice until nucleic acid extraction was performed (within the following 2 h).</p> <h3>Nucleic Acid Extraction, Preparation of pCCFOS1 Libraries, and Naïve Screening</h3> <p class="mb15">The DNA samples from B1A and B1B were the same as those used in a previous work (<a href="#B50">Méndez-García et al., 2014</a>), which were obtained using the Power Soil DNA extraction kit (Cambio, Cambridge, United Kingdom) according to the manufacturer’s guidelines. Prior to clone library construction, the metagenomics DNA was concentrated by first adding 50 μl of 3 M sodium acetate solution to 50 μl DNA extract. Precipitation was conducted by the addition of 1.25 ml of ethanol and incubation at room temperature for 10 s. Precipitated DNA was pelleted by centrifugation at 20,000 <i>g</i> for 10 min. The resulting pellets were washed with 500 μl of 70% (v/v) ethanol twice, and the traces of ethanol were evaporated by incubation under a fume hood at room temperature for 10 min. The resulting pellets were then dissolved in 20 μl of sterile nuclease-free water. Before cloning in the large-insert pCCFOS1 fosmid libraries using the CopyControl Fosmid Library Kit (Epicentre Biotechnologies, Madison, WI, United States) and the <i>E. coli</i> EPI300-T1<i><sup>R</sup></i> strain, the DNA (10 μg) that was unsheared by gel electrophoresis was subjected to shearing by pipetting through a 200 μl pipette tip 100 times, following the recommendations of the supplier (Epicentre Biotechnologies, Madison, WI, United States) to reach an approximately size of 30,000 bp. Cells of each pCCFOS1 fosmid library were suspended in glycerol to a final concentration of 20% (v/v) and stored at −80°C until further use. We generated subsets of 94,000 and 81,000 clones for the B1A and B1B samples, respectively. Restriction analysis of 10 randomly selected clones from each library revealed average insert sizes of 34,000 bp (for the B1A samples) and 39,500 bp (for the B1B samples), which included nearly 3.2 Gbp of community genomes per sample. This size is within the range of the average size range of DNA inserts in positive clones found in this study (see below).</p> <p class="mb0">Fosmid clones were plated onto large (22.5 × 22.5 cm) Petri plates with Luria Bertani (LB) agar containing chloramphenicol (12.5 μg/ml) and induction solution (Epicentre Biotechnologies; WI, United States) at a quantity recommended by the supplier to induce a high fosmid copy number. Clones were scored by the ability to hydrolyze α-naphthyl acetate (α-NA) and tributyrin, as previously described (<a href="#B60">Reyes-Duarte et al., 2012</a>). Positive clones presumptively containing carboxylesterases and lipases with the α/β hydrolase fold were selected, and their DNA inserts were sequenced using a MiSeq Sequencing System (Illumina, San Diego, CA, United States) with a 2 × 150-bp sequencing v2 kit at Lifesequencing S.L. (Valencia, Spain). Before sequencing, fosmid DNA was extracted from the fosmid clones containing the metagenomic segments using the QUIAGEN Large-Construct Kit (QUIAGEN, Hilden, Germany), according to the manufacturer’s protocol. Upon the completion of sequencing, the reads were quality-filtered and assembled to generate non-redundant meta-sequences, and genes were predicted and annotated as described previously (<a href="#B57">Placido et al., 2015</a>).</p> <h3>Selection of Genes Encoding Enzymes by Homology Sequence Analysis</h3> <p class="mb0">The predicted protein-coding genes obtained in a previous study (<a href="#B50">Méndez-García et al., 2014</a>) after the sequencing of DNA material from resident microbial communities in each of the samples (B1A and B1B) with a Roche 454 GS FLX Ti sequencer (Roche Applied Science, Penzberg, Germany) were used in this study. The meta-sequences are available from the National Center for Biotechnology Information (NCBI) non-redundant public database with the IDs PRJNA193663 (for B1A) and PRJNA193664 (for B1B). Protein-coding genes identified from metagenomes (sequence-based screening) and from the DNA inserts of positive clones (naïve screen) were screened (score >45; e-value <10e<sup>–3</sup>) using BLASTP and PSI-BLAST searching (<a href="#B3">Altschul et al., 1997</a>) for enzymes of interest against the <i>ESTerases and alpha/beta-Hydrolase Enzymes and Relatives</i> (ESTHER) and LED databases (<a href="#B25">Fischer and Pleiss, 2003</a>; <a href="#B7">Barth et al., 2004</a>; <a href="#B8">Bauer et al., 2020</a>).</p> <h3>Gene Expression and Protein Purification</h3> <p class="mb0">The experimental procedures used for the cloning, expression, and purification of selected proteins (either from naïve or homology sequence screening) in the Ek/LIC 46 vector and <i>E. coli</i> strain BL21 (DE3) were performed as described previously (<a href="#B2">Alcaide et al., 2015</a>). The primers used for amplification are listed in <a href="#S9">Supplementary Material</a>. All proteins studied here were N-terminally His<sub>6</sub>-tagged, and the soluble His-tagged proteins were produced and purified at room temperature after binding to a nickel–nitrilotriacetic acid (Ni–NTA) His-Bind resin (from Merck Life Science S.L.U., Madrid, Spain) as described previously (<a href="#B29">Giunta et al., 2020</a>), with slight modifications (the expression culture was scaled up to 1 L using 50 ml pre-inoculum). The purity was assessed as >98% using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE; <a href="#S9">Supplementary Figure 1</a>) in a Bio-Rad Mini Protein system (<a href="#B46">Laemmli, 1970</a>). Protein concentrations were determined according to the Bradford method with bovine serum albumin as the standard (<a href="#B12">Bradford, 1976</a>). A total of approximately 0.8–37 mg of purified recombinant proteins was obtained from each 1 L culture on average, as follows: Est<sub><i>A</i>1</sub> (6.4 mg/L), Est<sub><i>A</i>2</sub> (25 mg/L), Est<sub><i>A</i>3</sub> (13 mg/L), Est<sub><i>A</i>4</sub> (37 mg/L), Est<sub><i>A</i>5</sub> (41 mg/L), Est<sub><i>A</i>6</sub> (7 mg/L), Est<sub><i>A</i>7</sub> (0.8 mg/L), Est<sub><i>A</i>8</sub> (19 mg/L), Est<sub><i>B</i>1</sub> (1.0 mg/L), and Est<sub><i>B</i>2</sub> (32 mg/L).</p> <h3>Enzyme Assays</h3> <p class="mb15">The hydrolysis of 2-naphthyl acrylate (ref. 577189), tri(propylene glycol) diacrylate (ref. 246832), dibenzyl terephthalate (ref. PH000126), and bis(2-hydroxyethyl)-terephthalate (BHET; ref. 465151) (all from Merck Life Science S.L.U., Madrid, Spain) was assessed using a pH indicator assay in 384-well plates (ref. 781162, Greiner Bio-One GmbH, Kremsmünster, Austria) at 30°C and pH 8.0 in a Synergy HT Multi-Mode Microplate Reader in continuous mode at 550 nm over 24 h [extinction coefficient (ε) of phenol red, 8,450 M<sup>–1</sup> cm<sup>–1</sup>]. The acid produced after ester bond cleavage by the hydrolytic enzyme induced a color change in the pH indicator that was measured spectrophotometrically at 550 nm. The experimental conditions were as detailed previously (<a href="#B29">Giunta et al., 2020</a>), with the absence of activity defined as at least a twofold background signal. For <i>V</i><sub><i>max</i></sub> determination, (protein): 270 μg/ml; (ester): 20 mM; reaction volume: 44 μl; T: 30°C; and pH: 8.0. Activity was calculated by determining the absorbance per minute from the generated slopes and applying the following equation:</p> <div class="equationImageholder pb0"> <math id="M1"><mrow><mrow><mi>R</mi><mi>a</mi><mi>t</mi><mpadded width="+2.8pt"><mi>e</mi></mpadded><mrow><mo>(</mo><mstyle displaystyle="true"><mfrac><mrow><mi mathvariant="normal">μ</mi><mi>m</mi><mi>o</mi><mi>l</mi></mrow><mrow><mi>min</mi><mo>⁡</mo><mrow><mi>m</mi><mpadded width="+2.8pt"><mi>g</mi></mpadded><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow></mrow></mfrac></mstyle><mo>)</mo></mrow></mrow><mo>=</mo><mrow><mrow><mrow><mstyle displaystyle="true"><mfrac><mfrac><mrow><mi mathvariant="normal">△</mi><mrow><mtext>Abs</mtext></mrow></mrow><mrow><mtext>min</mtext></mrow></mfrac><mrow><mpadded width="+2.8pt"><mn>8450</mn></mpadded><msup><mi mathvariant="normal">M</mi><mrow><mo>-</mo><mn>1</mn></mrow></msup><msup><mtext>cm</mtext><mrow><mo>-</mo><mn>1</mn></mrow></msup></mrow></mfrac></mstyle><mo>*</mo><mstyle displaystyle="true"><mfrac><mn>1</mn><mrow><mpadded width="+2.8pt"><mn>0.4</mn></mpadded><mi>c</mi><mi>m</mi></mrow></mfrac></mstyle><mo>*</mo><mpadded width="+2.8pt"><mstyle displaystyle="true"><mfrac><mrow><mpadded width="+2.8pt"><msup><mn>10</mn><mn>6</mn></msup></mpadded><mi mathvariant="normal">μ</mi><mi>M</mi></mrow><mrow><mpadded width="+2.8pt"><mn>1</mn></mpadded><mi mathvariant="normal">M</mi></mrow></mfrac></mstyle></mpadded><mo>*</mo><mpadded width="+2.8pt"><mn> 0.000044</mn></mpadded></mrow><mi>L</mi></mrow><mo>*</mo><mstyle displaystyle="true"><mfrac><mn>1</mn><mrow><mi>m</mi><mpadded width="+2.8pt"><mi>g</mi></mpadded><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mo>.</mo></mrow></mfrac></mstyle></mrow></mrow></math> </div> <p class="mb15">The activity toward the model esters <i>p</i>-nitrophenyl acetate (<i>p</i>NPC<sub>2</sub>), propionate (<i>p</i>NPC<sub>3</sub>), butyrate (<i>p</i>NPC<sub>4</sub>), octanoate (<i>p</i>NPC<sub>8</sub>), decanoate (<i>p</i>NPC<sub>10</sub>), and decanoate (<i>p</i>NPC<sub>12</sub>) was assessed in 50 mM Britton and Robinson (BR) buffer at pH 8.0 and 30°C by monitoring the production of 4-nitrophenol at 348 nm (pH-independent isosbestic point, ε = 4147 M<sup>–1</sup> cm<sup>–1</sup>) for over 5 min and determining the absorbance per minute from the generated slopes (<a href="#B61">Santiago et al., 2018</a>). The reactions were performed at 30°C in 96-well plates (ref. 655801, Greiner Bio-One GmbH, Kremsmünster, Austria) and contained 0.09 to 3 μg proteins and 0.8 mM esters in a total volume of 200 μl. The effect of pH on the activity was determined in 50 mM BR buffer at pH 4.0–12.0, as described previously. Similar assay conditions were used to assay the effects of temperature on the ester hydrolysis of <i>p</i>NPC<sub>3</sub>, but in this case, the reactions were performed in 50 mM BR buffer pH 7.0. Note that the BR buffer consists of a mixture of 0.04 M H<sub>3</sub>BO<sub>3</sub>, 0.04 M H<sub>3</sub>PO<sub>4</sub>, and 0.04 M CH<sub>3</sub>COOH that has been titrated to the desired pH with 0.2 M NaOH. All values were determined in triplicate and were corrected for non-enzymatic transformation. In all cases, the activity was calculated by determining the absorbance per minute from the generated slopes and applying the following equation:</p> <div class="equationImageholder pb0"> <math id="M2"><mrow><mrow><mi>R</mi><mi>a</mi><mi>t</mi><mpadded width="+2.8pt"><mi>e</mi></mpadded><mrow><mo>(</mo><mstyle displaystyle="true"><mfrac><mrow><mi mathvariant="normal">μ</mi><mi>m</mi><mi>o</mi><mi>l</mi></mrow><mrow><mi>min</mi><mo>⁡</mo><mrow><mi>m</mi><mpadded width="+2.8pt"><mi>g</mi></mpadded><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow></mrow></mfrac></mstyle><mo>)</mo></mrow></mrow><mo>=</mo><mrow><mrow><mrow><mstyle displaystyle="true"><mfrac><mfrac><mrow><mi mathvariant="normal">△</mi><mrow><mtext>Abs</mtext></mrow></mrow><mrow><mtext>min</mtext></mrow></mfrac><mrow><mpadded width="+2.8pt"><mn>4147</mn></mpadded><msup><mi mathvariant="normal">M</mi><mrow><mo>-</mo><mn>1</mn></mrow></msup><msup><mtext>cm</mtext><mrow><mo>-</mo><mn>1</mn></mrow></msup></mrow></mfrac></mstyle><mo>*</mo><mstyle displaystyle="true"><mfrac><mn>1</mn><mrow><mpadded width="+2.8pt"><mn>0.4</mn></mpadded><mi>c</mi><mi>m</mi></mrow></mfrac></mstyle><mo>*</mo><mpadded width="+2.8pt"><mstyle displaystyle="true"><mfrac><mrow><mpadded width="+2.8pt"><msup><mn>10</mn><mn>6</mn></msup></mpadded><mi mathvariant="normal">μ</mi><mi>M</mi></mrow><mrow><mpadded width="+2.8pt"><mn>1</mn></mpadded><mi mathvariant="normal">M</mi></mrow></mfrac></mstyle></mpadded><mo>*</mo><mpadded width="+2.8pt"><mn> 0.0002</mn></mpadded></mrow><mi>L</mi></mrow><mo>*</mo><mstyle displaystyle="true"><mfrac><mn>1</mn><mrow><mi>m</mi><mpadded width="+2.8pt"><mi>g</mi></mpadded><mi>p</mi><mi>r</mi><mi>o</mi><mi>t</mi><mi>e</mi><mi>i</mi><mi>n</mi></mrow></mfrac></mstyle></mrow></mrow></math> </div> <p class="mb15">Poly(propylene glycol) diacrylate (ref. 455024, Merck Life Science S.L.U., Madrid, Spain) and poly(DL-lactide) with an average molecular weight 2,000 (ref. AP224, PolySciTech, Akina, IN, United States) were assayed as described previously (<a href="#B37">Hajighasemi et al., 2018</a>). The hydrolysis of polyethylene terephthalate (PET) films (prepared as reported by <a href="#B11">Bollinger et al., 2020</a>) and particles, which were prepared using PET from a bottle (from a local shop – Granini brand), as described previously (<a href="#B58">Pütz, 2006</a>), was evaluated at 30°C and pH 8.0 with 270 μg protein/ml and 2 mg/ml plastic material, as previously reported (<a href="#B11">Bollinger et al., 2020</a>).</p> <p class="mb0">The effect of the inhibitors mercaptoethanol (ref. M7154) and iodoacetamide (ref. I1149), which were both from Merck Life Science S.L.U., Madrid, Spain, was tested as follows. A mixture containing the purified enzymes (final concentration of 1 mg/ml) in 190 μl of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.0 and the inhibitors (final concentration, 1–10 mM) was incubated for 5 min to 24 h at 30–45°C. The reaction was initiated by adding <i>p</i>NPC<sub>3</sub> (0.8 mM, final concentration), and the activity was measured for over 5 min as described above and compared to control samples without inhibitors.</p> <h3>Circular Dichroism to Estimate Thermal Denaturation</h3> <p class="mb0">Circular dichroism (CD) spectra were acquired between 190 and 270 nm with a Jasco J-720 spectropolarimeter equipped with a Peltier temperature controller in a 0.1-mm cell at 25°C. The spectra were analyzed, and denaturation temperature (T<sub><i>d</i></sub>) values were determined at 220 nm between 10 and 85°C at a rate of 30°C per hour in 40 mM HEPES buffer at pH 7.0. CD measurements were performed at pH 7.0 and not at the optimal pH (8.5–9.0) to ensure protein stability. A protein concentration of 0.5 mg/ml was used. T<sub><i>d</i></sub> (and the standard deviation of the linear fit) was calculated by fitting the ellipticity (mdeg) at 220 nm at each of the different temperatures using a 5-parameter sigmoid fit with SigmaPlot 13.0.</p> <h3>Codes and Accession Numbers</h3> <p class="mb0">The sequences were named based on the code “Est,” which refers to <u>Est</u>erase, followed by a letter indicating the origin of the sample, as follows: Est<sub><i>A</i></sub>, esterase from the uppermost oxic B1A strata; and Est<sub><i>B</i></sub>, esterase from the lowermost anoxic B1B sediment attached strata. The final number (subscript) is an arbitrary number representing the number of enzymes per site. Sequences encoding enzymes were deposited under the BioProject IDs PRJNA193663 (for B1A) and PRJNA193664 (for B1B) in the NCBI public database, with the accession numbers detailed in <a href="#T1">Table 1</a>.</p> <div class="DottedLine"></div> <div class="Imageheaders">TABLE 1</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" name="table1" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t001.jpg" alt="www.frontiersin.org" id="T1" loading="lazy"> </picture> </a> <p><strong>Table 1.</strong> General sequence-based characteristics of Los Rueldos esterases.</p> </div> <div class="clear"></div> <div class="DottedLine"></div> <h3>Three-Dimensional Modeling</h3> <p class="mb0">The models of the protein structures were predicted with AlphaFold 2.1.0 (<a href="#B44">Jumper et al., 2021</a>).</p> <a id="h4" name="h4"></a><h2>Results</h2> <h3 class="pt0">Enzyme Selection and Divergence at the Sequence Level</h3> <p class="mb15">Microbial communities inhabiting two distinct compartments within Los Rueldos AMD formation were screened for sequences encoding carboxylesterases or lipases. For that, we used two complementary metagenomics approaches, namely, naïve and homology sequence screens. First, a total of approximately 81,000 pCCFOS1 clones from each clone library (equivalent to 2.8 Gbp for B1A and 3.20 Gbp for B1B) were screened for esterase/lipase activity using plate-based screen with αNA and tributyrin as model substrates. We identified a total of 10 positive clones being active against both substrates in the B1A clone library, whereas no positives were found in the B1B library. The fosmids with insert lengths ranging from 16,545 to 41,280 bp were fully sequenced, from which 10 genes (one per positive clone), encoding presumptive esterases/lipases, were identified. In addition, we searched the predicted protein-coding genes obtained through next-generation sequencing for sequences encoding esterases and lipases by BLAST search against the ESTHER and LED databases. A total of 6 full-length sequences (B1A: 2; B1B: 4), with accession numbers EQD63018.1, EQD66234.1, EQD71191.1, EQD52136.1, EQD55146.1, and EQD26916.1, encoding potential enzymes were identified. Taken together, a total of 16 genes encoding hydrolases from the α/β-hydrolase fold superfamily, specifically, 12 from B1A (EstA<sub>1</sub> to EstA<sub>12</sub>) and 4 from B1B (EstB<sub>1</sub> to EstB<sub>4</sub>), were identified (<a href="#T1">Table 1</a>). As determined by Matcher (EMBOSS package), the pairwise amino acid sequence identity for 14 of the 16 α/β hydrolases ranged from 4.0 to 44.9%. This, together with the fact that only 2 out of 16 polypeptides were highly similar (EstA<sub>5</sub> and EstA<sub>6</sub> differ in only 2 amino acids: arginine 152 and alanine 179 in Est<sub><i>A</i>5</sub> are cysteine 152 and threonine 179 in Est<sub><i>A</i>6</sub>), suggests a large divergence at the sequence level within the enzymes examined, and that the diversity of polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins, but rather by diverse non-redundant sequences. Note that only 1 of 10 sequences selected after naïve screens was found in the metagenomic data generated after direct DNA sequencing (EstA<sub>11</sub>, which is 99% identical to GenBank accession no. EQD37671.1 from the Los Rueldos metagenome) (<a href="#B50">Méndez-García et al., 2014</a>). This demonstrates that both types of screens (naïve and <i>in silico</i>) are complementary tools for enzyme discovery. However, deeper metagenomic sequencing could potentially detect all enzymes isolated by naïve screens.</p> <p class="mb0">The deduced molecular mass and estimated isoelectric point (<i>p</i>I) values ranged from 23.19 to 101.53 kDa and from 4.62 to 10.04, respectively. Putative proteins exhibited a maximum amino acid sequence identity ranging from 39 to 100% to putative esterases/lipases in public databases (<a href="#T1">Table 1</a>). It is worth mentioning that EstA<sub>3</sub>, EstA<sub>8</sub>, and EstB<sub>3</sub> are related to presumptive esterase/lipase-like subfamily proteins of the Serine-Glycine-Asparagine-Histidine (SGNH) hydrolases, EstA<sub>11</sub> to presumptive glycoside-hydrolase family GH114 (N-terminal domain) and CE4_PelA_like hydrolases (C-terminal domain), and EstA<sub>12</sub> to presumptive sialate O-acetylesterases. A further TBLASTX search against metagenomics proteins deposited in databases revealed no similarity of 10 proteins with homologous AMD metagenome proteins. In contrast, five proteins (EstA<sub>3</sub> to EstA<sub>6</sub>, and EstA<sub>12</sub>) do share from 27 to 54% homology to three proteins from the Carnoules arsenic-contaminated mine drainage (GenBank: CBI07622.1, CBH97521.1, and CBI00527.1). This finding suggests that esterases/lipases from microbial communities from the Los Rueldos site are distantly related to proteins from other known homologous proteins from AMD formations with metagenome sequences available. It also reflects the large undiscovered pool of enzymes from bacterial species populating the Los Rueldos site.</p> <h3>Primary Structure Analysis</h3> <p class="mb0">Based on the comparison of the primary structures, 14 families of sequence-related esterases and lipases have been reported (<a href="#B4">Arpigny and Jaeger, 1999</a>; <a href="#B59">Rao et al., 2013</a>). Sequence analysis categorized 13 enzymes from Los Rueldos into some of these known subfamilies (<a href="#F1">Figure 1</a>) with most structurally similar homologs as follows: EstA<sub>9</sub> [27%; best hit in Protein Data Bank (PDB) 3DOH_A] and EstB<sub>4</sub> (41%; 3OM8_A) to Family I; EstA<sub>1</sub> (41%; 3V9A_A), EstA<sub>4</sub> (41%; 4YPV_A), EstA<sub>5</sub> (49%; 4YPV_A), and EstA<sub>6</sub> (49%; 4YPV_A) to Family IV; EstA<sub>7</sub> (53%; 4YPV_A) and EstB<sub>2</sub> (41%; 1AUO_A) to Family VI; EstA<sub>2</sub> (37%; 2OGT_A) to Family VII (EstA<sub>2</sub>); and EstA<sub>10</sub> (41%; 4IVK_A) to beta-lactamase like Family VIII. EstA<sub>3</sub> (43%; PDB code 3P94_A), EstA<sub>8</sub> (44%; PDB code 3P94_A), and EstB<sub>3</sub> (26%; PDB code 3KVN_X) belong to Family II GDSL, but the structural alignment also confirms that they contain a domain that displays the characteristic α–β–α globular fold of the SGNH hydrolase family. In addition, EstB<sub>3</sub> also contains a passenger domain providing the driving force for passenger translocation (<a href="#B65">Van den Berg, 2010</a>). Three of the sequences could not be assigned to these subfamilies. First, EstA<sub>11</sub> contains a 300 amino acid long N-terminal domain most similar to glycoside-hydrolase family 114 and a 616 amino acid long C-terminal domain most similar to the carbohydrate esterase 4 (CE4) superfamily that includes chitin deacetylases (EC 3.5.1.41), N-acetylglucosamine deacetylases (EC 3.5.1.-), and acetylxylan esterases (EC 3.1.1.72), which catalyze the N- or O-deacetylation of substrates such as acetylated chitin, peptidoglycan, and acetylated xylan. Its N-terminal domain is most structurally similar (26%) to that of the glycosidase 2AAM_A and its C-terminal domain is structurally similar to the polysaccharide deacetylase from <i>Bacillus cereus</i> (4HD5). Second, EstA<sub>12</sub> is associated with acetylxylan esterases (EC 3.1.1.72), with most similar (21%) structural homolog in PDB being 1ZMB_A. Third, EstB<sub>1</sub> shows homology to small serine alpha/beta-hydrolase/acyl-peptidase (58%; 2FUK_A). The tentative amino acids participating in the typical catalytic triad of esterases and lipases are summarized in <a href="#T1">Table 1</a>. Together, the analysis of the primary sequence suggests that the diversity of esterases was not dominated by a particular type of protein or a highly similar cluster of proteins, but rather by diverse non-redundant sequences belonging to different microbial groups and distinct esterase/lipase subfamilies.</p> <div class="DottedLine"></div> <div class="Imageheaders">FIGURE 1</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" name="figure1" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g001.jpg" alt="www.frontiersin.org" id="F1" loading="lazy"> </picture> </a> <p><strong>Figure 1.</strong> The unrooted circular neighbor-joining tree indicating phylogenetic positions of polypeptide sequences of Los Rueldos esterases. Positioning is referred to homologous proteins with unambiguous categorization into lipase/esterase families (from Family I to XIV) according to Arpigny and Jaeger and further classifications (<a href="#B4">Arpigny and Jaeger, 1999</a>; <a href="#B59">Rao et al., 2013</a>). GS-F, genome sequences assigned to an esterase/lipase family (in bold letters). Sequences from Los Rueldos that correspond to proteins that could not be produced as soluble active proteins using Ek/LIC 46 vector and <i>E. coli</i> strain BL21 (DE3) as a host are indicated in gray color, while those being active and soluble are indicated in bold.</p> </div> <div class="clear"></div> <div class="DottedLine"></div> <h3>Source Organisms of Selected Polypeptides</h3> <p class="mb0">A search of oligonucleotide patterns against the GOHTAM database (<a href="#B52">Ménigaud et al., 2012</a>) and TBLASTX analysis revealed compositional similarities between the DNA fragment containing the genes for EstA<sub>1</sub>, EstA<sub>2</sub>, EstA<sub>10</sub>, and EstB<sub>4</sub>, with genomic sequences of bacteria from the phylum <i>Actinomycetota</i>. Among them, only unambiguous affiliations at lower levels could be achieved for fragments containing Est<sub><i>A</i>1</sub>, Est<sub><i>A</i>2</sub>, and EstA<sub>10</sub> that may most likely belong to bacteria from the genera <i>Acidithrix</i> (EstA<sub>1</sub> and EstA<sub>10</sub>) and <i>Acidimicrobium</i>/<i>Ferrimicrobium</i> (EstA<sub>2</sub>), both from the family <i>Acidimicrobiaceae</i> within the order <i>Acidimicrobiales</i>. Note that EstA<sub>1</sub> and EstA<sub>10</sub> showed 99–100% sequence identity with uncharacterized esterases and lipases (WP_052605564.1 and WP_052605292.1) from <i>Acidithrix ferrooxidans</i>, and EstA<sub>2</sub> showed 94% sequence identity with an uncharacterized esterase-lipase (NNN14078.1) from <i>Acidimicrobiaceae</i>. EstA<sub>3</sub> was most likely derived from an uncultured bacterium assigned to the phylum <i>Acidobacteria</i> with ambiguous affiliation below the phylum level. The genes for EstA<sub>4</sub> to EstA<sub>9</sub>, EstA<sub>11</sub>, and EstB<sub>1</sub> to EstB<sub>3</sub> were associated with uncultured bacteria of the <i>Proteobacteria</i> phylum, with ambiguous affiliations at a lower taxonomic level, except for EstA<sub>4</sub>, which was most likely derived from a bacterium of the genus <i>Acidiphilium</i> from the family <i>Acetobacteraceae</i> within the order <i>Rhodospirillales</i> (best hit OYV70855.1 from <i>Acidiphilium</i> sp., 79% homology). All these bacterial groups have been detected in biofilms thriving in the Los Rueldos mine (<a href="#B50">Méndez-García et al., 2014</a>). No clear affiliation, other than Bacteria, could be found for EstA<sub>12</sub>. Note that in some cases no clear affiliation to a taxon of source organism below the level of the phylum could be established, either because of the short fragment length or the low compositional similarity between the metagenomic fragments and the sequences of related bacterial chromosomes and plasmids do not allow proper assignations.</p> <h3>Enzyme Characterization</h3> <p class="mb15">From the 16 sequences selected, 8 from B1A and 2 from B1B were successfully cloned, expressed, and purified as soluble active proteins when expressed in pET Ek/LIC 46 vector and <i>E. coli</i> BL21 as the host. These proteins were herein referred to as EstA<sub>1</sub> to EstA<sub>8</sub>, EstB<sub>1</sub>, and EstB<sub>2</sub>. The remaining six (EstA<sub>9</sub>-EstA<sub>12</sub> and EstB<sub>3</sub>-EstB<sub>4</sub>) could not be produced in soluble active form (they formed inclusion bodies) in the expression system applied herein, which consists of the use of the Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Ek/LIC 46 vector and <i>E. coli</i> strain BL21 (DE3) as a host, and their properties are not described herein. Refining the expression conditions, which included variations in the expression conditions (16, 30, and 37°C) and IPTG concentration (from 0.1 to 2 mM), resulted in unsuccessful production of sufficient active protein material for characterization. Further efforts may be needed with different expression vectors, which is beyond the scope of the present study.</p> <p class="mb0">The substrate profile of all α/β hydrolases was first examined using a set of esters commonly used to characterize esterases and lipases, namely, <i>p</i>NP esters such as <i>p</i>NPC<sub>2</sub>, <i>p</i>NPC<sub>3</sub>, <i>p</i>NPC<sub>4</sub>, <i>p</i>NPC<sub>8</sub>, <i>p</i>NPC<sub>10</sub>, and <i>p</i>NPC<sub>12</sub>. All ester hydrolases preferred short-chain-length pNP-esters, particularly <i>p</i>NPC<sub>2</sub> (EstA<sub>3</sub>), <i>p</i>NPC<sub>3</sub> (EstA<sub>2</sub>, EstA<sub>4</sub>, EstA<sub>5</sub>, EstA<sub>6</sub>, EstB<sub>1</sub>, and EstB<sub>2</sub>), and <i>p</i>NPC<sub>4</sub> (EstA<sub>1</sub>, EstA<sub>7</sub>, and EstA<sub>8</sub>) (<a href="#T2">Table 2</a>). Within all six <i>p</i>NP ester tested, all but one (EstA<sub>3</sub>) enzyme was able to hydrolyze up to <i>p</i>NPC<sub>12</sub>, albeit at a much lower level (from 62- to 3,900-fold) compared to shorter derivatives. Considering the preferred <i>p</i>NP esters, the maximum specific activity ranged from 3.06 ± 0.03 to 679.8 ± 9.8 U/mg. We further test the possibility that the enzymes hydrolyze substrates other than <i>p</i>NP esters, particularly, plastic substrates and esters relevant to plastics. Using previously described conditions (<a href="#B37">Hajighasemi et al., 2018</a>; <a href="#B11">Bollinger et al., 2020</a>), we did not find that any of the enzymes hydrolyzed large plastic materials such as poly(propylene glycol) diacrylate, poly(DL-lactide), amorphous PET film, and PET nanoparticles. However, by using a pH-indicator assay, we found that the enzymes were able to hydrolyze other terephthalate esters and acrylate esters. Thus, as shown in <a href="#T3">Table 3</a>, six of the enzymes hydrolyzed esters relevant to acrylic acid plastics, e.g., 2-naphthyl acrylate and tripropylene glycol diacrylate, a commonly used material principally exploited to prepare thermally stable polymers (<a href="#B38">He et al., 2017</a>). These substrates, herein found to be converted at a maximum rate of 3,915 ± 48 U/g, are rarely hydrolyzed by esterases and lipases. There are only two examples reported, namely, human salivary pseudocholinesterase and cholesterol esterase (<a href="#B24">Finer and Santerre, 2004</a>; <a href="#B13">Cai et al., 2014</a>). In addition, one of the enzymes (Est<sub><i>A</i>8</sub>) was capable of hydrolyzing dibenzyl terephthalate (432.2 ± 27.5 U/g), an intermediate produced during chemical PET recycling with benzyl alcohol in the presence of a catalyst (<a href="#B19">Donahue et al., 2003</a>). No esterase or lipase has been reported to date that degrades this substrate. In addition, six of the esterases (Est<sub><i>A</i>1</sub>, Est<sub><i>A</i>2</sub>, Est<sub><i>A</i>5</sub>, Est<sub><i>A</i>6</sub>, Est<sub><i>A</i>8</sub>, and Est<sub><i>B</i>2</sub>) efficiently hydrolyzed BHET (from 5.0 ± 1.0 to 336.9 ± 3.6 U/g), an intermediate in the degradation of PET (<a href="#B68">Yoshida et al., 2016</a>). High performance liquid chromatography (HPLC) analysis, performed as described (<a href="#B11">Bollinger et al., 2020</a>), confirmed the hydrolysis of BHET to mono-(2-hydroxyethyl)-terephthalic acid (MHET) and not to terephthalic acid. To conclude, the enzymes reported herein from the Los Rueldos AMD formation showed high activity for converting and recycling components of synthetic plastics, namely, acrylic- and terephthalate-based plastics, and could be of potential use in developing plastic degradation strategies.</p> <div class="DottedLine"></div> <div class="Imageheaders">TABLE 2</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" name="table2" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t002.jpg" alt="www.frontiersin.org" id="T2" loading="lazy"> </picture> </a> <p><strong>Table 2.</strong> Specific activity (U/mg pure protein) for each of the enzymes tested over a set of p-nitrophenyl (<i>p</i>NP) esters of different lengths.</p> </div> <div class="clear"></div> <div class="DottedLine mb15"></div> <div class="Imageheaders">TABLE 3</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" name="table3" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-t003.jpg" alt="www.frontiersin.org" id="T3" loading="lazy"> </picture> </a> <p><strong>Table 3.</strong> Specific activity (U/g pure protein) for each of the enzymes able to hydrolyze a set of structurally different plastic-related esters.</p> </div> <div class="clear"></div> <div class="DottedLine"></div> <p class="mb15 w100pc float_left mt15">Using <i>p</i>NPC<sub>3</sub> as a substrate, the purified proteins were most active at temperatures ranging from 30 to 65°C (<a href="#F2">Figure 2</a>). The average annual temperature in Los Rueldos is 13.8 ± 0.6°C, which varied from 10 ± 0.6°C to 17.1 ± 0.6 (<a href="#B50">Méndez-García et al., 2014</a>), with a temperature of 17°C when samples were taken (July). At this value, the enzymes retained from 20 to 61% of the activity shown at the optimal temperature (<a href="#F2">Figure 2</a>).</p> <div class="DottedLine"></div> <div class="Imageheaders">FIGURE 2</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" name="figure2" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g002.jpg" alt="www.frontiersin.org" id="F2" loading="lazy"> </picture> </a> <p><strong>Figure 2.</strong> pH and thermal profiles of the purified enzymes. The data represent the relative percentages (%) of specific activity (U/g) in triplicates compared with the maximum activity using <i>p</i>NPC<sub>3</sub> as substrate. For raw data see <a href="#S9">Supplementary Table 1</a>.</p> </div> <div class="clear"></div> <div class="DottedLine"></div> <p class="mb15 w100pc float_left mt15">Using <i>p</i>NPC<sub>3</sub> as a substrate, all enzymes showed an optimum pH for activity from neutral to slightly basic, which varied from 7.0 to 9.0 (<a href="#F2">Figure 2</a>). This finding suggests that these proteins are most likely intracellularly produced, consistent with the absence of signal peptides in their sequences. Even though the enzymes showed a slightly basic optimum pH, all retained 33–68% of their activity at pH 5.5. Interestingly, Est<sub><i>A6</i></sub> shows two activity peaks, one at pH 5.5 and one at pH 9.0, while Est<sub><i>A5</i></sub>, which only differs in two amino acids, has an optimum pH of 9.0 (<a href="#F2">Figure 2</a>).</p> <p class="mb0">Sequence analysis revealed that EstA<sub>5</sub> and EstA<sub>6</sub> which have their origins in a bacterium of the phylum <i>Proteobacteria</i>, differ in only 2 amino acids (99.4% identity). Positions 152 and 172 are occupied by Arg and Ala in EstA<sub>5</sub> and by Cys and Thr in EstA<sub>6</sub>, respectively. Notably, EstA<sub>5</sub> was most active at 30°C, retaining more than 80% of the activity at temperatures from 20 to 45°C (<a href="#F2">Figure 2</a>). The optimum temperature for activity increased up to 45°C for EstA<sub>6</sub>, which maintained more than 80% of its activity in the range from 30 to 60°C. Analysis by circular dichroism revealed that Est<sub><i>A</i>5</sub> showed a sigmoidal curve from which a temperature of denaturation of 60.4 ± 0.2°C could be obtained (<a href="#F3">Figure 3</a>). However, the curve for Est<sub><i>A</i>6</sub> shows two transitions, one with a denaturation temperature of 48.1 ± 0.8°C and a second at 75.7 ± 0.2°C. The presence of these two phases may therefore indicate that the presence of these two amino acids may contribute to protein stability and its denaturation under distinct conditions. This result may explain the higher optimum temperature for the activity of this enzyme compared to Est<sub><i>A</i>5</sub>, and the stabilization effect of Cys152 and Thr172. This difference in thermostability between Est<sub><i>A</i>5</sub> and Est<sub><i>A</i>6</sub> can probably be explained by the difference in amino acid 152, since Est<sub><i>A</i>6</sub> has a Cys that would allow it to make a possible disulfide bridge with Cys181, giving it greater thermostability than Est<sub><i>A</i>5</sub> (since Est<sub><i>A</i>5</sub> has an Arg at position 152 instead of a Cys), as shown by examination of the 3D models (<a href="#S9">Supplementary Figure 2</a> and <a href="#F4">Figure 4</a>). It is plausible that this difference may also be responsible for the different pH profiles of both enzymes (<a href="#F2">Figure 2</a>). If the disulfide bridge was present in Est<sub><i>A</i>6</sub>, it could be removed by reduction or chemical modification. Activity tests revealed that both enzymes are resistant to the reducing agent beta-mercaptoethanol, with no activity lost even after 24 h of incubation at 30–45°C in the presence of a 1–10 mM inhibitor. By contrast, in the presence of the cysteine alkylating agent iodoacetamide, both enzymes were rapidly inactivated after 5 min. Thus, we could not verify the presumptive formation of the disulfide bridge in Est<sub><i>A</i>6</sub>, and further studies are needed to test this assumption.</p> <div class="DottedLine"></div> <div class="Imageheaders">FIGURE 3</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" name="figure3" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g003.jpg" alt="www.frontiersin.org" id="F3" loading="lazy"> </picture> </a> <p><strong>Figure 3.</strong> The thermal denaturation curve of Est<sub><i>A</i>5</sub> (filled circle) and Est<sub><i>A</i>6</sub> (open circle) at pH 7.0. The datasets were obtained by measuring the ellipticity changes at 220 nm obtained at different temperatures. For raw data see <a href="#S9">Supplementary Table 2</a>.</p> </div> <div class="clear"></div> <div class="DottedLine mb15"></div> <div class="Imageheaders">FIGURE 4</div> <div class="FigureDesc"> <a href="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" name="figure4" target="_blank"> <picture> <source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=480&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" media="(max-width: 563px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=370&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" media="(max-width: 1024px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=290&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" media="(max-width: 1441px)"><source type="image/webp" srcset="https://images-provider.frontiersin.org/api/ipx/w=410&f=webp/https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" media=""><source type="image/jpg" srcset="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" media=""> <img src="https://www.frontiersin.org/files/Articles/868839/fmicb-13-868839-HTML/image_m/fmicb-13-868839-g004.jpg" alt="www.frontiersin.org" id="F4" loading="lazy"> </picture> </a> <p><strong>Figure 4.</strong> Three-dimensional comparison of the Est<sub><i>A</i>5</sub> and Est<sub><i>A</i>6</sub> hydrolases <b>(A)</b>. Zoom into the region that is different between both proteins, where the possible interaction of Cys152 and Cys181 in Est<sub><i>A</i>6</sub> can be seen in panel <b>(B)</b>. As shown, position 152 is occupied by Arg in Est<sub><i>A</i>5</sub> instead. Figure has been created using PMOL(TM) 2.2.3.</p> </div> <div class="clear"></div> <div class="DottedLine"></div> <a id="h5" name="h5"></a><h2>Discussion</h2> <p class="mb15">The effects of environmental constraints as prime forces shaping AMD populations have only begun to be elucidated through omics studies (<a href="#B51">Méndez-García et al., 2015</a>). These effects are also of high interest in the context of the isolation and characterization of novel enzymes, for which limited data are available. However, the difficulty of cultivating organisms inhabiting AMD sites, which is due to their longer generation times, lower biomass yields, and cultivation conditions that are not yet fully understood, requires different strategies to overcome the problems associated not only with their cultivation, but also with the isolation of enzymes. Metagenomic approaches allow the screening of enzymes from such extreme environments. However, by using these tools, we have thus far explored only a small fraction of the enormous diversity on the planet, especially that of organisms inhabiting extremely acidic environments (<a href="#B21">Ferrer et al., 2015</a>), again indicating the importance of establishing enzyme screening programs for AMD sites. The particular characteristics of the Los Rueldos AMD site (<a href="#B51">Méndez-García et al., 2015</a>) that make it an interesting study site include the following. First, it is populated by a larger diversity of <i>Bacteria</i> and <i>Archaea</i> compared to other AMD sites, containing a total of 39 different species. Second, it has high microbial heterogeneity in local microniches defined by its O<sub>2</sub> concentration gradients and spatial and biofilm architecture. As an example, only 1 of 18 species inhabiting the two distinct compartments in a stratified streamer investigated herein, namely, the oxic uppermost (B1A) and anoxic lowermost (B1B) sediment-attached strata, was shared. Therefore, it is plausible that Los Rueldos may also contain a greater diversity of microbial products such as enzymes.</p> <p class="mb15">We have sought to address this possibility by screening for esterases and lipases from the α/β-hydrolase fold superfamily in microbial communities inhabiting the Los Rueldos AMD site. These enzymes are desired tools for biocatalysis in a variety of industrial sectors (<a href="#B16">Daiha et al., 2015</a>; <a href="#B21">Ferrer et al., 2015</a>). Microorganisms that can survive under low pH values similar to those in Los Rueldos (pH ∼2) could be good sources of enzymes that can be used, for example, as additives in detergents, for the biobleaching of pulp and paper, in the clean-up of effluent streams from the textile processing industry, and in the degradation of plastics (<a href="#B35">Gomes et al., 2003</a>; <a href="#B1">Adrio and Demain, 2014</a>; <a href="#B53">Nchedo Ariole and George-West, 2020</a>) and other polymers (<a href="#B27">Fütterer et al., 2004</a>).</p> <p class="mb15">We used two complementary approaches for enzyme mining. A sequence-based metagenomic approach that searched for homologous enzymes in the metagenomic sequence data and function-driven screens in which expression libraries were used to identify, by using specific colorimetric substrates (<a href="#B22">Ferrer et al., 2016</a>; <a href="#B56">Peña-García et al., 2016</a>), clones containing enzymes of interest that could be missed in shallow metagenomics sequencing. By using both approaches, we identified 16 sequences that were potentially encoding esterases and lipases. The amino acid sequences were distantly related to sequences found in other AMD formations, which was in agreement with the distinct Los Rueldos-specific populations (<a href="#B50">Méndez-García et al., 2014</a>). Indeed, we only observed some degree of sequence identity (27–54%) to 3 homologs from the Carnoulès (lead-zinc) mine, France (<a href="#B10">Bertin et al., 2011</a>) in 6 of 16 sequences. In addition, the large differences among the recovered enzymes may correspond to the high population diversity that characterizes the Los Rueldos site (<a href="#B50">Méndez-García et al., 2014</a>).</p> <p class="mb15">Notably, activity-based screens did not yield any active clones from the library created from the anoxic lowermost strata (B1B), while they yielded 10 active clones from the library created from the oxic uppermost strata (B1A). Thus, we searched for such enzymes in B1B by screening sequence data generated in a previous study (<a href="#B50">Méndez-García et al., 2014</a>). It is plausible that the presence of low-O<sub>2</sub>-adapted microbial species in B1B, in contrast to the aerobic species in B1A, may account for the low efficiency of heterologous gene expression after cloning of the genetic material in the <i>E. coli</i> host and, possibly, the lower efficiency of the screening tests in the B1B library compared to that obtained for B1A. However, the fact that similar proportions of identified proteins (by naïve and sequence screening) in B1A (7 of 12) and B1B (2 of 4) could be produced as soluble active proteins when expressed in <i>E. coli</i> suggests that this may not be the only reason explaining the absence of positive clones in the B1B library. We cannot rule out that the native promoters of the partial genes from microorganisms inhabiting B1B cloned in the pCCFOS1 fosmids were inactive in <i>E. coli</i>, resulting in failed active clones on the plates. The data provided in <a href="#T2">Tables 2</a>, <a href="#T3">3</a> revealed that B1B enzymes were among the least-active enzymes among all hydrolases identified and characterized in the present study, and it is therefore also plausible that the low efficiency of the screening tests may have been due to the low activity level of enzymes from microorganisms inhabiting the anoxic B1B compartment. Additionally, it is plausible that different screening conditions (temperature, pH, inductor concentration, etc.) may be needed to detect other active proteins and that the enzymes from B1B would be more active under other assay conditions, the investigation of which is beyond the scope of the present study.</p> <p class="mb15">Regardless of the problems associated with the screening efficiency in different environments, including extreme AMD formations such as Los Rueldos, the analysis of the optimal pH profile of 10 out of the 16 hydrolases that could be produced in active form additionally revealed that their optimal pH was in the range from 7.0 to 9.0. This finding suggests that all hydrolases are presumptively produced intracellularly by acidophiles that thrive in the acidic Los Rueldos environment with a pH of 2.0. A similar phenotype has been found for other enzymes from AMD inhabitants, such as ATP-dependent DNA ligase from “<i>F. acidarmanus</i>” Fer1 (<a href="#B40">Jackson et al., 2007</a>) and ene-reductase from “<i>Ferrovum</i>” sp. JA12 (<a href="#B62">Scholtissek et al., 2016</a>), with pH optima of 6.0–7.0. However, we observed that most of the enzymes showed a slightly acid-stable phenotype, retaining ∼33–68% of activity at pH 5.5. It is plausible that the identification of enzymes with neutral-like pH optima is a consequence of screening tests performed at neutral pH, using a vector and host that allow mostly intracellular proteins to be produced and that presumably acid-stable enzymes could not be detected. In the future, performing naïve screens at such low pH values may help obtain additional active clones. However, while specific adaptations need to be explored in great detail, the retention of a high activity level at a slightly acidic pH might be attributed to the prevalence of acidic amino acids (negatively charged at a neutral pH) on the surfaces of these enzymes (<a href="#S9">Supplementary Figure 2</a>), as reported for other proteins from acidophiles (<a href="#B66">Wu et al., 2020</a>). Indeed, the relative frequencies of acidic residues in proteins in this study ranged from 67 to 54%, except for Est<sub><i>A</i>8</sub> (37%). As no major differences in pH profiles were observed when comparing the enzymes with the highest (Est<sub><i>A</i>1</sub>: 67%) and lowest (Est<sub><i>A</i>8</sub>: 37%) percentages of acidic residues, it is possible that other factors affect the activity and stability of the studied proteins from the Los Rueldos site. An example of this is the differences in stability against pH and temperature of Est<sub><i>A</i>5</sub> and Est<sub><i>A</i>6</sub>, which show very different features and have only a two amino acid difference despite having the same percentage of acidic residues.</p> <p class="mb15">Furthermore, the biochemical properties of the esterases reported in this study revealed that all enzymes showed an activity–stability trade-off characteristic of mesophilic-adapted enzymes (from 30 to 65°C), which is a phenotype that has also been found for enzymes from other AMD inhabitants (<a href="#B31">Golyshina et al., 2006</a>; <a href="#B40">Jackson et al., 2007</a>; <a href="#B54">Ohara et al., 2014</a>; <a href="#B62">Scholtissek et al., 2016</a>). It is noticeable, however, that 5 of 10 characterized enzymes retained at least 50% of their maximal activity at temperatures as low as 12°C. The fact that the Los Rueldos site is characterized by a relatively low temperature compared to other AMD sites (<a href="#B50">Méndez-García et al., 2014</a>) may account for this low-temperature-active phenotype. However, the lack of biochemical information on enzymes from other AMD sites does not allow us to validate this assumption.</p> <p class="mb15">Finally, it should be emphasized that the activity levels of the characterized enzymes (maximum for the best <i>p</i>NP substrates: approximately 680 to 3 U/mg, depending on the hydrolase) were in the range of other reported enzymes of different origins with esterase and lipase activities (<a href="#B21">Ferrer et al., 2015</a>; <a href="#B48">Martínez-Martínez et al., 2018</a>). The data suggest that the low-O<sub>2</sub>-adapted microbial species developed in the anoxic lowermost (B1B) sediment-attached strata do contain less-active enzymes than those developed in the oxic uppermost (B1A) strata under the conditions used herein. Whether this is typical <i>in vivo</i> or is a result of bias due to the assay conditions would require the characterization of a larger number of enzymes from both microenvironments in the Los Rueldos AMD system. The capacity of six of the enzymes from the Los Rueldos AMD formation, and thus the bacteria that contain them, for degrading acrylic- and terephthalate-like esters is noticeable. These enzymes could potentially be of use in developing plastic degradation strategies that have yet to be explored. In this context, the taxonomic distribution of top protein hits and the results of genome linguistics analysis suggested that the metagenomic fragments containing the six characterized enzymes that can potentially degrade plastic substrates most likely belong to <i>Actinobacteria</i> (genera related to <i>Acidithrix</i> and <i>Acidimicrobium</i>/<i>Ferrimicrobium</i>), <i>Acidobacteria</i>, and <i>Proteobacteria</i> (some are related to <i>Acidiphilium</i>). These are groups of acidophiles that have been largely neglected with respect to enzyme discovery.</p> <p class="mb15">Polyethylene terephthalate-degrading bioprospecting has shown that only a tiny fraction of carboxylic ester hydrolases can degrade PET and its intermediates BHET and MHET, including Actinomyces (i.e., <i>Thermobifida</i>, <i>Thermomonospora</i>, and <i>Saccharomonospora</i>), <i>Bacillus</i>, <i>Firmicutes</i> (e.g., <i>Clostridium</i>), <i>Bacteroidetes</i>, <i>Proteobacteria</i> (e.g., <i>Pseudomonas</i>, <i>Enterobacteria</i>, and <i>Ideonella sakaiensis</i>), and fungi [<i>Fusarium</i> and <i>Thermomyces</i> (<i>Humicola</i>)] (for a recent example, see <a href="#B68">Yoshida et al., 2016</a>; <a href="#B17">Danso et al., 2018</a>; <a href="#B11">Bollinger et al., 2020</a>; <a href="#B67">Yan et al., 2021</a>). In this study, we found that bacteria from the genera <i>Acidithrix</i> (the host of Est<sub><i>A</i>1</sub>), <i>Acidimicrobium</i>/<i>Ferrimicrobium</i> (the host of Est<sub><i>A</i>2</sub>), and unknown genera from the phylum <i>Proteobacteria</i> (the hosts of Est<sub><i>A</i>5</sub>, Est<sub><i>A</i>6</sub>, Est<sub><i>A</i>8</sub>, and Est<sub><i>B</i>2</sub>) could degrade BHET and could potentially degrade PET or PET oligomers under conditions yet to be explored, as no PET hydrolysis was detected under the assay conditions employed herein. Thus, the metagenomics approach applied herein expands the range of microorganisms containing enzymes supporting BHET hydrolysis and, possibly, PET depolymerization. For effective PET hydrolysis, in addition to a high degradation rate at 40–70°C, a broad range of pH stability (toward both the alkaline and acidic ranges) is one of the prerequisites of applied enzymes (<a href="#B49">Maurya et al., 2020</a>), and these features were characteristic of some of the esterases from Los Rueldos reported herein. Further studies will reveal the catalytic efficiency and stability of hydrolases from Los Rueldos AMD systems to establish PET degradation systems or to support these systems in combination with other known PET-degrading enzymes.</p> <p class="mb0">It is plausible that the capacity to degrade plastic substrates comes from the adaptation of the active sites of enzymes to metabolize microbial polymeric substances that are naturally occurring in AMDs. Thus, in Los Rueldos, as in other AMDs such as the Richmond Mine at Iron Mountain (<a href="#B41">Jiao et al., 2010</a>, <a href="#B42">2011</a>; <a href="#B47">Martínez-Martínez et al., 2013</a>), the organisms present might contribute to the use/degradation of extracellular polymeric substances (EPS), which requires a broad range of enzymes (<a href="#B26">Flemming and Wingender, 2010</a>).</p> <a id="h6" name="h6"></a><h2>Data Availability Statement</h2> <p class="mb0">The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/<a href="#S9">Supplementary Material</a>.</p> <a id="h7" name="h7"></a><h2>Author Contributions</h2> <p class="mb0">MF and AP conceived this study. MM-M contributed to gene cloning and expression. PV, MM-M, LF-L, and MF performed biochemical data and interpreted the data. SR and VG performed the 3D modeling. CM-G and MF contributed sample processing and library construction. OG contributed the phylogenetic analysis. MF drafted and revised the manuscript. All authors discussed, read, approved the manuscript, and authorized its submission for publication.</p> <a id="fun1" name="fun1"></a><h2>Funding</h2> <p class="mb0">This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We acknowledge financial support under Grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET, GA No. 604814) (MF), BIO2017-85522-R (MF), PID2020-112758RB-I00 (MF), PID2019-106370RB-I00 (VG), and PDC2021-121534-I00 (MF) from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (<a href="https://doi.org/10.13039/501100011033">Doi: 10.13039/501100011033</a>), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU/PRTR”), and Grant 2020AEP061 (MF) from the Agencia Estatal CSIC. OG acknowledges the support of the Centre for Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) <i>via</i> the Welsh Government. SR thanks the Spanish Ministry of Science and Innovation for a Ph.D. fellowship (FPU19/00608).</p> <a id="conf1" name="conf1"></a><h2>Conflict of Interest</h2> <p class="mb0">The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p> <a id="disclaimer1" name="disclaimer1"></a><h2>Publisher’s Note</h2> <p class="mb0">All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p> <a id="ack1" name="ack1"></a><h2>Acknowledgments</h2> <p class="mb0">We acknowledge David Almendral for supporting the circular dichroism analysis and Rafael Bargiela for preparation of <a href="#F2">Figure 2</a>. We also acknowledge the support of Cristina Coscolín for supporting the BHET degradation tests and Jesús Sánchez for the support in the study of acid mine drainage (AMD) systems and the evaluation and review of this work.</p> <a id="S9" name="S9"></a><h2>Supplementary Material</h2> <p class="mb0">The Supplementary Material for this article can be found online at: <a href="https://www.frontiersin.org/articles/10.3389/fmicb.2022.868839/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fmicb.2022.868839/full#supplementary-material</a></p> <a id="refer1" name="refer1"></a><h2>References</h2> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B1" id="B1"></a>Adrio, J. L., and Demain, A. L. (2014). Microbial enzymes: tools for biotechnological processes. <i>Biomolecules</i> 4, 117–139. doi: 10.3390/biom4010117</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/24970208" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3390/biom4010117" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+enzymes%3A+tools+for+biotechnological+processes%2E&journal=Biomolecules&author=Adrio+J.+L.&author=Demain+A.+L.&publication_year=2014&volume=4&pages=117–139" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B2" id="B2"></a>Alcaide, M., Tchigvintsev, A., Martinez-Martinez, M., Popovic, A., Reva, O. N., Lafraya, A., et al. (2015). Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp <i>Rimicaris exoculata</i> by using functional metagenomics. <i>Appl. Environ. Microbiol</i>. 81, 2125–2136. doi: 10.1128/AEM.03387-14</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/25595762" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.03387-14" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Identification+and+characterization+of+carboxyl+esterases+of+gill+chamber-associated+microbiota+in+the+deep-sea+shrimp+Rimicaris+exoculata+by+using+functional+metagenomics%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Alcaide+M.&author=Tchigvintsev+A.&author=Martinez-Martinez+M.&author=Popovic+A.&author=Reva+O.+N.&author=Lafraya+A.&publication_year=2015&volume=81&pages=2125–2136" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B3" id="B3"></a>Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. <i>Nucleic Acids Res</i>. 25, 3389–3402. doi: 10.1093/nar/25.17.3389</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/9254694" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1093/nar/25.17.3389" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Gapped+BLAST+and+PSI-BLAST%3A+a+new+generation+of+protein+database+search+programs%2E&journal=Nucleic+Acids+Res%2E&author=Altschul+S.+F.&author=Madden+T.+L.&author=Schäffer+A.+A.&author=Zhang+J.&author=Zhang+Z.&author=Miller+W.&publication_year=1997&volume=25&pages=3389–3402" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B4" id="B4"></a>Arpigny, J. L., and Jaeger, K. E. (1999). Bacterial lipolytic enzymes: classification and properties. <i>Biochem. J.</i> 343, 177–183. doi: 10.1042/bj3430177</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1042/bj3430177" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Bacterial+lipolytic+enzymes%3A+classification+and+properties%2E&journal=Biochem%2E+J%2E&author=Arpigny+J.+L.&author=Jaeger+K.+E.&publication_year=1999&volume=343&pages=177–183" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B5" id="B5"></a>Baker, B. J., Comolli, L. R., Dick, G. J., Hauser, L. J., Hyatt, D., Dill, B. D., et al. (2010). Enigmatic, ultrasmall, uncultivated Archaea. <i>Proc. Natl. Acad. Sci. U.S.A.</i> 107, 8806–8811. doi: 10.1073/pnas.0914470107</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/20421484" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1073/pnas.0914470107" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Enigmatic%2C+ultrasmall%2C+uncultivated+Archaea%2E&journal=Proc%2E+Natl%2E+Acad%2E+Sci%2E+U%2ES%2EA%2E&author=Baker+B.+J.&author=Comolli+L.+R.&author=Dick+G.+J.&author=Hauser+L.+J.&author=Hyatt+D.&author=Dill+B.+D.&publication_year=2010&volume=107&pages=8806–8811" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B6" id="B6"></a>Baker, B. J., Tyson, G. W., Webb, R. I., Flanagan, J., Hugenholtz, P., Allen, E. E., et al. (2006). Lineages of acidophilic archaea revealed by community genomic analysis. <i>Science</i> 314, 1933–1935. doi: 10.1126/science.1132690</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/17185602" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1126/science.1132690" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Lineages+of+acidophilic+archaea+revealed+by+community+genomic+analysis%2E&journal=Science&author=Baker+B.+J.&author=Tyson+G.+W.&author=Webb+R.+I.&author=Flanagan+J.&author=Hugenholtz+P.&author=Allen+E.+E.&publication_year=2006&volume=314&pages=1933–1935" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B7" id="B7"></a>Barth, S., Fischer, M., Schmid, R. D., and Pleiss, J. (2004). The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions. <i>Bioinformatics</i> 20, 2845–2847. doi: 10.1093/bioinformatics/bth284</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/15117755" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1093/bioinformatics/bth284" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+database+of+epoxide+hydrolases+and+haloalkane+dehalogenases%3A+one+structure%2C+many+functions%2E&journal=Bioinformatics&author=Barth+S.&author=Fischer+M.&author=Schmid+R.+D.&author=Pleiss+J.&publication_year=2004&volume=20&pages=2845–2847" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B8" id="B8"></a>Bauer, T. L., Buchholz, P. C. F., and Pleiss, J. (2020). The modular structure of α/β-hydrolases. <i>FEBS J.</i> 287, 1035–1053. doi: 10.1111/febs.15071</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/31545554" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1111/febs.15071" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+modular+structure+of+α%2Fβ-hydrolases%2E&journal=FEBS+J%2E&author=Bauer+T.+L.&author=Buchholz+P.+C.+F.&author=Pleiss+J.&publication_year=2020&volume=287&pages=1035–1053" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B9" id="B9"></a>Baweja, M., Nain, L., Kawarabayasi, Y., and Shukla, P. (2016). Current technological improvements in enzymes toward their biotechnological applications. <i>Front. Microbiol.</i> 7:965. doi: 10.3389/fmicb.2016.00965</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/27379087" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fmicb.2016.00965" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Current+technological+improvements+in+enzymes+toward+their+biotechnological+applications%2E&journal=Front%2E+Microbiol%2E&author=Baweja+M.&author=Nain+L.&author=Kawarabayasi+Y.&author=Shukla+P.&publication_year=2016&volume=7" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B10" id="B10"></a>Bertin, P. N., Heinrich-Salmeron, A., Pelletier, E., Goulhen-Chollet, F., Arsène-Ploetze, F., Gallien, S., et al. (2011). Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics. <i>ISME J</i>. 5, 1735–1747. doi: 10.1038/ismej.2011.51</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/21562598" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/ismej.2011.51" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Metabolic+diversity+among+main+microorganisms+inside+an+arsenic-rich+ecosystem+revealed+by+meta-+and+proteo-genomics%2E&journal=ISME+J%2E&author=Bertin+P.+N.&author=Heinrich-Salmeron+A.&author=Pelletier+E.&author=Goulhen-Chollet+F.&author=Arsène-Ploetze+F.&author=Gallien+S.&publication_year=2011&volume=5&pages=1735–1747" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B11" id="B11"></a>Bollinger, A., Thies, S., Knieps-Grünhagen, E., Gertzen, C., Kobus, S., Höppner, A., et al. (2020). A Novel polyester hydrolase from the marine bacterium <i>Pseudomonas aestusnigri</i> Structural and functional insights. <i>Front. Microbiol.</i> 11:114. doi: 10.3389/fmicb.2020.00114</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/32117139" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fmicb.2020.00114" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+Novel+polyester+hydrolase+from+the+marine+bacterium+Pseudomonas+aestusnigri+Structural+and+functional+insights%2E&journal=Front%2E+Microbiol%2E&author=Bollinger+A.&author=Thies+S.&author=Knieps-Grünhagen+E.&author=Gertzen+C.&author=Kobus+S.&author=Höppner+A.&publication_year=2020&volume=11" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B12" id="B12"></a>Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. <i>Anal. Biochem</i>. 72, 248–254. doi: 10.1006/abio.1976.9999</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/942051" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1006/abio.1976.9999" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+rapid+and+sensitive+method+for+the+quantification+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%2E&journal=Anal%2E+Biochem%2E&author=Bradford+M.+M.&publication_year=1976&volume=72&pages=248–254" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B13" id="B13"></a>Cai, K., Delaviz, Y., Banh, M., Guo, Y., and Santerre, J. P. (2014). Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process. <i>Dent Mater</i>. 30, 848–860. doi: 10.1016/j.dental.2014.05.031</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/24950808" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1016/j.dental.2014.05.031" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Biodegradation+of+composite+resin+with+ester+linkages%3A+identifying+human+salivary+enzyme+activity+with+a+potential+role+in+the+esterolytic+process%2E&journal=Dent+Mater%2E&author=Cai+K.&author=Delaviz+Y.&author=Banh+M.&author=Guo+Y.&author=Santerre+J.+P.&publication_year=2014&volume=30&pages=848–860" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B14" id="B14"></a>Chen, L. X., Huang, L. N., Méndez-García, C., Kuang, J. L., Hua, Z. S., Liu, J., et al. (2016). Microbial communities, processes and functions in acid mine drainage ecosystems. <i>Curr. Opin. Biotechnol</i>. 38, 150–158. doi: 10.1016/j.copbio.2016.01.013</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26921733" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1016/j.copbio.2016.01.013" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+communities%2C+processes+and+functions+in+acid+mine+drainage+ecosystems%2E&journal=Curr%2E+Opin%2E+Biotechnol%2E&author=Chen+L.+X.&author=Huang+L.+N.&author=Méndez-García+C.&author=Kuang+J.+L.&author=Hua+Z.+S.&author=Liu+J.&publication_year=2016&volume=38&pages=150–158" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B15" id="B15"></a>Chen, L. X., Méndez-García, C., Dombrowski, N., Servín-Garcidueñas, L. E., Eloe-Fadrosh, E. A., Fang, B. Z., et al. (2018). Metabolic versatility of small archaea Micrarchaeota and Parvarchaeota. <i>ISME J.</i> 12, 756–775. doi: 10.1038/s41396-017-0002-z</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/29222443" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/s41396-017-0002-z" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Metabolic+versatility+of+small+archaea+Micrarchaeota+and+Parvarchaeota%2E&journal=ISME+J%2E&author=Chen+L.+X.&author=Méndez-García+C.&author=Dombrowski+N.&author=Servín-Garcidueñas+L.+E.&author=Eloe-Fadrosh+E.+A.&author=Fang+B.+Z.&publication_year=2018&volume=12&pages=756–775" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B16" id="B16"></a>Daiha Kde, G., Angeli, R., de Oliveira, S. D., and Almeida, R. V. (2015). Are lipases still important biocatalysts? a study of scientific publications and patents for technological forecasting. <i>PLoS One</i> 10:e0131624. doi: 10.1371/journal.pone.0131624</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26111144" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1371/journal.pone.0131624" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Are+lipases+still+important+biocatalysts%B4+a+study+of+scientific+publications+and+patents+for+technological+forecasting%2E&journal=PLoS+One&author=Daiha&author=Kde+G.&author=Angeli+R.&author=de+Oliveira+S.+D.&author=Almeida+R.+V.&publication_year=2015&volume=10" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B17" id="B17"></a>Danso, D., Schmeisser, C., Chow, J., Zimmermann, W., Wei, R., Leggewie, C., et al. (2018). New insights into the function and global distribution of polyethylene terephthalate (PET)-degrading bacteria and enzymes in marine and terrestrial metagenomes. <i>Appl. Environ. Microbiol.</i> 84, e2773–e2717. doi: 10.1128/AEM.02773-17</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/29427431" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.02773-17" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=New+insights+into+the+function+and+global+distribution+of+polyethylene+terephthalate+%28PET%29-degrading+bacteria+and+enzymes+in+marine+and+terrestrial+metagenomes%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Danso+D.&author=Schmeisser+C.&author=Chow+J.&author=Zimmermann+W.&author=Wei+R.&author=Leggewie+C.&publication_year=2018&volume=84&pages=e2773–e2717" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B18" id="B18"></a>Delavat, F., Phalip, V., Forster, A., Plewniak, F., Lett, M. C., and Lièvremont, D. (2012). Amylases without known homologues discovered in an acid mine drainage: significance and impact. <i>Sci. Rep</i>. 2:354. doi: 10.1038/srep00354</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/22482035" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/srep00354" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Amylases+without+known+homologues+discovered+in+an+acid+mine+drainage%3A+significance+and+impact%2E&journal=Sci%2E+Rep%2E&author=Delavat+F.&author=Phalip+V.&author=Forster+A.&author=Plewniak+F.&author=Lett+M.+C.&author=Lièvremont+D.&publication_year=2012&volume=2" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B19" id="B19"></a>Donahue, Craig, J., Exline, Jennifer, A., and Warner Cynthia. (2003). Chemical recycling of pop bottles: the synthesis of dibenzyl terephthalate from the plastic polyethylene terephthalate. <i>J. Chem. Educ.</i> 80:79. doi: 10.1021/ed080p79</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1021/ed080p79" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Chemical+recycling+of+pop+bottles%3A+the+synthesis+of+dibenzyl+terephthalate+from+the+plastic+polyethylene+terephthalate%2E&journal=J%2E+Chem%2E+Educ%2E&author=Donahue&author=Craig+J.&author=Exline&author=Jennifer+A.&publication_year=2003&volume=80" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B20" id="B20"></a>Dopson, M., Baker-Austin, C., Hind, A., Bowman, J. P., and Bond, P. L. (2004). Characterization of <i>Ferroplasma</i> isolates and <i>Ferroplasma acidarmanus</i> sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments. <i>Appl. Environ. Microbiol</i>. 70, 2079–2088. doi: 10.1128/AEM.70.4.2079-2088.2004</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/15066799" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.70.4.2079-2088.2004" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Characterization+of+Ferroplasma+isolates+and+Ferroplasma+acidarmanus+sp%2E+nov%2E%2C+extreme+acidophiles+from+acid+mine+drainage+and+industrial+bioleaching+environments%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Dopson+M.&author=Baker-Austin+C.&author=Hind+A.&author=Bowman+J.+P.&author=Bond+P.+L.&publication_year=2004&volume=70&pages=2079–2088" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B21" id="B21"></a>Ferrer, M., Bargiela, R., Martínez-Martínez, M., Mir, J., Koch, R., Golyshina, O. V., et al. (2015). Biodiversity for biocatalysis: a review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes. <i>Biocatal. Biotransform</i>. 33, 235–249. doi: 10.3109/10242422.2016.1151416</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.3109/10242422.2016.1151416" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Biodiversity+for+biocatalysis%3A+a+review+of+the+α%2Fβ-hydrolase+fold+superfamily+of+esterases-lipases+discovered+in+metagenomes%2E&journal=Biocatal%2E+Biotransform%2E&author=Ferrer+M.&author=Bargiela+R.&author=Martínez-Martínez+M.&author=Mir+J.&author=Koch+R.&author=Golyshina+O.+V.&publication_year=2015&volume=33&pages=235–249" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B22" id="B22"></a>Ferrer, M., Martínez-Martínez, M., Bargiela, R., Streit, W. R., Golyshina, O. V., and Golyshin, P. N. (2016). Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. <i>Microb Biotechnol</i>. 9, 22–34. doi: 10.1111/1751-7915.12309</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26275154" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1111/1751-7915.12309" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Estimating+the+success+of+enzyme+bioprospecting+through+metagenomics%3A+current+status+and+future+trends%2E&journal=Microb+Biotechnol%2E&author=Ferrer+M.&author=Martínez-Martínez+M.&author=Bargiela+R.&author=Streit+W.+R.&author=Golyshina+O.+V.&author=Golyshin+P.+N.&publication_year=2016&volume=9&pages=22–34" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B23" id="B23"></a>Ferrer, M., Méndez-García, C., Bargiela, R., Chow, J., Alonso, S., García-Moyano, A., et al. (2019). Decoding the ocean’s microbiological secrets for marine enzyme biodiscovery. <i>FEMS Microbiol. Lett</i>. 366, 0378–1097. doi: 10.1093/femsle/fny285</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/30534987" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1093/femsle/fny285" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Decoding+the+ocean’s+microbiological+secrets+for+marine+enzyme+biodiscovery%2E&journal=FEMS+Microbiol%2E+Lett%2E&author=Ferrer+M.&author=Méndez-García+C.&author=Bargiela+R.&author=Chow+J.&author=Alonso+S.&author=García-Moyano+A.&publication_year=2019&volume=366&pages=0378–1097" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B24" id="B24"></a>Finer, Y., and Santerre, J. P. (2004). Salivary esterase activity and its association with the biodegradation of dental composites. <i>J. Dent Res</i>. 83, 22–26. doi: 10.1177/154405910408300105</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/14691108" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1177/154405910408300105" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Salivary+esterase+activity+and+its+association+with+the+biodegradation+of+dental+composites%2E&journal=J%2E+Dent+Res%2E&author=Finer+Y.&author=Santerre+J.+P.&publication_year=2004&volume=83&pages=22–26" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B25" id="B25"></a>Fischer, M., and Pleiss, J. (2003). The Lipase Engineering Database: a navigation and analysis tool for protein families. <i>Nucleic Acids Res</i>. 31, 319–321. doi: 10.1093/nar/gkg015</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/12520012" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1093/nar/gkg015" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+Lipase+Engineering+Database%3A+a+navigation+and+analysis+tool+for+protein+families%2E&journal=Nucleic+Acids+Res%2E&author=Fischer+M.&author=Pleiss+J.&publication_year=2003&volume=31&pages=319–321" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B26" id="B26"></a>Flemming, H. C., and Wingender, J. (2010). The biofilm matrix. <i>Nat. Rev. Microbiol.</i> 8, 623–633. doi: 10.3390/nano10081527</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/32759748" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3390/nano10081527" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+biofilm+matrix%2E&journal=Nat%2E+Rev%2E+Microbiol%2E&author=Flemming+H.+C.&author=Wingender+J.&publication_year=2010&volume=8&pages=623–633" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B27" id="B27"></a>Fütterer, O., Angelov, A., Liesegang, H., Gottschalk, G., Schleper, C., Schepers, B., et al. (2004). Genome sequence of <i>Picrophilus torridus</i> and its implications for life around pH 0. <i>Proc. Natl. Acad. Sci. U.S.A</i>. 101, 9091–9096. doi: 10.1073/pnas.0401356101</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/15184674" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1073/pnas.0401356101" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Genome+sequence+of+Picrophilus+torridus+and+its+implications+for+life+around+pH+0%2E&journal=Proc%2E+Natl%2E+Acad%2E+Sci%2E+U%2ES%2EA%2E&author=Fütterer+O.&author=Angelov+A.&author=Liesegang+H.&author=Gottschalk+G.&author=Schleper+C.&author=Schepers+B.&publication_year=2004&volume=101&pages=9091–9096" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B28" id="B28"></a>Gavrilov, S. N., Korzhenkov, A. A., Kublanov, I. V., Bargiela, R., Zamana, L. V., Popova, A. A., et al. (2019). Microbial communities of polymetallic deposits’ acidic ecosystems of continental climatic zone with high temperature contrasts. <i>Front. Microbiol</i>. 10:1573. doi: 10.3389/fmicb.2019.01573</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/31379766" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fmicb.2019.01573" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+communities+of+polymetallic+deposits’+acidic+ecosystems+of+continental+climatic+zone+with+high+temperature+contrasts%2E&journal=Front%2E+Microbiol%2E&author=Gavrilov+S.+N.&author=Korzhenkov+A.+A.&author=Kublanov+I.+V.&author=Bargiela+R.&author=Zamana+L.+V.&author=Popova+A.+A.&publication_year=2019&volume=10" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B29" id="B29"></a>Giunta, C. I., Cea-Rama, I., Alonso, S., Briand, M. L., Bargiela, R., Coscolín, C., et al. (2020). Tuning the properties of natural promiscuous enzymes by engineering their nano-environment. <i>ACS Nano</i> 14, 17652–17664. doi: 10.1021/acsnano.0c08716</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/33306346" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1021/acsnano.0c08716" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Tuning+the+properties+of+natural+promiscuous+enzymes+by+engineering+their+nano-environment%2E&journal=ACS+Nano&author=Giunta+C.+I.&author=Cea-Rama+I.&author=Alonso+S.&author=Briand+M.+L.&author=Bargiela+R.&author=Coscolín+C.&publication_year=2020&volume=14&pages=17652–17664" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B30" id="B30"></a>Golyshina, O. V. (2011). Environmental, biogeographic, and biochemical patterns of archaea of the family <i>Ferroplasmaceae</i>. <i>Appl. Environ. Microbiol</i>. 77, 5071–5078. doi: 10.1128/AEM.00726-11</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/21685165" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.00726-11" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Environmental%2C+biogeographic%2C+and+biochemical+patterns+of+archaea+of+the+family+Ferroplasmaceae%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Golyshina+O.+V.&publication_year=2011&volume=77&pages=5071–5078" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B31" id="B31"></a>Golyshina, O. V., Golyshin, P. N., Timmis, K. N., and Ferrer, M. (2006). The ‘pH optimum anomaly’ of intracellular enzymes of <i>Ferroplasma acidiphilum</i>. <i>Environ. Microbiol</i>. 8, 416–425. doi: 10.1111/j.1462-2920.2005.00907.x</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/16478448" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1111/j.1462-2920.2005.00907.x" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+‘pH+optimum+anomaly’+of+intracellular+enzymes+of+Ferroplasma+acidiphilum%2E&journal=Environ%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Golyshin+P.+N.&author=Timmis+K.+N.&author=Ferrer+M.&publication_year=2006&volume=8&pages=416–425" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B32" id="B32"></a>Golyshina, O. V., Lünsdorf, H., Kublanov, I. V., Goldenstein, N. I., Hinrichs, K. U., and Golyshin, P. N. (2016). The novel extremely acidophilic, cell-wall-deficient archaeon <i>Cuniculiplasma divulgatum</i> gen.Nov.sp nov.represents a new family, <i>Cuniculiplasmataceae</i> fam. nov.of the order Thermoplasmatales. <i>Int. J. Syst. Evol. Microbiol</i>. 66, 332–340. doi: 10.1099/ijsem.0.000725</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26518885" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1099/ijsem.0.000725" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+novel+extremely+acidophilic%2C+cell-wall-deficient+archaeon+Cuniculiplasma+divulgatum+gen%2ENov%2Esp+nov%2Erepresents+a+new+family%2C+Cuniculiplasmataceae+fam%2E+nov%2Eof+the+order+Thermoplasmatales%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Lünsdorf+H.&author=Kublanov+I.+V.&author=Goldenstein+N.+I.&author=Hinrichs+K.+U.&author=Golyshin+P.+N.&publication_year=2016&volume=66&pages=332–340" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B33" id="B33"></a>Golyshina, O. V., Pivovarova, T. A., Karavaiko, G. I., Kondratéva, T. F., Moore, E. R., Abraham, W. R., et al. (2000). <i>Ferroplasma acidiphilum</i> gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the <i>Ferroplasmaceae</i> fam. nov., comprising a distinct lineage of the Archaea. <i>Int. J. Syst. Evol. Microbiol</i>. 3, 997–1006. doi: 10.1099/00207713-50-3-997</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/10843038" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1099/00207713-50-3-997" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Ferroplasma+acidiphilum+gen%2E+nov%2E%2C+sp%2E+nov%2E%2C+an+acidophilic%2C+autotrophic%2C+ferrous-iron-oxidizing%2C+cell-wall-lacking%2C+mesophilic+member+of+the+Ferroplasmaceae+fam%2E+nov%2E%2C+comprising+a+distinct+lineage+of+the+Archaea%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Pivovarova+T.+A.&author=Karavaiko+G.+I.&author=Kondratéva+T.+F.&author=Moore+E.+R.&author=Abraham+W.+R.&publication_year=2000&volume=3&pages=997–1006" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B34" id="B34"></a>Golyshina, O. V., Yakimov, M. M., Lünsdorf, H., Ferrer, M., Nimtz, M., Timmis, K. N., et al. (2009). <i>Acidiplasma aeolicum</i> gen. nov., sp. nov., a euryarchaeon of the family Ferroplasmaceae isolated from a hydrothermal pool, and transfer of <i>Ferroplasma cupricumulans</i> to <i>Acidiplasma cupricumulans</i> comb. nov. <i>Int. J. Syst. Evol. Microbiol</i>. 59, 2815–2823. doi: 10.1099/ijs.0.009639-0</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/19628615" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1099/ijs.0.009639-0" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Acidiplasma+aeolicum+gen%2E+nov%2E%2C+sp%2E+nov%2E%2C+a+euryarchaeon+of+the+family+Ferroplasmaceae+isolated+from+a+hydrothermal+pool%2C+and+transfer+of+Ferroplasma+cupricumulans+to+Acidiplasma+cupricumulans+comb%2E+nov%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Yakimov+M.+M.&author=Lünsdorf+H.&author=Ferrer+M.&author=Nimtz+M.&author=Timmis+K.+N.&publication_year=2009&volume=59&pages=2815–2823" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B35" id="B35"></a>Gomes, I., Gomes, J., and Steiner, W. (2003). Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium <i>Rhodothermus marinus</i>: production and partial characterization. <i>Bioresour. Technol</i>. 90, 207–214. doi: 10.1016/s0960-8524(03)00110-x</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1016/s0960-8524(03)00110-x" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Highly+thermostable+amylase+and+pullulanase+of+the+extreme+thermophilic+eubacterium+Rhodothermus+marinus%3A+production+and+partial+characterization%2E&journal=Bioresour%2E+Technol%2E&author=Gomes+I.&author=Gomes+J.&author=Steiner+W.&publication_year=2003&volume=90&pages=207–214" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B36" id="B36"></a>Guazzaroni, M. E., Morgante, V., Mirete, S., and González-Pastor, J. E. (2013). Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment. <i>Environ. Microbiol</i>. 15, 1088–1102. doi: 10.1111/1462-2920.12021</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/23145860" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1111/1462-2920.12021" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Novel+acid+resistance+genes+from+the+metagenome+of+the+Tinto+River%2C+an+extremely+acidic+environment%2E&journal=Environ%2E+Microbiol%2E&author=Guazzaroni+M.+E.&author=Morgante+V.&author=Mirete+S.&author=González-Pastor+J.+E.&publication_year=2013&volume=15&pages=1088–1102" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B37" id="B37"></a>Hajighasemi, M., Tchigvintsev, A., Nocek, B., Flick, R., Popovic, A., Hai, T., et al. (2018). Screening and characterization of novel polyesterases from environmental metagenomes with high hydrolytic activity against synthetic polyesters. <i>Environ. Sci. Technol</i>. 52, 12388–12401. doi: 10.1021/acs.est.8b04252</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/30284819" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1021/acs.est.8b04252" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Screening+and+characterization+of+novel+polyesterases+from+environmental+metagenomes+with+high+hydrolytic+activity+against+synthetic+polyesters%2E&journal=Environ%2E+Sci%2E+Technol%2E&author=Hajighasemi+M.&author=Tchigvintsev+A.&author=Nocek+B.&author=Flick+R.&author=Popovic+A.&author=Hai+T.&publication_year=2018&volume=52&pages=12388–12401" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B38" id="B38"></a>He, Y., Zhang, F., Saleh, E., Vaithilingam, J., Aboulkhair, N., Begines, B., et al. (2017). A tripropylene glycol diacrylate-based polymeric support ink for material jetting. <i>Addit. Manuf</i>. 16, 153–161. doi: 10.1016/j.addma.2017.06.001</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1016/j.addma.2017.06.001" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+tripropylene+glycol+diacrylate-based+polymeric+support+ink+for+material+jetting%2E&journal=Addit%2E+Manuf%2E&author=He+Y.&author=Zhang+F.&author=Saleh+E.&author=Vaithilingam+J.&author=Aboulkhair+N.&author=Begines+B.&publication_year=2017&volume=16&pages=153–161" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B39" id="B39"></a>Huang, L.-N., Kuang, J.-L., and Shu, W.-S. (2016). Microbial ecology and evolution in the acid mine drainage model system. <i>Trends Microbiol.</i> 24, 581–593. doi: 10.1016/j.tim.2016.03.004</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/27050827" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1016/j.tim.2016.03.004" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+ecology+and+evolution+in+the+acid+mine+drainage+model+system%2E&journal=Trends+Microbiol%2E&author=Huang+L.-N.&author=Kuang+J.-L.&author=Shu+W.-S.&publication_year=2016&volume=24&pages=581–593" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B40" id="B40"></a>Jackson, B. R., Noble, C., Lavesa-Curto, M., Bond, P. L., and Bowater, R. P. (2007). Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “<i>Ferroplasma acidarmanus</i>” Fer1. <i>Extremophiles</i> 11, 315–327. doi: 10.1007/s00792-006-0041-2</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/17136487" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1007/s00792-006-0041-2" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Characterization+of+an+ATP-dependent+DNA+ligase+from+the+acidophilic+archaeon+“Ferroplasma+acidarmanus”+Fer1%2E&journal=Extremophiles&author=Jackson+B.+R.&author=Noble+C.&author=Lavesa-Curto+M.&author=Bond+P.+L.&author=Bowater+R.+P.&publication_year=2007&volume=11&pages=315–327" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B41" id="B41"></a>Jiao, Y., Cody, G. D., Harding, A. K., Wilmes, P., Schrenk, M., Wheeler, K. E., et al. (2010). Characterization of extracellular polymeric substances from acidophilic microbial biofilms. <i>Appl. Environ. Microbiol</i>. 76, 2916–2922. doi: 10.1128/AEM.02289-09</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/20228116" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.02289-09" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Characterization+of+extracellular+polymeric+substances+from+acidophilic+microbial+biofilms%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Jiao+Y.&author=Cody+G.+D.&author=Harding+A.+K.&author=Wilmes+P.&author=Schrenk+M.&author=Wheeler+K.+E.&publication_year=2010&volume=76&pages=2916–2922" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B42" id="B42"></a>Jiao, Y., D’haeseleer, P., Dill, B. D., Shah, M., Verberkmoes, N. C., Hettich, R. L., et al. (2011). Identification of biofilm matrix associated proteins from an acid mine drainage microbial community. <i>Appl. Environ. Microbiol</i>. 77, 5230–5237. doi: 10.1128/AEM.03005-10</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/21685158" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.03005-10" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Identification+of+biofilm+matrix+associated+proteins+from+an+acid+mine+drainage+microbial+community%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Jiao+Y.&author=D’haeseleer+P.&author=Dill+B.+D.&author=Shah+M.&author=Verberkmoes+N.+C.&author=Hettich+R.+L.&publication_year=2011&volume=77&pages=5230–5237" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B43" id="B43"></a>Johnson, D. B., and Quatrini, R. (2020). Acidophile microbiology in space and time. <i>Curr. Issues Mol. Biol.</i> 39, 63–76. doi: 10.21775/cimb.039.063</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/32083998" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.21775/cimb.039.063" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Acidophile+microbiology+in+space+and+time%2E&journal=Curr%2E+Issues+Mol%2E+Biol%2E&author=Johnson+D.+B.&author=Quatrini+R.&publication_year=2020&volume=39&pages=63–76" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B44" id="B44"></a>Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021). Highly accurate protein structure prediction with AlphaFold. <i>Nature</i> 596, 583–589. doi: 10.1038/s41586-021-03819-2</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/34265844" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/s41586-021-03819-2" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Highly+accurate+protein+structure+prediction+with+AlphaFold%2E&journal=Nature&author=Jumper+J.&author=Evans+R.&author=Pritzel+A.&author=Green+T.&author=Figurnov+M.&author=Ronneberger+O.&publication_year=2021&volume=596&pages=583–589" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B45" id="B45"></a>Korzhenkov, A. A., Toshchakov, S. V., Bargiela, R., Gibbard, H., Ferrer, M., Teplyuk, A. V., et al. (2019). Archaea dominate the microbial community in an ecosystem with low-to-moderate temperature and extreme acidity. <i>Microbiome</i> 7:11. doi: 10.1186/s40168-019-0623-8</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/30691532" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1186/s40168-019-0623-8" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Archaea+dominate+the+microbial+community+in+an+ecosystem+with+low-to-moderate+temperature+and+extreme+acidity%2E&journal=Microbiome&author=Korzhenkov+A.+A.&author=Toshchakov+S.+V.&author=Bargiela+R.&author=Gibbard+H.&author=Ferrer+M.&author=Teplyuk+A.+V.&publication_year=2019&volume=7" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B46" id="B46"></a>Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. <i>Nature</i> 227, 680–685. doi: 10.1038/227680a0</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/5432063" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/227680a0" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4%2E&journal=Nature&author=Laemmli+U.+K.&publication_year=1970&volume=227&pages=680–685" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B47" id="B47"></a>Martínez-Martínez, M., Alcaide, M., Tchigvintsev, A., Reva, O., Polaina, J., and Bargiela, R. (2013). Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach. <i>Appl. Environ. Microbiol</i>. 79, 3553–3562. doi: 10.1128/AEM.00240-13</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/23542620" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1128/AEM.00240-13" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Biochemical+diversity+of+carboxyl+esterases+and+lipases+from+Lake+Arreo+%28Spain%29%3A+a+metagenomic+approach%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Martínez-Martínez+M.&author=Alcaide+M.&author=Tchigvintsev+A.&author=Reva+O.&author=Polaina+J.&author=Bargiela+R.&publication_year=2013&volume=79&pages=3553–3562" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B48" id="B48"></a>Martínez-Martínez, M., Coscolín, C., Santiago, G., Chow, J., Stogios, P. J., and Bargiela, R. (2018). The Inmare Consortium.Determinants and prediction of esterase substrate promiscuity patterns. <i>ACS Chem. Biol.</i> 13, 225–234. doi: 10.1021/acschembio.7b00996</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/29182315" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1021/acschembio.7b00996" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=The+Inmare+Consortium%2EDeterminants+and+prediction+of+esterase+substrate+promiscuity+patterns%2E&journal=ACS+Chem%2E+Biol%2E&author=Martínez-Martínez+M.&author=Coscolín+C.&author=Santiago+G.&author=Chow+J.&author=Stogios+P.+J.&author=Bargiela+R.&publication_year=2018&volume=13&pages=225–234" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B49" id="B49"></a>Maurya, A., Bhattacharya, A., and Khare, S. K. (2020). Enzymatic remediation of polyethylene terephthalate (PET)-based polymers for effective management of plastic wastes: an Overview. <i>Front. Bioeng. Biotechnol</i>. 8:602325. doi: 10.3389/fbioe.2020.602325</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/33330434" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fbioe.2020.602325" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Enzymatic+remediation+of+polyethylene+terephthalate+%28PET%29-based+polymers+for+effective+management+of+plastic+wastes%3A+an+Overview%2E&journal=Front%2E+Bioeng%2E+Biotechnol%2E&author=Maurya+A.&author=Bhattacharya+A.&author=Khare+S.+K.&publication_year=2020&volume=8" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B50" id="B50"></a>Méndez-García, C., Mesa, V., Sprenger, R. R., Richter, M., Diez, M. S., and Solano, J. (2014). Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage. <i>ISME J</i>. 8, 1259–1274. doi: 10.1038/ismej.2013.242</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/24430486" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1038/ismej.2013.242" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+stratification+in+low+pH+oxic+and+suboxic+macroscopic+growths+along+an+acid+mine+drainage%2E&journal=ISME+J%2E&author=Méndez-García+C.&author=Mesa+V.&author=Sprenger+R.+R.&author=Richter+M.&author=Diez+M.+S.&author=Solano+J.&publication_year=2014&volume=8&pages=1259–1274" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B51" id="B51"></a>Méndez-García, C., Peláez, A. I., Mesa, V., Sánchez, J., Golyshina, O. V., and Ferrer, M. (2015). Microbial diversity and metabolic networks in acid mine drainage habitats. <i>Front. Microbiol.</i> 6:475. doi: 10.3389/fmicb.2015.00475</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26074887" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fmicb.2015.00475" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+diversity+and+metabolic+networks+in+acid+mine+drainage+habitats%2E&journal=Front%2E+Microbiol%2E&author=Méndez-García+C.&author=Peláez+A.+I.&author=Mesa+V.&author=Sánchez+J.&author=Golyshina+O.+V.&author=Ferrer+M.&publication_year=2015&volume=6" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B52" id="B52"></a>Ménigaud, S., Mallet, L., Picord, G., Churlaud, C., Borrel, A., and Deschavanne, P. (2012). GOHTAM: a website for ’Genomic Origin of Horizontal Transfers. <i>Align. Metagenom. Bioinform</i>. 28, 1270–1271. doi: 10.1093/bioinformatics/bts118</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/22426345" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1093/bioinformatics/bts118" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=GOHTAM%3A+a+website+for+’Genomic+Origin+of+Horizontal+Transfers%2E&journal=Align%2E+Metagenom%2E+Bioinform%2E&author=Ménigaud+S.&author=Mallet+L.&author=Picord+G.&author=Churlaud+C.&author=Borrel+A.&author=Deschavanne+P.&publication_year=2012&volume=28&pages=1270–1271" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B53" id="B53"></a>Nchedo Ariole, C., and George-West, O. (2020). Bioplastic degradation potential of microorganisms isolated from the soil. <i>Am. J. Chem. Eng</i>. 4:1. doi: 10.11648/j.ajcbe.20200401.11</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.11648/j.ajcbe.20200401.11" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Bioplastic+degradation+potential+of+microorganisms+isolated+from+the+soil%2E&journal=Am%2E+J%2E+Chem%2E+Eng%2E&author=Nchedo+Ariole+C.&author=George-West+O.&publication_year=2020&volume=4" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B54" id="B54"></a>Ohara, K., Unno, H., Oshima, Y., Hosoya, M., Fujino, N., Hirooka, K., et al. (2014). Structural insights into the low pH adaptation of a unique carboxylesterase from <i>Ferroplasma</i>: altering the pH optima of two carboxylesterases. <i>J. Biol. Chem</i>. 289, 24499–24510. doi: 10.1074/jbc.M113.521856</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/25043762" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1074/jbc.M113.521856" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Structural+insights+into+the+low+pH+adaptation+of+a+unique+carboxylesterase+from+Ferroplasma%3A+altering+the+pH+optima+of+two+carboxylesterases%2E&journal=J%2E+Biol%2E+Chem%2E&author=Ohara+K.&author=Unno+H.&author=Oshima+Y.&author=Hosoya+M.&author=Fujino+N.&author=Hirooka+K.&publication_year=2014&volume=289&pages=24499–24510" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B55" id="B55"></a>Pellis, A., Cantone, S., Ebert, C., and Gardossi, L. (2017). Evolving biocatalysis to meet bioeconomy challenges and opportunities. <i>New Biotechnol</i>. 40, 154–169. doi: 10.1016/j.nbt.2017.07.005</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/28743564" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1016/j.nbt.2017.07.005" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Evolving+biocatalysis+to+meet+bioeconomy+challenges+and+opportunities%2E&journal=New+Biotechnol%2E&author=Pellis+A.&author=Cantone+S.&author=Ebert+C.&author=Gardossi+L.&publication_year=2017&volume=40&pages=154–169" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B56" id="B56"></a>Peña-García, C., Martínez-Martínez, M., Reyes-Duarte, D., and Ferrer, M. (2016). High throughput screening of esterases, lipases and phospholipases in mutant and metagenomic libraries: a review. <i>Comb. Chem. High Throughput Screen</i> 19, 605–615. doi: 10.2174/1386207319666151110123927</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26552433" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.2174/1386207319666151110123927" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=High+throughput+screening+of+esterases%2C+lipases+and+phospholipases+in+mutant+and+metagenomic+libraries%3A+a+review%2E&journal=Comb%2E+Chem%2E+High+Throughput+Screen&author=Peña-García+C.&author=Martínez-Martínez+M.&author=Reyes-Duarte+D.&author=Ferrer+M.&publication_year=2016&volume=19&pages=605–615" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B57" id="B57"></a>Placido, A., Hai, T., Ferrer, M., Chernikova, T. N., Distaso, M., and Armstrong, D. (2015). Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase. <i>Appl. Microbiol. Biotechnol.</i> 99, 10031–10046. doi: 10.1007/s00253-015-6873-x</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26266751" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1007/s00253-015-6873-x" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Diversity+of+hydrolases+from+hydrothermal+vent+sediments+of+the+Levante+Bay%2C+Vulcano+Island+%28Aeolian+archipelago%29+identified+by+activity-based+metagenomics+and+biochemical+characterization+of+new+esterases+and+an+arabinopyranosidase%2E&journal=Appl%2E+Microbiol%2E+Biotechnol%2E&author=Placido+A.&author=Hai+T.&author=Ferrer+M.&author=Chernikova+T.+N.&author=Distaso+M.&author=Armstrong+D.&publication_year=2015&volume=99&pages=10031–10046" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B58" id="B58"></a>Pütz, A. (2006). <i>Isolierung, Identifizierung und Biochemische Charakterisierung Dialkylphthalat Spaltender Esterasen.</i> [Ph D thesis]. Düsseldorf: HeinrichHeine-Universität, 39–40.</p> <p class="ReferencesCopy2"><a href="http://scholar.google.com/scholar_lookup?&journal=Isolierung%2C+Identifizierung+und+Biochemische+Charakterisierung+Dialkylphthalat+Spaltender+Esterasen%2E&author=Pütz+A.&publication_year=2006&pages=39–40" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B59" id="B59"></a>Rao, L., Xue, Y., Zheng, Y., Lu, J. R., and Ma, Y. A. (2013). A novel alkaliphilic bacillus esterase belongs to the 13(th) bacterial lipolytic enzyme family. <i>PLoS One</i> 8:e60645. doi: 10.1371/journal.pone.0060645</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/23577139" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1371/journal.pone.0060645" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+novel+alkaliphilic+bacillus+esterase+belongs+to+the+13%28th%29+bacterial+lipolytic+enzyme+family%2E&journal=PLoS+One&author=Rao+L.&author=Xue+Y.&author=Zheng+Y.&author=Lu+J.+R.&author=Ma+Y.+A.&publication_year=2013&volume=8" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B60" id="B60"></a>Reyes-Duarte, D., Ferrer, M., and García-Arellano, H. (2012). Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries. <i>Methods Mol. Biol</i>. 861, 101–113. doi: 10.1007/978-1-61779-600-5_6</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1007/978-1-61779-600-5_6" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Functional-based+screening+methods+for+lipases%2C+esterases%2C+and+phospholipases+in+metagenomic+libraries%2E&journal=Methods+Mol%2E+Biol%2E&author=Reyes-Duarte+D.&author=Ferrer+M.&author=García-Arellano+H.&publication_year=2012&volume=861&pages=101–113" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B61" id="B61"></a>Santiago, G., Martínez-Martínez, M., Alonso, S., Bargiela, R., Coscolín, C., Golyshin, P. N., et al. (2018). Rational engineering of multiple active sites in an ester hydrolase. <i>Biochemistry</i> 57, 2245–2255. doi: 10.1021/acs.biochem.8b00274</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/29600855" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1021/acs.biochem.8b00274" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Rational+engineering+of+multiple+active+sites+in+an+ester+hydrolase%2E&journal=Biochemistry&author=Santiago+G.&author=Martínez-Martínez+M.&author=Alonso+S.&author=Bargiela+R.&author=Coscolín+C.&author=Golyshin+P.+N.&publication_year=2018&volume=57&pages=2245–2255" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B62" id="B62"></a>Scholtissek, A., Ullrich, S. R., Mühling, M., Schlömann, M., Paul, C. E., and Tischler, D. (2016). A thermophilic-like ene-reductase originating from an acidophilic iron oxidizer. <i>Appl. Microbiol. Biotechnol</i>. 101:2. doi: 10.1007/s00253-016-7782-3</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/27542380" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1007/s00253-016-7782-3" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+thermophilic-like+ene-reductase+originating+from+an+acidophilic+iron+oxidizer%2E&journal=Appl%2E+Microbiol%2E+Biotechnol%2E&author=Scholtissek+A.&author=Ullrich+S.+R.&author=Mühling+M.&author=Schlömann+M.&author=Paul+C.+E.&author=Tischler+D.&publication_year=2016&volume=101" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B63" id="B63"></a>Sharma, A., Kawarabayasi, Y., and Satyanarayana, T. (2012). Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications. <i>Extremophiles</i> 16, 1–19. doi: 10.1007/s00792-011-0402-3</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/22080280" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1007/s00792-011-0402-3" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Acidophilic+bacteria+and+archaea%3A+acid+stable+biocatalysts+and+their+potential+applications%2E&journal=Extremophiles&author=Sharma+A.&author=Kawarabayasi+Y.&author=Satyanarayana+T.&publication_year=2012&volume=16&pages=1–19" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B64" id="B64"></a>Thapa, S., Mishra, J., Arora, N., Mishra, P., Li, H., O’Hair, J., et al. (2020). Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation. <i>Rev. Environ. Sci. Biotechnol.</i> 19, 621–648. doi: 10.1007/s11157-020-09536-y</p> <p class="ReferencesCopy2"><a href="https://doi.org/10.1007/s11157-020-09536-y" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Microbial+cellulolytic+enzymes%3A+diversity+and+biotechnology+with+reference+to+lignocellulosic+biomass+degradation%2E&journal=Rev%2E+Environ%2E+Sci%2E+Biotechnol%2E&author=Thapa+S.&author=Mishra+J.&author=Arora+N.&author=Mishra+P.&author=Li+H.&author=O’Hair+J.&publication_year=2020&volume=19&pages=621–648" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B65" id="B65"></a>Van den Berg, B. (2010). Crystal structure of a full-length autotransporter. <i>J. Mol. Biol</i>. 396, 627–633. doi: 10.1016/j.jmb.2009.12.061</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/20060837" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1016/j.jmb.2009.12.061" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Crystal+structure+of+a+full-length+autotransporter%2E&journal=J%2E+Mol%2E+Biol%2E&author=Van+den+Berg+B.&publication_year=2010&volume=396&pages=627–633" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B66" id="B66"></a>Wu, X., Zhang, Q., Zhang, L., Liu, S., Chen, G., Zhang, H., et al. (2020). Insights into the role of exposed surface charged residues in the alkali-tolerance of GH11 xylanase. <i>Front. Microbiol.</i> 8:872. doi: 10.3389/fmicb.2020.00872</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/32457729" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.3389/fmicb.2020.00872" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Insights+into+the+role+of+exposed+surface+charged+residues+in+the+alkali-tolerance+of+GH11+xylanase%2E&journal=Front%2E+Microbiol%2E&author=Wu+X.&author=Zhang+Q.&author=Zhang+L.&author=Liu+S.&author=Chen+G.&author=Zhang+H.&publication_year=2020&volume=8" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B67" id="B67"></a>Yan, F., Wei, R., Cui, Q., Bornscheuer, U. T., and Liu, Y. J. (2021). Thermophilic whole-cell degradation of polyethylene terephthalate using engineered <i>Clostridium thermocellum</i>. <i>Microb Biotechnol.</i> 14, 374–385. doi: 10.1111/1751-7915.13580</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/32343496" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1111/1751-7915.13580" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=Thermophilic+whole-cell+degradation+of+polyethylene+terephthalate+using+engineered+Clostridium+thermocellum%2E&journal=Microb+Biotechnol%2E&author=Yan+F.&author=Wei+R.&author=Cui+Q.&author=Bornscheuer+U.+T.&author=Liu+Y.+J.&publication_year=2021&volume=14&pages=374–385" target="_blank">Google Scholar</a></p> </div> <div class="References" style="margin-bottom:0.5em;"> <p class="ReferencesCopy1"><a name="B68" id="B68"></a>Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., et al. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). <i>Science</i> 351, 1196–1199. doi: 10.1126/science.aad6359</p> <p class="ReferencesCopy2"><a href="https://pubmed.ncbi.nlm.nih.gov/26965627" target="_blank">PubMed Abstract</a> | <a href="https://doi.org/10.1126/science.aad6359" target="_blank">CrossRef Full Text</a> | <a href="http://scholar.google.com/scholar_lookup?&title=A+bacterium+that+degrades+and+assimilates+poly%28ethylene+terephthalate%29%2E&journal=Science&author=Yoshida+S.&author=Hiraga+K.&author=Takehana+T.&author=Taniguchi+I.&author=Yamaji+H.&author=Maeda+Y.&publication_year=2016&volume=351&pages=1196–1199" target="_blank">Google Scholar</a></p> </div> </div> <div class="thinLineM20"></div> <div class="AbstractSummary"> <p><span>Keywords</span>: acidophiles, acidophilic bacteria, acid mine drainage, biodiversity, extremozymes, esterase, metagenomics, plastic</p> <p><span>Citation:</span> Vidal P, Martínez-Martínez M, Fernandez-Lopez L, Roda S, Méndez-García C, Golyshina OV, Guallar V, Peláez AI and Ferrer M (2022) Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation. <i>Front. Microbiol.</i> 13:868839. doi: 10.3389/fmicb.2022.868839</p> <p id="timestamps"> <span>Received:</span> 03 February 2022; <span>Accepted:</span> 14 March 2022;<br><span>Published:</span> 19 May 2022.</p> <div> <p>Edited by:</p> <a href="https://loop.frontiersin.org/people/111623/overview">Rolf Daniel</a>, University of Göttingen, Germany</div> <div> <p>Reviewed by:</p> <a href="https://loop.frontiersin.org/people/231858/overview">Wolfgang R. Streit</a>, University of Hamburg, Germany<br> <a href="https://loop.frontiersin.org/people/1576541/overview">Jiujun Cheng</a>, University of Waterloo, Canada</div> <p><span>Copyright</span> © 2022 Vidal, Martínez-Martínez, Fernandez-Lopez, Roda, Méndez-García, Golyshina, Guallar, Peláez and Ferrer. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p> <p><span>*Correspondence:</span> Manuel Ferrer, <a id="encmail">bWZlcnJlckBpY3AuY3NpYy5lcw==</a></p> <p><span><sup>†</sup>Present address:</span> Mónica Martínez-Martínez, Departamento de Ciencias de la Salud, Facultad de Ciencias Biomédicas, Universidad Europea de Madrid, Villaviciosa de Odón, Spain</p> <div class="clear"></div> </div></div></div> <p class="AbstractSummary__disclaimer"><span>Disclaimer: </span> All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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src="https://loop.frontiersin.org/images/profile/111623/32" alt="Rolf Daniel" class="Avatar__img is-inside-mask"></figure> <div class="ArticleDetailsEditors__ediorInfo__info"><div class="ArticleDetailsEditors__ediorInfo__name notranslate"> Rolf Daniel </div> <div class="ArticleDetailsEditors__ediorInfo__affiliation notranslate"> University of Göttingen, Germany </div></div></a></div></div> <div class="ArticleDetailsEditors"><div class="ArticleDetailsEditors__editors"><div class="ArticleDetailsEditors__title">Reviewed by</div> <a href="https://loop.frontiersin.org/people/231858/overview" data-event="editorInfo-a-wolfgangRStreit" class="ArticleDetailsEditors__ediorInfo"><figure class="Avatar Avatar--size-32"><img src="https://loop.frontiersin.org/images/profile/231858/32" alt="Wolfgang R. Streit" class="Avatar__img is-inside-mask"></figure> <div class="ArticleDetailsEditors__ediorInfo__info"><div class="ArticleDetailsEditors__ediorInfo__name notranslate"> Wolfgang R. Streit </div> <div class="ArticleDetailsEditors__ediorInfo__affiliation notranslate"> University of Hamburg, Germany </div></div></a><a href="https://loop.frontiersin.org/people/1576541/overview" data-event="editorInfo-a-jiujunCheng" class="ArticleDetailsEditors__ediorInfo"><figure class="Avatar Avatar--size-32"><img src="https://loop.frontiersin.org/images/profile/1576541/32" alt="Jiujun Cheng" class="Avatar__img is-inside-mask"></figure> <div class="ArticleDetailsEditors__ediorInfo__info"><div class="ArticleDetailsEditors__ediorInfo__name notranslate"> Jiujun Cheng </div> <div class="ArticleDetailsEditors__ediorInfo__affiliation notranslate"> University of Waterloo, Canada </div></div></a></div></div> <div class="ArticleDetailsGlossary ArticleDetailsGlossary--open"><button class="ArticleDetailsGlossary__header"><div class="ArticleDetailsGlossary__header__title">Table of contents</div> <div class="ArticleDetailsGlossary__header__arrow"></div></button> <div class="ArticleDetailsGlossary__content"><ul class="flyoutJournal"> <li><a href="#h1">Abstract</a></li> <li><a href="#h2">Introduction</a></li> <li><a href="#h3">Materials and Methods</a></li> <li><a href="#h4">Results</a></li> <li><a href="#h5">Discussion</a></li> <li><a href="#h6">Data Availability Statement</a></li> <li><a href="#h7">Author Contributions</a></li> <li><a href="#fun1">Funding</a></li> <li><a href="#conf1">Conflict of Interest</a></li> <li><a href="#disclaimer1">Publisher’s Note</a></li> <li><a href="#ack1">Acknowledgments</a></li> <li><a href="#S9">Supplementary Material</a></li> <li><a href="#refer1">References</a></li> </ul> </div></div>  <div class="ActionsDropDown"><button aria-label="Open dropdown" data-event="actionsDropDown-button-toggle" class="ActionsDropDown__button ActionsDropDown__button--typeOutline ActionsDropDown__button--iconQuote"><span class="ActionsDropDown__button__label">Export citation</span></button> <div class="ActionsDropDown__menuWrapper"> <ul 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Indexed in PubMed Central (PMC), Scopus and the DOAJ, the journal explores the habitable world and the potential of microbial life.\u003C\u002Fp\u003E\n\n\u003Cp\u003ELed by Field Chief Editor Prof Paul D. Cotter (Co-founder and CTO of SeqBiome; Senior Principal Research Office, Teagasc Food Research Centre), this multidisciplinary journal welcomes submissions across the entire spectrum of microbiology and advances our understanding of the role microbes play in human lives, from health care to climate change.\u003C\u002Fp\u003E\n\n\u003Cp\u003ETopics include, but are not limited to:\u003C\u002Fp\u003E\n\n\u003Cul\u003E\n \u003Cli\u003Eancient DNA and forensic microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Eantimicrobials, resistance and chemotherapy\u003C\u002Fli\u003E\n \u003Cli\u003Eaquatic microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Ebiology of archaea\u003C\u002Fli\u003E\n \u003Cli\u003Eextreme microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Efood microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Einfectious agents and disease\u003C\u002Fli\u003E\n \u003Cli\u003Emicrobe and virus interactions with plants\u003C\u002Fli\u003E\n \u003Cli\u003Emicrobial physiology and metabolism\u003C\u002Fli\u003E\n \u003Cli\u003Emicrobial symbioses\u003C\u002Fli\u003E\n \u003Cli\u003Emicrobiological chemistry and geomicrobiology\u003C\u002Fli\u003E\n \u003Cli\u003Emicrobiotechnology\u003C\u002Fli\u003E\n \u003Cli\u003Emicroorganisms in vertebrate digestive systems\u003C\u002Fli\u003E\n \u003Cli\u003Ephage biology\u003C\u002Fli\u003E\n \u003Cli\u003Esystems microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Eterrestrial microbiology\u003C\u002Fli\u003E\n \u003Cli\u003Evirology.\u003C\u002Fli\u003E\n\u003C\u002Ful\u003E\n\n\u003Cp\u003EManuscripts that focus solely on clinical aspects of diseases, such as treatment outcomes, clinical trials, patient management, and epidemiological studies, are not suitable for publication in this journal. Additionally, studies that are purely descriptive, such as the characterization of bacterial isolates or the description of new methods, without providing significant biological insights, are not within the scope of this journal.\u003C\u002Fp\u003E\n\n\u003Cp\u003EFrontiers in Microbiology is committed to advancing developments in microorganism interactions by allowing unrestricted access to articles and communicating scientific knowledge to researchers and the public alike, to enable the scientific breakthroughs of the future.\u003C\u002Fp\u003E",palette:"cyan",impactFactor:"5.2",citeScore:"7.8",citations:"761000",showTagline:e,twitter:"@FrontMicrobiol",__typename:"Journal"},currentFrontiersJournal:{id:t,name:q,slug:u,printISSN:e,shortName:J,electronicISSN:K,abbreviation:aa,specialtyId:e,publicationDate:e,isOnline:h,isOpenForSubmissions:h,spaceId:c,field:{id:ab,domainId:c,__typename:ac},__typename:a},articleHubSlug:f,articleHubPage:L,currentArticle:{id:868839,doi:ad,title:M,acceptanceDate:new Date(1647253506000),receptionDate:new Date(1643889577000),publicationDate:new Date(1652918400000),isPublished:h,abstract:ae,researchTopic:{id:20327,title:"Functional Metagenomics for Enzyme Discovery",articlesCount:N,isMagazinePage:j,slug:"functional-metagenomics-for-enzyme-discovery",isOpenForSubmission:j},articleType:{id:24,name:"Original Research"},stage:{id:O,name:f},keywords:["acidophiles","Acidophilic bacteria","acid mine drainage","Biodiversity","extremozymes","esterase","Metagenomics","Plastic"],authors:[{id:m,firstName:af,lastName:"Vidal",givenNames:af,isCorresponding:j,isProfilePublic:j,userId:m,affiliations:[{organizationName:y,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:m,firstName:ag,lastName:"Martínez-Martínez",givenNames:ag,isCorresponding:j,isProfilePublic:j,userId:m,affiliations:[{organizationName:y,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:ah,firstName:ai,lastName:"Fernandez-Lopez",givenNames:ai,isCorresponding:j,isProfilePublic:h,userId:ah,affiliations:[{organizationName:y,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:aj,firstName:ak,lastName:"Roda",givenNames:ak,isCorresponding:j,isProfilePublic:h,userId:aj,affiliations:[{organizationName:al,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:am,firstName:an,lastName:"Méndez-García",givenNames:an,isCorresponding:j,isProfilePublic:h,userId:am,affiliations:[{organizationName:ao,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:ap,firstName:aq,lastName:"Golyshina",givenNames:aq,isCorresponding:j,isProfilePublic:h,userId:ap,affiliations:[{organizationName:"Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University",countryName:"United Kingdom",cityName:f,stateName:f,zipCode:f}]},{id:ar,firstName:as,lastName:"Guallar",givenNames:as,isCorresponding:j,isProfilePublic:h,userId:ar,affiliations:[{organizationName:al,countryName:n,cityName:f,stateName:f,zipCode:f},{organizationName:"Institució Catalana de Recerca i Estudis Avançats",countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:at,firstName:au,lastName:"Peláez",givenNames:au,isCorresponding:j,isProfilePublic:h,userId:at,affiliations:[{organizationName:ao,countryName:n,cityName:f,stateName:f,zipCode:f}]},{id:av,firstName:aw,lastName:"Ferrer",givenNames:aw,isCorresponding:j,isProfilePublic:h,userId:av,affiliations:[{organizationName:y,countryName:n,cityName:f,stateName:f,zipCode:f}]}],editors:[{id:ax,firstName:ay,lastName:"Daniel",givenNames:ay,isCorresponding:j,isProfilePublic:h,userId:ax,affiliations:[{organizationName:"University of Göttingen",countryName:az,cityName:f,stateName:f,zipCode:f}]}],reviewers:[{id:aA,firstName:"Wolfgang",lastName:"Streit",givenNames:"Wolfgang R.",isCorresponding:j,isProfilePublic:h,userId:aA,affiliations:[{organizationName:"University of Hamburg",countryName:az,cityName:f,stateName:f,zipCode:f}]},{id:aB,firstName:aC,lastName:"Cheng",givenNames:aC,isCorresponding:j,isProfilePublic:h,userId:aB,affiliations:[{organizationName:"University of Waterloo",countryName:"Canada",cityName:f,stateName:f,zipCode:f}]}],journal:{id:t,slug:u,name:q,shortName:J,electronicISSN:K,field:{id:ab,domainId:c,__typename:ac},specialtyId:e,journalSectionPaths:[{section:aD,__typename:"journal_journalSectionPath"}],__typename:a},section:aD,impactMetrics:{views:aE,downloads:757,citations:P},volume:Q,articleVolume:"Volume 13 - 2022",relatedArticles:[],isPublishedV2:j,contents:{titleHtml:M,fullTextHtml:"\u003Cdiv class=\"JournalAbstract\"\u003E\r\n\u003Ca id=\"h1\" name=\"h1\"\u003E\u003C\u002Fa\u003E\r\n\r\n\u003Cdiv class=\"authors\"\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\r\n\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg\" alt=\"\\r\\nPaula Vidal\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\"\u003EPaula Vidal\u003Csup\u003E1\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg\" alt=\"Mnica Martínez-Martínez&#x;\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\"\u003EMónica Martínez-Martínez\u003Csup\u003E1†\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F1685724\" class=\"user-id-1685724\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F1685724\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Laura Fernandez-Lopez\"\u003ELaura Fernandez-Lopez\u003C\u002Fa\u003E\u003Csup\u003E1\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F1685246\" class=\"user-id-1685246\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F1685246\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Sergi Roda\"\u003ESergi Roda\u003C\u002Fa\u003E\u003Csup\u003E2\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F217882\" class=\"user-id-217882\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F217882\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Celia Mndez-García\"\u003ECelia Méndez-García\u003C\u002Fa\u003E\u003Csup\u003E3\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F224799\" class=\"user-id-224799\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F224799\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Olga V. Golyshina\"\u003EOlga V. Golyshina\u003C\u002Fa\u003E\u003Csup\u003E4\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F517366\" class=\"user-id-517366\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F517366\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Víctor Guallar,\"\u003EVíctor Guallar\u003C\u002Fa\u003E\u003Csup\u003E2,5\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F93877\" class=\"user-id-93877\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F93877\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Ana I. Pelez\"\u003EAna I. Peláez\u003C\u002Fa\u003E\u003Csup\u003E3\u003C\u002Fsup\u003E\u003C\u002Fspan\u003E\u003Cspan class=\"author-wrapper notranslate\"\u003E\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F236735\" class=\"user-id-236735\"\u003E\u003Cimg class=\"pr5\" src=\"https:\u002F\u002Floop.frontiersin.org\u002Fimages\u002Fprofile\u002F236735\u002F74\" onerror=\"this.onerror=null;this.src='https:\u002F\u002Floop.frontiersin.org\u002Fcdn\u002Fimages\u002Fprofile\u002Fdefault_32.jpg';\" alt=\"Manuel Ferrer*\\r\\n\"\u003EManuel Ferrer\u003C\u002Fa\u003E\u003Csup\u003E1*\u003C\u002Fsup\u003E\r\n\u003C\u002Fspan\u003E\u003C\u002Fdiv\u003E\r\n\u003Cul class=\"notes\"\u003E\r\n\u003Cli\u003E\u003Cspan\u003E\u003Csup\u003E1\u003C\u002Fsup\u003E\u003C\u002Fspan\u003EInstitute of Catalysis, Department of Applied Biocatalysis, Consejo Superior de Investigaciones Científicas, Madrid, Spain\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Cspan\u003E\u003Csup\u003E2\u003C\u002Fsup\u003E\u003C\u002Fspan\u003EDepartment of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Cspan\u003E\u003Csup\u003E3\u003C\u002Fsup\u003E\u003C\u002Fspan\u003EÁrea de Microbiología, Departamento Biología Funcional e Instituto de Biotecnología de Asturias, Universidad de Oviedo, Oviedo, Spain\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Cspan\u003E\u003Csup\u003E4\u003C\u002Fsup\u003E\u003C\u002Fspan\u003ECentre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, United Kingdom\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Cspan\u003E\u003Csup\u003E5\u003C\u002Fsup\u003E\u003C\u002Fspan\u003EInstitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp class=\"mb0\"\u003EAcid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and \u003Ci\u003Ein silico\u003C\u002Fi\u003E metagenomic approaches, we retrieved 16 esterases from the α\u002Fβ-hydrolase fold superfamily with the closest match from uncultured acidophilic \u003Ci\u003EAcidobacteria, Actinobacteria (Acidithrix, Acidimicrobium\u003C\u002Fi\u003E, and \u003Ci\u003EFerrimicrobium), Acidiphilium\u003C\u002Fi\u003E, and other \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in \u003Ci\u003EEscherichia coli\u003C\u002Fi\u003E. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U\u002Fg at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"JournalFullText\"\u003E\r\n\u003Ca id=\"h2\" name=\"h2\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EIntroduction\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb15\"\u003EIn biotechnology, there is high interest in finding enzymes with new or improved properties (\u003Ca href=\"#B55\"\u003EPellis et al., 2017\u003C\u002Fa\u003E; \u003Ca href=\"#B23\"\u003EFerrer et al., 2019\u003C\u002Fa\u003E). This interest is especially increased in relation to enzymes from extremophiles, which are microorganisms that have evolved to thrive in extreme environments (\u003Ca href=\"#B9\"\u003EBaweja et al., 2016\u003C\u002Fa\u003E), as they can efficiently operate under multiple conditions requested by industry. One example application is the eco-friendly bioconversion of cellulosic biomass by extremozymes, which produces green products and has less substrate loss (\u003Ca href=\"#B64\"\u003EThapa et al., 2020\u003C\u002Fa\u003E). Furthermore, plastic disposal is one of the major problems currently faced by the environment, as enormous quantities of synthetic plastics are non-degradable. Researchers are constantly exploring new ways to degrade plastics, and one of these ways involves using enzymes from microorganisms or microbial communities, including some that inhabit extreme environments (\u003Ca href=\"#B53\"\u003ENchedo Ariole and George-West, 2020\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EAcid mine drainage (AMD) systems deserve special attention as a source of extremozymes. AMDs are extremely acidic runoff formations that originate from the microbial oxidation of pyrite and other sulfide minerals, which results in the production of sulfuric acid and metal-rich solutions (\u003Ca href=\"#B51\"\u003EMéndez-García et al., 2015\u003C\u002Fa\u003E; \u003Ca href=\"#B43\"\u003EJohnson and Quatrini, 2020\u003C\u002Fa\u003E). AMD systems are common in our planet, although only a limited number of them have been microbiologically characterized (\u003Ca href=\"#B51\"\u003EMéndez-García et al., 2015\u003C\u002Fa\u003E; \u003Ca href=\"#B43\"\u003EJohnson and Quatrini, 2020\u003C\u002Fa\u003E). Although it has recently been demonstrated that some of these AMD formations, such as the Los Rueldos mercury mine in northwestern Spain (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E), appear to be populated by a great diversity of prokaryotes, the majority of them are inhabited by a restricted set of acidophilic bacteria and archaea (\u003Ca href=\"#B20\"\u003EDopson et al., 2004\u003C\u002Fa\u003E; \u003Ca href=\"#B30\"\u003EGolyshina, 2011\u003C\u002Fa\u003E; \u003Ca href=\"#B51\"\u003EMéndez-García et al., 2015\u003C\u002Fa\u003E; \u003Ca href=\"#B14\"\u003EChen et al., 2016\u003C\u002Fa\u003E; \u003Ca href=\"#B43\"\u003EJohnson and Quatrini, 2020\u003C\u002Fa\u003E), whose variety and abundance depend on geochemical constraints (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E, \u003Ca href=\"#B51\"\u003E2015\u003C\u002Fa\u003E; \u003Ca href=\"#B39\"\u003EHuang et al., 2016\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EMajor bacterial lineages detected in AMD systems include the phyla \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E (\u003Ci\u003EAcidithiobacillus, Acidiphilium\u003C\u002Fi\u003E, \u003Ci\u003EAcidocella\u003C\u002Fi\u003E, \u003Ci\u003EAcidicaldus\u003C\u002Fi\u003E, \u003Ci\u003EAcidomonas\u003C\u002Fi\u003E, \u003Ci\u003EAcidisphaera\u003C\u002Fi\u003E, “\u003Ci\u003EFerrovum\u003C\u002Fi\u003E,” \u003Ci\u003EAcidibacter\u003C\u002Fi\u003E, and \u003Ci\u003EMetallibacterium\u003C\u002Fi\u003E spp.), \u003Ci\u003ENitrospirae\u003C\u002Fi\u003E (\u003Ci\u003ELeptospirillum\u003C\u002Fi\u003E spp. such as \u003Ci\u003ELeptospirillum ferrooxidans\u003C\u002Fi\u003E, \u003Ci\u003ELeptospirillum ferriphilum\u003C\u002Fi\u003E, and “\u003Ci\u003ELeptospirillum ferrodiazotrophum\u003C\u002Fi\u003E”), \u003Ci\u003EActinobacteria\u003C\u002Fi\u003E, \u003Ci\u003EFirmicutes\u003C\u002Fi\u003E (\u003Ci\u003ESulfobacillus\u003C\u002Fi\u003E spp., and \u003Ci\u003EAlicyclobacillus\u003C\u002Fi\u003E spp.), \u003Ci\u003EAcidobacteria\u003C\u002Fi\u003E, \u003Ci\u003ESaccharibacteria\u003C\u002Fi\u003E (TM7) and other candidate phyla radiation (CPR) organisms. Archaea include the phyla \u003Ci\u003EEuryarchaeota\u003C\u002Fi\u003E (\u003Ci\u003EFerroplasma\u003C\u002Fi\u003E spp. such as \u003Ci\u003EFerroplasma acidiphilum\u003C\u002Fi\u003E and “\u003Ci\u003EFerroplasma acidarmanus\u003C\u002Fi\u003E,” \u003Ci\u003EAcidiplasma cupricumulans\u003C\u002Fi\u003E, and \u003Ci\u003ECuniculiplasma divulgatum\u003C\u002Fi\u003E), \u003Ci\u003EThaumarchaeota\u003C\u002Fi\u003E, and the Candidate divisions “\u003Ci\u003EMicrarchaeota\u003C\u002Fi\u003E” and “\u003Ci\u003EParvarchaeota\u003C\u002Fi\u003E” (\u003Ca href=\"#B33\"\u003EGolyshina et al., 2000\u003C\u002Fa\u003E, \u003Ca href=\"#B34\"\u003E2009\u003C\u002Fa\u003E, \u003Ca href=\"#B32\"\u003E2016\u003C\u002Fa\u003E; \u003Ca href=\"#B20\"\u003EDopson et al., 2004\u003C\u002Fa\u003E; \u003Ca href=\"#B6\"\u003EBaker et al., 2006\u003C\u002Fa\u003E, \u003Ca href=\"#B5\"\u003E2010\u003C\u002Fa\u003E; \u003Ca href=\"#B30\"\u003EGolyshina, 2011\u003C\u002Fa\u003E; \u003Ca href=\"#B14\"\u003EChen et al., 2016\u003C\u002Fa\u003E, \u003Ca href=\"#B15\"\u003E2018\u003C\u002Fa\u003E; \u003Ca href=\"#B28\"\u003EGavrilov et al., 2019\u003C\u002Fa\u003E; \u003Ca href=\"#B45\"\u003EKorzhenkov et al., 2019\u003C\u002Fa\u003E). These microorganisms are expected to be reservoirs of enzymes selected to resist acidic harsh conditions (at least regarding extracellular products) (\u003Ca href=\"#B63\"\u003ESharma et al., 2012\u003C\u002Fa\u003E), some of which might be of biotechnological relevance (\u003Ca href=\"#B35\"\u003EGomes et al., 2003\u003C\u002Fa\u003E; \u003Ca href=\"#B1\"\u003EAdrio and Demain, 2014\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EIn this category, esterases and lipases from the α\u002Fβ-hydrolase fold superfamily are appropriate biocatalysts for use in a modern circular bioeconomy because of their abundance (at least one per genome; \u003Ca href=\"#B21\"\u003EFerrer et al., 2015\u003C\u002Fa\u003E); the extensive knowledge that has been accumulated after the analysis of the biochemical features, sequences, and structures of more than 280,638 such proteins (\u003Ca href=\"#B8\"\u003EBauer et al., 2020\u003C\u002Fa\u003E); their ease of identification (multiple available screening methods; \u003Ca href=\"#B60\"\u003EReyes-Duarte et al., 2012\u003C\u002Fa\u003E); and their outstanding properties in terms of stability, reactivity, and scalability, which make them third-choice tools for the functionalization and modification of low-reactivity hydrocarbon-like blocks, oils, and fats (\u003Ca href=\"#B16\"\u003EDaiha et al., 2015\u003C\u002Fa\u003E). Genomics and metagenomics can potentially make accessible an enormous reserve of such important biocatalysts in organisms or microbial communities inhabiting any environment, including AMD systems. However, only 239 of the 280,638 sequences available at the Lipase Engineering (LED) Database (\u003Ca href=\"#B8\"\u003EBauer et al., 2020\u003C\u002Fa\u003E) have been retrieved from cultured microorganisms (listed above) and uncultured microorganisms that are inhabitants of AMD systems, including \u003Ci\u003EAlicyclobacillus\u003C\u002Fi\u003E spp., 118 in total; \u003Ci\u003ESulfobacillus\u003C\u002Fi\u003E spp., 53; \u003Ci\u003EAcidobacteria\u003C\u002Fi\u003E, 34; \u003Ci\u003EAcidithiobacillus\u003C\u002Fi\u003E, 13; \u003Ci\u003ELeptospirillum\u003C\u002Fi\u003E, 9; “\u003Ci\u003EFerrovum\u003C\u002Fi\u003E,” 5; \u003Ci\u003EAcidocella\u003C\u002Fi\u003E, 3; and \u003Ci\u003EFerroplasma\u003C\u002Fi\u003E, \u003Ci\u003EAciditrix\u003C\u002Fi\u003E, \u003Ci\u003EAcidiphilium\u003C\u002Fi\u003E, and \u003Ci\u003EMetallibacterium\u003C\u002Fi\u003E, with 1 each. Among these biocatalysts, only a low-pH optimum carboxylesterase from \u003Ci\u003EF. acidiphilum\u003C\u002Fi\u003E (\u003Ca href=\"#B54\"\u003EOhara et al., 2014\u003C\u002Fa\u003E) has been characterized. This limits the assessment of the biotechnological potential of acidophiles living in AMD systems, at least regarding esterases and lipases. The minimal enzyme-level information that is known about these systems is restricted to two endo-acting amylases with no similarity to any known protein and two genes conferring metal and acid resistance from the microbial community inhabiting the AMD systems of the Carnoulès (lead–zinc) mine in France (\u003Ca href=\"#B18\"\u003EDelavat et al., 2012\u003C\u002Fa\u003E) and the Tinto River in southwestern Spain (\u003Ca href=\"#B18\"\u003EDelavat et al., 2012\u003C\u002Fa\u003E; \u003Ca href=\"#B36\"\u003EGuazzaroni et al., 2013\u003C\u002Fa\u003E), respectively.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003ETo fill this knowledge gap, we initiated a metagenomic investigation to isolate carboxylesterases from a recently discovered and microbiologically characterized AMD formation, namely, the Los Rueldos mercury mine in northwestern Spain (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). By applying homology searches in metagenomic sequences and naïve screening in clone libraries with enzyme substrates, we discovered a number of such enzymes whose characteristics are reported herein. Both function- and DNA sequence-based metagenomic methods are complementary, with each having advantages and disadvantages. Bioinformatics methods allow a rapid process of enzyme searching. However, in prokaryotic genomes, >30% of genes remain annotated as “hypothetical, conserved hypothetical or with general prediction,” and large numbers of genes may have non-specific annotations (such as putative hydrolases). The analysis of biochemical functions is likely to provide a superior approach to avoid this limitation, especially when screening novel enzymes. However, only a few hundred specific enzymatic assays exist, with a limited number of them applied in a high-throughput manner for the naïve screening of metagenomics libraries.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EAlthough the \u003Ci\u003Ein vivo\u003C\u002Fi\u003E roles and expression levels of the genes encoding the hydrolases presented in this study are unknown, their sequences and results of biochemical analyses shed new light on the enzymology of the microbial inhabitants of the Los Rueldos AMD formation, which have been neglected in enzyme prospecting.\u003C\u002Fp\u003E\r\n\u003Ca id=\"h3\" name=\"h3\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EMaterials and Methods\u003C\u002Fh2\u003E\r\n\u003Ch3 class=\"pt0\"\u003EGeneral Experimental Procedures\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe source and brand of each of the esters (purity ≥99%) used in this study was Merck Life Science S.L.U., Madrid, Spain. The oligonucleotides used for DNA amplification were synthesized by Sigma Genosys, Ltd. (Pampisford, Cambridgeshire, United Kingdom). The \u003Ci\u003EEscherichia coli\u003C\u002Fi\u003E EPI-300-T1R strain used for pCCFOS1 fosmid library construction and screening was from Epicentre Biotechnologies (Madison, WI, United States). The \u003Ci\u003EE. coli\u003C\u002Fi\u003E strain GigaSingles used for gene cloning and \u003Ci\u003EE. coli\u003C\u002Fi\u003E strain BL21 (DE3) used for gene expression were from Novagen (Darmstadt, Germany).\u003C\u002Fp\u003E\r\n\u003Ch3\u003ESampling Site and Sample Collection\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe Los Rueldos gallery is located along the northwestern slope of the Morgao Valley (2 km northeast of the town of Mieres and 20 km southeast of Oviedo, which is the capital city of Asturias in northwestern Spain; 43°15′47″N, 5°46′9″W). It is a 70 m-long gallery with 10–30 cm depths in the shallower areas and 40–70 cm depths in the deeper sections (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). Microorganisms are developed along the AMD system (pH ∼2), forming a bedded acidic biofilm with uppermost oxic (B1A) and lowermost anoxic (B1B) strata. The DNA samples from B1A and B1B (see below) samples collected and used in this study were the same as those in the previous work (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). Briefly, samples were collected in sterile 50 ml tubes at two sampling sites determined by the presence of each different macroscopic microbial growth morphology (B1A: up to 2 cm deep; B1B: from 2 to 15 cm deep) and kept on ice until nucleic acid extraction was performed (within the following 2 h).\u003C\u002Fp\u003E\r\n\u003Ch3\u003ENucleic Acid Extraction, Preparation of pCCFOS1 Libraries, and Naïve Screening\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb15\"\u003EThe DNA samples from B1A and B1B were the same as those used in a previous work (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E), which were obtained using the Power Soil DNA extraction kit (Cambio, Cambridge, United Kingdom) according to the manufacturer’s guidelines. Prior to clone library construction, the metagenomics DNA was concentrated by first adding 50 μl of 3 M sodium acetate solution to 50 μl DNA extract. Precipitation was conducted by the addition of 1.25 ml of ethanol and incubation at room temperature for 10 s. Precipitated DNA was pelleted by centrifugation at 20,000 \u003Ci\u003Eg\u003C\u002Fi\u003E for 10 min. The resulting pellets were washed with 500 μl of 70% (v\u002Fv) ethanol twice, and the traces of ethanol were evaporated by incubation under a fume hood at room temperature for 10 min. The resulting pellets were then dissolved in 20 μl of sterile nuclease-free water. Before cloning in the large-insert pCCFOS1 fosmid libraries using the CopyControl Fosmid Library Kit (Epicentre Biotechnologies, Madison, WI, United States) and the \u003Ci\u003EE. coli\u003C\u002Fi\u003E EPI300-T1\u003Ci\u003E\u003Csup\u003ER\u003C\u002Fsup\u003E\u003C\u002Fi\u003E strain, the DNA (10 μg) that was unsheared by gel electrophoresis was subjected to shearing by pipetting through a 200 μl pipette tip 100 times, following the recommendations of the supplier (Epicentre Biotechnologies, Madison, WI, United States) to reach an approximately size of 30,000 bp. Cells of each pCCFOS1 fosmid library were suspended in glycerol to a final concentration of 20% (v\u002Fv) and stored at −80°C until further use. We generated subsets of 94,000 and 81,000 clones for the B1A and B1B samples, respectively. Restriction analysis of 10 randomly selected clones from each library revealed average insert sizes of 34,000 bp (for the B1A samples) and 39,500 bp (for the B1B samples), which included nearly 3.2 Gbp of community genomes per sample. This size is within the range of the average size range of DNA inserts in positive clones found in this study (see below).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EFosmid clones were plated onto large (22.5 × 22.5 cm) Petri plates with Luria Bertani (LB) agar containing chloramphenicol (12.5 μg\u002Fml) and induction solution (Epicentre Biotechnologies; WI, United States) at a quantity recommended by the supplier to induce a high fosmid copy number. Clones were scored by the ability to hydrolyze α-naphthyl acetate (α-NA) and tributyrin, as previously described (\u003Ca href=\"#B60\"\u003EReyes-Duarte et al., 2012\u003C\u002Fa\u003E). Positive clones presumptively containing carboxylesterases and lipases with the α\u002Fβ hydrolase fold were selected, and their DNA inserts were sequenced using a MiSeq Sequencing System (Illumina, San Diego, CA, United States) with a 2 × 150-bp sequencing v2 kit at Lifesequencing S.L. (Valencia, Spain). Before sequencing, fosmid DNA was extracted from the fosmid clones containing the metagenomic segments using the QUIAGEN Large-Construct Kit (QUIAGEN, Hilden, Germany), according to the manufacturer’s protocol. Upon the completion of sequencing, the reads were quality-filtered and assembled to generate non-redundant meta-sequences, and genes were predicted and annotated as described previously (\u003Ca href=\"#B57\"\u003EPlacido et al., 2015\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Ch3\u003ESelection of Genes Encoding Enzymes by Homology Sequence Analysis\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe predicted protein-coding genes obtained in a previous study (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E) after the sequencing of DNA material from resident microbial communities in each of the samples (B1A and B1B) with a Roche 454 GS FLX Ti sequencer (Roche Applied Science, Penzberg, Germany) were used in this study. The meta-sequences are available from the National Center for Biotechnology Information (NCBI) non-redundant public database with the IDs PRJNA193663 (for B1A) and PRJNA193664 (for B1B). Protein-coding genes identified from metagenomes (sequence-based screening) and from the DNA inserts of positive clones (naïve screen) were screened (score >45; e-value <10e\u003Csup\u003E–3\u003C\u002Fsup\u003E) using BLASTP and PSI-BLAST searching (\u003Ca href=\"#B3\"\u003EAltschul et al., 1997\u003C\u002Fa\u003E) for enzymes of interest against the \u003Ci\u003EESTerases and alpha\u002Fbeta-Hydrolase Enzymes and Relatives\u003C\u002Fi\u003E (ESTHER) and LED databases (\u003Ca href=\"#B25\"\u003EFischer and Pleiss, 2003\u003C\u002Fa\u003E; \u003Ca href=\"#B7\"\u003EBarth et al., 2004\u003C\u002Fa\u003E; \u003Ca href=\"#B8\"\u003EBauer et al., 2020\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Ch3\u003EGene Expression and Protein Purification\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe experimental procedures used for the cloning, expression, and purification of selected proteins (either from naïve or homology sequence screening) in the Ek\u002FLIC 46 vector and \u003Ci\u003EE. coli\u003C\u002Fi\u003E strain BL21 (DE3) were performed as described previously (\u003Ca href=\"#B2\"\u003EAlcaide et al., 2015\u003C\u002Fa\u003E). The primers used for amplification are listed in \u003Ca href=\"#S9\"\u003ESupplementary Material\u003C\u002Fa\u003E. All proteins studied here were N-terminally His\u003Csub\u003E6\u003C\u002Fsub\u003E-tagged, and the soluble His-tagged proteins were produced and purified at room temperature after binding to a nickel–nitrilotriacetic acid (Ni–NTA) His-Bind resin (from Merck Life Science S.L.U., Madrid, Spain) as described previously (\u003Ca href=\"#B29\"\u003EGiunta et al., 2020\u003C\u002Fa\u003E), with slight modifications (the expression culture was scaled up to 1 L using 50 ml pre-inoculum). The purity was assessed as >98% using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE; \u003Ca href=\"#S9\"\u003ESupplementary Figure 1\u003C\u002Fa\u003E) in a Bio-Rad Mini Protein system (\u003Ca href=\"#B46\"\u003ELaemmli, 1970\u003C\u002Fa\u003E). Protein concentrations were determined according to the Bradford method with bovine serum albumin as the standard (\u003Ca href=\"#B12\"\u003EBradford, 1976\u003C\u002Fa\u003E). A total of approximately 0.8–37 mg of purified recombinant proteins was obtained from each 1 L culture on average, as follows: Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E (6.4 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E (25 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E3\u003C\u002Fsub\u003E (13 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E4\u003C\u002Fsub\u003E (37 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E (41 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E (7 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E7\u003C\u002Fsub\u003E (0.8 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E (19 mg\u002FL), Est\u003Csub\u003E\u003Ci\u003EB\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E (1.0 mg\u002FL), and Est\u003Csub\u003E\u003Ci\u003EB\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E (32 mg\u002FL).\u003C\u002Fp\u003E\r\n\u003Ch3\u003EEnzyme Assays\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb15\"\u003EThe hydrolysis of 2-naphthyl acrylate (ref. 577189), tri(propylene glycol) diacrylate (ref. 246832), dibenzyl terephthalate (ref. PH000126), and bis(2-hydroxyethyl)-terephthalate (BHET; ref. 465151) (all from Merck Life Science S.L.U., Madrid, Spain) was assessed using a pH indicator assay in 384-well plates (ref. 781162, Greiner Bio-One GmbH, Kremsmünster, Austria) at 30°C and pH 8.0 in a Synergy HT Multi-Mode Microplate Reader in continuous mode at 550 nm over 24 h [extinction coefficient (ε) of phenol red, 8,450 M\u003Csup\u003E–1\u003C\u002Fsup\u003E cm\u003Csup\u003E–1\u003C\u002Fsup\u003E]. The acid produced after ester bond cleavage by the hydrolytic enzyme induced a color change in the pH indicator that was measured spectrophotometrically at 550 nm. The experimental conditions were as detailed previously (\u003Ca href=\"#B29\"\u003EGiunta et al., 2020\u003C\u002Fa\u003E), with the absence of activity defined as at least a twofold background signal. For \u003Ci\u003EV\u003C\u002Fi\u003E\u003Csub\u003E\u003Ci\u003Emax\u003C\u002Fi\u003E\u003C\u002Fsub\u003E determination, (protein): 270 μg\u002Fml; (ester): 20 mM; reaction volume: 44 μl; T: 30°C; and pH: 8.0. Activity was calculated by determining the absorbance per minute from the generated slopes and applying the following equation:\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"equationImageholder pb0\"\u003E\r\n\u003Cmath id=\"M1\"\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmi\u003ER\u003C\u002Fmi\u003E\u003Cmi\u003Ea\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Ee\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmrow\u003E\u003Cmo\u003E(\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmi mathvariant=\"normal\"\u003Eμ\u003C\u002Fmi\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003El\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmi\u003Emin\u003C\u002Fmi\u003E\u003Cmo\u003E⁡\u003C\u002Fmo\u003E\u003Cmrow\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Eg\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ep\u003C\u002Fmi\u003E\u003Cmi\u003Er\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmi\u003Ee\u003C\u002Fmi\u003E\u003Cmi\u003Ei\u003C\u002Fmi\u003E\u003Cmi\u003En\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E)\u003C\u002Fmo\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003Cmo\u003E=\u003C\u002Fmo\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmi mathvariant=\"normal\"\u003E△\u003C\u002Fmi\u003E\u003Cmrow\u003E\u003Cmtext\u003EAbs\u003C\u002Fmtext\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmtext\u003Emin\u003C\u002Fmtext\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E8450\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmsup\u003E\u003Cmi mathvariant=\"normal\"\u003EM\u003C\u002Fmi\u003E\u003Cmrow\u003E\u003Cmo\u003E-\u003C\u002Fmo\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmsup\u003E\u003Cmsup\u003E\u003Cmtext\u003Ecm\u003C\u002Fmtext\u003E\u003Cmrow\u003E\u003Cmo\u003E-\u003C\u002Fmo\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmsup\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E0.4\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ec\u003C\u002Fmi\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmsup\u003E\u003Cmn\u003E10\u003C\u002Fmn\u003E\u003Cmn\u003E6\u003C\u002Fmn\u003E\u003C\u002Fmsup\u003E\u003C\u002Fmpadded\u003E\u003Cmi mathvariant=\"normal\"\u003Eμ\u003C\u002Fmi\u003E\u003Cmi\u003EM\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmi mathvariant=\"normal\"\u003EM\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003C\u002Fmpadded\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E 0.000044\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003C\u002Fmrow\u003E\u003Cmi\u003EL\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003Cmrow\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Eg\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ep\u003C\u002Fmi\u003E\u003Cmi\u003Er\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmo\u003E.\u003C\u002Fmo\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmath\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cp class=\"mb15\"\u003EThe activity toward the model esters \u003Ci\u003Ep\u003C\u002Fi\u003E-nitrophenyl acetate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E2\u003C\u002Fsub\u003E), propionate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E), butyrate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E4\u003C\u002Fsub\u003E), octanoate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E8\u003C\u002Fsub\u003E), decanoate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E10\u003C\u002Fsub\u003E), and decanoate (\u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E12\u003C\u002Fsub\u003E) was assessed in 50 mM Britton and Robinson (BR) buffer at pH 8.0 and 30°C by monitoring the production of 4-nitrophenol at 348 nm (pH-independent isosbestic point, ε = 4147 M\u003Csup\u003E–1\u003C\u002Fsup\u003E cm\u003Csup\u003E–1\u003C\u002Fsup\u003E) for over 5 min and determining the absorbance per minute from the generated slopes (\u003Ca href=\"#B61\"\u003ESantiago et al., 2018\u003C\u002Fa\u003E). The reactions were performed at 30°C in 96-well plates (ref. 655801, Greiner Bio-One GmbH, Kremsmünster, Austria) and contained 0.09 to 3 μg proteins and 0.8 mM esters in a total volume of 200 μl. The effect of pH on the activity was determined in 50 mM BR buffer at pH 4.0–12.0, as described previously. Similar assay conditions were used to assay the effects of temperature on the ester hydrolysis of \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E, but in this case, the reactions were performed in 50 mM BR buffer pH 7.0. Note that the BR buffer consists of a mixture of 0.04 M H\u003Csub\u003E3\u003C\u002Fsub\u003EBO\u003Csub\u003E3\u003C\u002Fsub\u003E, 0.04 M H\u003Csub\u003E3\u003C\u002Fsub\u003EPO\u003Csub\u003E4\u003C\u002Fsub\u003E, and 0.04 M CH\u003Csub\u003E3\u003C\u002Fsub\u003ECOOH that has been titrated to the desired pH with 0.2 M NaOH. All values were determined in triplicate and were corrected for non-enzymatic transformation. In all cases, the activity was calculated by determining the absorbance per minute from the generated slopes and applying the following equation:\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"equationImageholder pb0\"\u003E\r\n\u003Cmath id=\"M2\"\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmi\u003ER\u003C\u002Fmi\u003E\u003Cmi\u003Ea\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Ee\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmrow\u003E\u003Cmo\u003E(\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmi mathvariant=\"normal\"\u003Eμ\u003C\u002Fmi\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003El\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmi\u003Emin\u003C\u002Fmi\u003E\u003Cmo\u003E⁡\u003C\u002Fmo\u003E\u003Cmrow\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Eg\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ep\u003C\u002Fmi\u003E\u003Cmi\u003Er\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmi\u003Ee\u003C\u002Fmi\u003E\u003Cmi\u003Ei\u003C\u002Fmi\u003E\u003Cmi\u003En\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E)\u003C\u002Fmo\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003Cmo\u003E=\u003C\u002Fmo\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmrow\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmi mathvariant=\"normal\"\u003E△\u003C\u002Fmi\u003E\u003Cmrow\u003E\u003Cmtext\u003EAbs\u003C\u002Fmtext\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmtext\u003Emin\u003C\u002Fmtext\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E4147\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmsup\u003E\u003Cmi mathvariant=\"normal\"\u003EM\u003C\u002Fmi\u003E\u003Cmrow\u003E\u003Cmo\u003E-\u003C\u002Fmo\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmsup\u003E\u003Cmsup\u003E\u003Cmtext\u003Ecm\u003C\u002Fmtext\u003E\u003Cmrow\u003E\u003Cmo\u003E-\u003C\u002Fmo\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmsup\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E0.4\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ec\u003C\u002Fmi\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmsup\u003E\u003Cmn\u003E10\u003C\u002Fmn\u003E\u003Cmn\u003E6\u003C\u002Fmn\u003E\u003C\u002Fmsup\u003E\u003C\u002Fmpadded\u003E\u003Cmi mathvariant=\"normal\"\u003Eμ\u003C\u002Fmi\u003E\u003Cmi\u003EM\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmrow\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003Cmi mathvariant=\"normal\"\u003EM\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003C\u002Fmpadded\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmn\u003E 0.0002\u003C\u002Fmn\u003E\u003C\u002Fmpadded\u003E\u003C\u002Fmrow\u003E\u003Cmi\u003EL\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003Cmo\u003E*\u003C\u002Fmo\u003E\u003Cmstyle displaystyle=\"true\"\u003E\u003Cmfrac\u003E\u003Cmn\u003E1\u003C\u002Fmn\u003E\u003Cmrow\u003E\u003Cmi\u003Em\u003C\u002Fmi\u003E\u003Cmpadded width=\"+2.8pt\"\u003E\u003Cmi\u003Eg\u003C\u002Fmi\u003E\u003C\u002Fmpadded\u003E\u003Cmi\u003Ep\u003C\u002Fmi\u003E\u003Cmi\u003Er\u003C\u002Fmi\u003E\u003Cmi\u003Eo\u003C\u002Fmi\u003E\u003Cmi\u003Et\u003C\u002Fmi\u003E\u003Cmi\u003Ee\u003C\u002Fmi\u003E\u003Cmi\u003Ei\u003C\u002Fmi\u003E\u003Cmi\u003En\u003C\u002Fmi\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmfrac\u003E\u003C\u002Fmstyle\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmrow\u003E\u003C\u002Fmath\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cp class=\"mb15\"\u003EPoly(propylene glycol) diacrylate (ref. 455024, Merck Life Science S.L.U., Madrid, Spain) and poly(DL-lactide) with an average molecular weight 2,000 (ref. AP224, PolySciTech, Akina, IN, United States) were assayed as described previously (\u003Ca href=\"#B37\"\u003EHajighasemi et al., 2018\u003C\u002Fa\u003E). The hydrolysis of polyethylene terephthalate (PET) films (prepared as reported by \u003Ca href=\"#B11\"\u003EBollinger et al., 2020\u003C\u002Fa\u003E) and particles, which were prepared using PET from a bottle (from a local shop – Granini brand), as described previously (\u003Ca href=\"#B58\"\u003EPütz, 2006\u003C\u002Fa\u003E), was evaluated at 30°C and pH 8.0 with 270 μg protein\u002Fml and 2 mg\u002Fml plastic material, as previously reported (\u003Ca href=\"#B11\"\u003EBollinger et al., 2020\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EThe effect of the inhibitors mercaptoethanol (ref. M7154) and iodoacetamide (ref. I1149), which were both from Merck Life Science S.L.U., Madrid, Spain, was tested as follows. A mixture containing the purified enzymes (final concentration of 1 mg\u002Fml) in 190 μl of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.0 and the inhibitors (final concentration, 1–10 mM) was incubated for 5 min to 24 h at 30–45°C. The reaction was initiated by adding \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E (0.8 mM, final concentration), and the activity was measured for over 5 min as described above and compared to control samples without inhibitors.\u003C\u002Fp\u003E\r\n\u003Ch3\u003ECircular Dichroism to Estimate Thermal Denaturation\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003ECircular dichroism (CD) spectra were acquired between 190 and 270 nm with a Jasco J-720 spectropolarimeter equipped with a Peltier temperature controller in a 0.1-mm cell at 25°C. The spectra were analyzed, and denaturation temperature (T\u003Csub\u003E\u003Ci\u003Ed\u003C\u002Fi\u003E\u003C\u002Fsub\u003E) values were determined at 220 nm between 10 and 85°C at a rate of 30°C per hour in 40 mM HEPES buffer at pH 7.0. CD measurements were performed at pH 7.0 and not at the optimal pH (8.5–9.0) to ensure protein stability. A protein concentration of 0.5 mg\u002Fml was used. T\u003Csub\u003E\u003Ci\u003Ed\u003C\u002Fi\u003E\u003C\u002Fsub\u003E (and the standard deviation of the linear fit) was calculated by fitting the ellipticity (mdeg) at 220 nm at each of the different temperatures using a 5-parameter sigmoid fit with SigmaPlot 13.0.\u003C\u002Fp\u003E\r\n\u003Ch3\u003ECodes and Accession Numbers\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe sequences were named based on the code “Est,” which refers to \u003Cu\u003EEst\u003C\u002Fu\u003Eerase, followed by a letter indicating the origin of the sample, as follows: Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E\u003C\u002Fsub\u003E, esterase from the uppermost oxic B1A strata; and Est\u003Csub\u003E\u003Ci\u003EB\u003C\u002Fi\u003E\u003C\u002Fsub\u003E, esterase from the lowermost anoxic B1B sediment attached strata. The final number (subscript) is an arbitrary number representing the number of enzymes per site. Sequences encoding enzymes were deposited under the BioProject IDs PRJNA193663 (for B1A) and PRJNA193664 (for B1B) in the NCBI public database, with the accession numbers detailed in \u003Ca href=\"#T1\"\u003ETable 1\u003C\u002Fa\u003E.\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003ETABLE 1\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" name=\"table1\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t001.jpg\" alt=\"www.frontiersin.org\" id=\"T1\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003ETable 1.\u003C\u002Fstrong\u003E General sequence-based characteristics of Los Rueldos esterases.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Ch3\u003EThree-Dimensional Modeling\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EThe models of the protein structures were predicted with AlphaFold 2.1.0 (\u003Ca href=\"#B44\"\u003EJumper et al., 2021\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Ca id=\"h4\" name=\"h4\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EResults\u003C\u002Fh2\u003E\r\n\u003Ch3 class=\"pt0\"\u003EEnzyme Selection and Divergence at the Sequence Level\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb15\"\u003EMicrobial communities inhabiting two distinct compartments within Los Rueldos AMD formation were screened for sequences encoding carboxylesterases or lipases. For that, we used two complementary metagenomics approaches, namely, naïve and homology sequence screens. First, a total of approximately 81,000 pCCFOS1 clones from each clone library (equivalent to 2.8 Gbp for B1A and 3.20 Gbp for B1B) were screened for esterase\u002Flipase activity using plate-based screen with αNA and tributyrin as model substrates. We identified a total of 10 positive clones being active against both substrates in the B1A clone library, whereas no positives were found in the B1B library. The fosmids with insert lengths ranging from 16,545 to 41,280 bp were fully sequenced, from which 10 genes (one per positive clone), encoding presumptive esterases\u002Flipases, were identified. In addition, we searched the predicted protein-coding genes obtained through next-generation sequencing for sequences encoding esterases and lipases by BLAST search against the ESTHER and LED databases. A total of 6 full-length sequences (B1A: 2; B1B: 4), with accession numbers EQD63018.1, EQD66234.1, EQD71191.1, EQD52136.1, EQD55146.1, and EQD26916.1, encoding potential enzymes were identified. Taken together, a total of 16 genes encoding hydrolases from the α\u002Fβ-hydrolase fold superfamily, specifically, 12 from B1A (EstA\u003Csub\u003E1\u003C\u002Fsub\u003E to EstA\u003Csub\u003E12\u003C\u002Fsub\u003E) and 4 from B1B (EstB\u003Csub\u003E1\u003C\u002Fsub\u003E to EstB\u003Csub\u003E4\u003C\u002Fsub\u003E), were identified (\u003Ca href=\"#T1\"\u003ETable 1\u003C\u002Fa\u003E). As determined by Matcher (EMBOSS package), the pairwise amino acid sequence identity for 14 of the 16 α\u002Fβ hydrolases ranged from 4.0 to 44.9%. This, together with the fact that only 2 out of 16 polypeptides were highly similar (EstA\u003Csub\u003E5\u003C\u002Fsub\u003E and EstA\u003Csub\u003E6\u003C\u002Fsub\u003E differ in only 2 amino acids: arginine 152 and alanine 179 in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E are cysteine 152 and threonine 179 in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E), suggests a large divergence at the sequence level within the enzymes examined, and that the diversity of polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins, but rather by diverse non-redundant sequences. Note that only 1 of 10 sequences selected after naïve screens was found in the metagenomic data generated after direct DNA sequencing (EstA\u003Csub\u003E11\u003C\u002Fsub\u003E, which is 99% identical to GenBank accession no. EQD37671.1 from the Los Rueldos metagenome) (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). This demonstrates that both types of screens (naïve and \u003Ci\u003Ein silico\u003C\u002Fi\u003E) are complementary tools for enzyme discovery. However, deeper metagenomic sequencing could potentially detect all enzymes isolated by naïve screens.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EThe deduced molecular mass and estimated isoelectric point (\u003Ci\u003Ep\u003C\u002Fi\u003EI) values ranged from 23.19 to 101.53 kDa and from 4.62 to 10.04, respectively. Putative proteins exhibited a maximum amino acid sequence identity ranging from 39 to 100% to putative esterases\u002Flipases in public databases (\u003Ca href=\"#T1\"\u003ETable 1\u003C\u002Fa\u003E). It is worth mentioning that EstA\u003Csub\u003E3\u003C\u002Fsub\u003E, EstA\u003Csub\u003E8\u003C\u002Fsub\u003E, and EstB\u003Csub\u003E3\u003C\u002Fsub\u003E are related to presumptive esterase\u002Flipase-like subfamily proteins of the Serine-Glycine-Asparagine-Histidine (SGNH) hydrolases, EstA\u003Csub\u003E11\u003C\u002Fsub\u003E to presumptive glycoside-hydrolase family GH114 (N-terminal domain) and CE4_PelA_like hydrolases (C-terminal domain), and EstA\u003Csub\u003E12\u003C\u002Fsub\u003E to presumptive sialate O-acetylesterases. A further TBLASTX search against metagenomics proteins deposited in databases revealed no similarity of 10 proteins with homologous AMD metagenome proteins. In contrast, five proteins (EstA\u003Csub\u003E3\u003C\u002Fsub\u003E to EstA\u003Csub\u003E6\u003C\u002Fsub\u003E, and EstA\u003Csub\u003E12\u003C\u002Fsub\u003E) do share from 27 to 54% homology to three proteins from the Carnoules arsenic-contaminated mine drainage (GenBank: CBI07622.1, CBH97521.1, and CBI00527.1). This finding suggests that esterases\u002Flipases from microbial communities from the Los Rueldos site are distantly related to proteins from other known homologous proteins from AMD formations with metagenome sequences available. It also reflects the large undiscovered pool of enzymes from bacterial species populating the Los Rueldos site.\u003C\u002Fp\u003E\r\n\u003Ch3\u003EPrimary Structure Analysis\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EBased on the comparison of the primary structures, 14 families of sequence-related esterases and lipases have been reported (\u003Ca href=\"#B4\"\u003EArpigny and Jaeger, 1999\u003C\u002Fa\u003E; \u003Ca href=\"#B59\"\u003ERao et al., 2013\u003C\u002Fa\u003E). Sequence analysis categorized 13 enzymes from Los Rueldos into some of these known subfamilies (\u003Ca href=\"#F1\"\u003EFigure 1\u003C\u002Fa\u003E) with most structurally similar homologs as follows: EstA\u003Csub\u003E9\u003C\u002Fsub\u003E [27%; best hit in Protein Data Bank (PDB) 3DOH_A] and EstB\u003Csub\u003E4\u003C\u002Fsub\u003E (41%; 3OM8_A) to Family I; EstA\u003Csub\u003E1\u003C\u002Fsub\u003E (41%; 3V9A_A), EstA\u003Csub\u003E4\u003C\u002Fsub\u003E (41%; 4YPV_A), EstA\u003Csub\u003E5\u003C\u002Fsub\u003E (49%; 4YPV_A), and EstA\u003Csub\u003E6\u003C\u002Fsub\u003E (49%; 4YPV_A) to Family IV; EstA\u003Csub\u003E7\u003C\u002Fsub\u003E (53%; 4YPV_A) and EstB\u003Csub\u003E2\u003C\u002Fsub\u003E (41%; 1AUO_A) to Family VI; EstA\u003Csub\u003E2\u003C\u002Fsub\u003E (37%; 2OGT_A) to Family VII (EstA\u003Csub\u003E2\u003C\u002Fsub\u003E); and EstA\u003Csub\u003E10\u003C\u002Fsub\u003E (41%; 4IVK_A) to beta-lactamase like Family VIII. EstA\u003Csub\u003E3\u003C\u002Fsub\u003E (43%; PDB code 3P94_A), EstA\u003Csub\u003E8\u003C\u002Fsub\u003E (44%; PDB code 3P94_A), and EstB\u003Csub\u003E3\u003C\u002Fsub\u003E (26%; PDB code 3KVN_X) belong to Family II GDSL, but the structural alignment also confirms that they contain a domain that displays the characteristic α–β–α globular fold of the SGNH hydrolase family. In addition, EstB\u003Csub\u003E3\u003C\u002Fsub\u003E also contains a passenger domain providing the driving force for passenger translocation (\u003Ca href=\"#B65\"\u003EVan den Berg, 2010\u003C\u002Fa\u003E). Three of the sequences could not be assigned to these subfamilies. First, EstA\u003Csub\u003E11\u003C\u002Fsub\u003E contains a 300 amino acid long N-terminal domain most similar to glycoside-hydrolase family 114 and a 616 amino acid long C-terminal domain most similar to the carbohydrate esterase 4 (CE4) superfamily that includes chitin deacetylases (EC 3.5.1.41), N-acetylglucosamine deacetylases (EC 3.5.1.-), and acetylxylan esterases (EC 3.1.1.72), which catalyze the N- or O-deacetylation of substrates such as acetylated chitin, peptidoglycan, and acetylated xylan. Its N-terminal domain is most structurally similar (26%) to that of the glycosidase 2AAM_A and its C-terminal domain is structurally similar to the polysaccharide deacetylase from \u003Ci\u003EBacillus cereus\u003C\u002Fi\u003E (4HD5). Second, EstA\u003Csub\u003E12\u003C\u002Fsub\u003E is associated with acetylxylan esterases (EC 3.1.1.72), with most similar (21%) structural homolog in PDB being 1ZMB_A. Third, EstB\u003Csub\u003E1\u003C\u002Fsub\u003E shows homology to small serine alpha\u002Fbeta-hydrolase\u002Facyl-peptidase (58%; 2FUK_A). The tentative amino acids participating in the typical catalytic triad of esterases and lipases are summarized in \u003Ca href=\"#T1\"\u003ETable 1\u003C\u002Fa\u003E. Together, the analysis of the primary sequence suggests that the diversity of esterases was not dominated by a particular type of protein or a highly similar cluster of proteins, but rather by diverse non-redundant sequences belonging to different microbial groups and distinct esterase\u002Flipase subfamilies.\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003EFIGURE 1\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" name=\"figure1\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g001.jpg\" alt=\"www.frontiersin.org\" id=\"F1\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003EFigure 1.\u003C\u002Fstrong\u003E The unrooted circular neighbor-joining tree indicating phylogenetic positions of polypeptide sequences of Los Rueldos esterases. Positioning is referred to homologous proteins with unambiguous categorization into lipase\u002Festerase families (from Family I to XIV) according to Arpigny and Jaeger and further classifications (\u003Ca href=\"#B4\"\u003EArpigny and Jaeger, 1999\u003C\u002Fa\u003E; \u003Ca href=\"#B59\"\u003ERao et al., 2013\u003C\u002Fa\u003E). GS-F, genome sequences assigned to an esterase\u002Flipase family (in bold letters). Sequences from Los Rueldos that correspond to proteins that could not be produced as soluble active proteins using Ek\u002FLIC 46 vector and \u003Ci\u003EE. coli\u003C\u002Fi\u003E strain BL21 (DE3) as a host are indicated in gray color, while those being active and soluble are indicated in bold.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Ch3\u003ESource Organisms of Selected Polypeptides\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb0\"\u003EA search of oligonucleotide patterns against the GOHTAM database (\u003Ca href=\"#B52\"\u003EMénigaud et al., 2012\u003C\u002Fa\u003E) and TBLASTX analysis revealed compositional similarities between the DNA fragment containing the genes for EstA\u003Csub\u003E1\u003C\u002Fsub\u003E, EstA\u003Csub\u003E2\u003C\u002Fsub\u003E, EstA\u003Csub\u003E10\u003C\u002Fsub\u003E, and EstB\u003Csub\u003E4\u003C\u002Fsub\u003E, with genomic sequences of bacteria from the phylum \u003Ci\u003EActinomycetota\u003C\u002Fi\u003E. Among them, only unambiguous affiliations at lower levels could be achieved for fragments containing Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E, and EstA\u003Csub\u003E10\u003C\u002Fsub\u003E that may most likely belong to bacteria from the genera \u003Ci\u003EAcidithrix\u003C\u002Fi\u003E (EstA\u003Csub\u003E1\u003C\u002Fsub\u003E and EstA\u003Csub\u003E10\u003C\u002Fsub\u003E) and \u003Ci\u003EAcidimicrobium\u003C\u002Fi\u003E\u002F\u003Ci\u003EFerrimicrobium\u003C\u002Fi\u003E (EstA\u003Csub\u003E2\u003C\u002Fsub\u003E), both from the family \u003Ci\u003EAcidimicrobiaceae\u003C\u002Fi\u003E within the order \u003Ci\u003EAcidimicrobiales\u003C\u002Fi\u003E. Note that EstA\u003Csub\u003E1\u003C\u002Fsub\u003E and EstA\u003Csub\u003E10\u003C\u002Fsub\u003E showed 99–100% sequence identity with uncharacterized esterases and lipases (WP_052605564.1 and WP_052605292.1) from \u003Ci\u003EAcidithrix ferrooxidans\u003C\u002Fi\u003E, and EstA\u003Csub\u003E2\u003C\u002Fsub\u003E showed 94% sequence identity with an uncharacterized esterase-lipase (NNN14078.1) from \u003Ci\u003EAcidimicrobiaceae\u003C\u002Fi\u003E. EstA\u003Csub\u003E3\u003C\u002Fsub\u003E was most likely derived from an uncultured bacterium assigned to the phylum \u003Ci\u003EAcidobacteria\u003C\u002Fi\u003E with ambiguous affiliation below the phylum level. The genes for EstA\u003Csub\u003E4\u003C\u002Fsub\u003E to EstA\u003Csub\u003E9\u003C\u002Fsub\u003E, EstA\u003Csub\u003E11\u003C\u002Fsub\u003E, and EstB\u003Csub\u003E1\u003C\u002Fsub\u003E to EstB\u003Csub\u003E3\u003C\u002Fsub\u003E were associated with uncultured bacteria of the \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E phylum, with ambiguous affiliations at a lower taxonomic level, except for EstA\u003Csub\u003E4\u003C\u002Fsub\u003E, which was most likely derived from a bacterium of the genus \u003Ci\u003EAcidiphilium\u003C\u002Fi\u003E from the family \u003Ci\u003EAcetobacteraceae\u003C\u002Fi\u003E within the order \u003Ci\u003ERhodospirillales\u003C\u002Fi\u003E (best hit OYV70855.1 from \u003Ci\u003EAcidiphilium\u003C\u002Fi\u003E sp., 79% homology). All these bacterial groups have been detected in biofilms thriving in the Los Rueldos mine (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). No clear affiliation, other than Bacteria, could be found for EstA\u003Csub\u003E12\u003C\u002Fsub\u003E. Note that in some cases no clear affiliation to a taxon of source organism below the level of the phylum could be established, either because of the short fragment length or the low compositional similarity between the metagenomic fragments and the sequences of related bacterial chromosomes and plasmids do not allow proper assignations.\u003C\u002Fp\u003E\r\n\u003Ch3\u003EEnzyme Characterization\u003C\u002Fh3\u003E\r\n\u003Cp class=\"mb15\"\u003EFrom the 16 sequences selected, 8 from B1A and 2 from B1B were successfully cloned, expressed, and purified as soluble active proteins when expressed in pET Ek\u002FLIC 46 vector and \u003Ci\u003EE. coli\u003C\u002Fi\u003E BL21 as the host. These proteins were herein referred to as EstA\u003Csub\u003E1\u003C\u002Fsub\u003E to EstA\u003Csub\u003E8\u003C\u002Fsub\u003E, EstB\u003Csub\u003E1\u003C\u002Fsub\u003E, and EstB\u003Csub\u003E2\u003C\u002Fsub\u003E. The remaining six (EstA\u003Csub\u003E9\u003C\u002Fsub\u003E-EstA\u003Csub\u003E12\u003C\u002Fsub\u003E and EstB\u003Csub\u003E3\u003C\u002Fsub\u003E-EstB\u003Csub\u003E4\u003C\u002Fsub\u003E) could not be produced in soluble active form (they formed inclusion bodies) in the expression system applied herein, which consists of the use of the Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Ek\u002FLIC 46 vector and \u003Ci\u003EE. coli\u003C\u002Fi\u003E strain BL21 (DE3) as a host, and their properties are not described herein. Refining the expression conditions, which included variations in the expression conditions (16, 30, and 37°C) and IPTG concentration (from 0.1 to 2 mM), resulted in unsuccessful production of sufficient active protein material for characterization. Further efforts may be needed with different expression vectors, which is beyond the scope of the present study.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EThe substrate profile of all α\u002Fβ hydrolases was first examined using a set of esters commonly used to characterize esterases and lipases, namely, \u003Ci\u003Ep\u003C\u002Fi\u003ENP esters such as \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E2\u003C\u002Fsub\u003E, \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E, \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E4\u003C\u002Fsub\u003E, \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E8\u003C\u002Fsub\u003E, \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E10\u003C\u002Fsub\u003E, and \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E12\u003C\u002Fsub\u003E. All ester hydrolases preferred short-chain-length pNP-esters, particularly \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E2\u003C\u002Fsub\u003E (EstA\u003Csub\u003E3\u003C\u002Fsub\u003E), \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E (EstA\u003Csub\u003E2\u003C\u002Fsub\u003E, EstA\u003Csub\u003E4\u003C\u002Fsub\u003E, EstA\u003Csub\u003E5\u003C\u002Fsub\u003E, EstA\u003Csub\u003E6\u003C\u002Fsub\u003E, EstB\u003Csub\u003E1\u003C\u002Fsub\u003E, and EstB\u003Csub\u003E2\u003C\u002Fsub\u003E), and \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E4\u003C\u002Fsub\u003E (EstA\u003Csub\u003E1\u003C\u002Fsub\u003E, EstA\u003Csub\u003E7\u003C\u002Fsub\u003E, and EstA\u003Csub\u003E8\u003C\u002Fsub\u003E) (\u003Ca href=\"#T2\"\u003ETable 2\u003C\u002Fa\u003E). Within all six \u003Ci\u003Ep\u003C\u002Fi\u003ENP ester tested, all but one (EstA\u003Csub\u003E3\u003C\u002Fsub\u003E) enzyme was able to hydrolyze up to \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E12\u003C\u002Fsub\u003E, albeit at a much lower level (from 62- to 3,900-fold) compared to shorter derivatives. Considering the preferred \u003Ci\u003Ep\u003C\u002Fi\u003ENP esters, the maximum specific activity ranged from 3.06 ± 0.03 to 679.8 ± 9.8 U\u002Fmg. We further test the possibility that the enzymes hydrolyze substrates other than \u003Ci\u003Ep\u003C\u002Fi\u003ENP esters, particularly, plastic substrates and esters relevant to plastics. Using previously described conditions (\u003Ca href=\"#B37\"\u003EHajighasemi et al., 2018\u003C\u002Fa\u003E; \u003Ca href=\"#B11\"\u003EBollinger et al., 2020\u003C\u002Fa\u003E), we did not find that any of the enzymes hydrolyzed large plastic materials such as poly(propylene glycol) diacrylate, poly(DL-lactide), amorphous PET film, and PET nanoparticles. However, by using a pH-indicator assay, we found that the enzymes were able to hydrolyze other terephthalate esters and acrylate esters. Thus, as shown in \u003Ca href=\"#T3\"\u003ETable 3\u003C\u002Fa\u003E, six of the enzymes hydrolyzed esters relevant to acrylic acid plastics, e.g., 2-naphthyl acrylate and tripropylene glycol diacrylate, a commonly used material principally exploited to prepare thermally stable polymers (\u003Ca href=\"#B38\"\u003EHe et al., 2017\u003C\u002Fa\u003E). These substrates, herein found to be converted at a maximum rate of 3,915 ± 48 U\u002Fg, are rarely hydrolyzed by esterases and lipases. There are only two examples reported, namely, human salivary pseudocholinesterase and cholesterol esterase (\u003Ca href=\"#B24\"\u003EFiner and Santerre, 2004\u003C\u002Fa\u003E; \u003Ca href=\"#B13\"\u003ECai et al., 2014\u003C\u002Fa\u003E). In addition, one of the enzymes (Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E) was capable of hydrolyzing dibenzyl terephthalate (432.2 ± 27.5 U\u002Fg), an intermediate produced during chemical PET recycling with benzyl alcohol in the presence of a catalyst (\u003Ca href=\"#B19\"\u003EDonahue et al., 2003\u003C\u002Fa\u003E). No esterase or lipase has been reported to date that degrades this substrate. In addition, six of the esterases (Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E, and Est\u003Csub\u003E\u003Ci\u003EB\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E) efficiently hydrolyzed BHET (from 5.0 ± 1.0 to 336.9 ± 3.6 U\u002Fg), an intermediate in the degradation of PET (\u003Ca href=\"#B68\"\u003EYoshida et al., 2016\u003C\u002Fa\u003E). High performance liquid chromatography (HPLC) analysis, performed as described (\u003Ca href=\"#B11\"\u003EBollinger et al., 2020\u003C\u002Fa\u003E), confirmed the hydrolysis of BHET to mono-(2-hydroxyethyl)-terephthalic acid (MHET) and not to terephthalic acid. To conclude, the enzymes reported herein from the Los Rueldos AMD formation showed high activity for converting and recycling components of synthetic plastics, namely, acrylic- and terephthalate-based plastics, and could be of potential use in developing plastic degradation strategies.\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003ETABLE 2\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" name=\"table2\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t002.jpg\" alt=\"www.frontiersin.org\" id=\"T2\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003ETable 2.\u003C\u002Fstrong\u003E Specific activity (U\u002Fmg pure protein) for each of the enzymes tested over a set of p-nitrophenyl (\u003Ci\u003Ep\u003C\u002Fi\u003ENP) esters of different lengths.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine mb15\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003ETABLE 3\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" name=\"table3\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-t003.jpg\" alt=\"www.frontiersin.org\" id=\"T3\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003ETable 3.\u003C\u002Fstrong\u003E Specific activity (U\u002Fg pure protein) for each of the enzymes able to hydrolyze a set of structurally different plastic-related esters.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cp class=\"mb15 w100pc float_left mt15\"\u003EUsing \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E as a substrate, the purified proteins were most active at temperatures ranging from 30 to 65°C (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E). The average annual temperature in Los Rueldos is 13.8 ± 0.6°C, which varied from 10 ± 0.6°C to 17.1 ± 0.6 (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E), with a temperature of 17°C when samples were taken (July). At this value, the enzymes retained from 20 to 61% of the activity shown at the optimal temperature (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003EFIGURE 2\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" name=\"figure2\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g002.jpg\" alt=\"www.frontiersin.org\" id=\"F2\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003EFigure 2.\u003C\u002Fstrong\u003E pH and thermal profiles of the purified enzymes. The data represent the relative percentages (%) of specific activity (U\u002Fg) in triplicates compared with the maximum activity using \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E as substrate. For raw data see \u003Ca href=\"#S9\"\u003ESupplementary Table 1\u003C\u002Fa\u003E.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cp class=\"mb15 w100pc float_left mt15\"\u003EUsing \u003Ci\u003Ep\u003C\u002Fi\u003ENPC\u003Csub\u003E3\u003C\u002Fsub\u003E as a substrate, all enzymes showed an optimum pH for activity from neutral to slightly basic, which varied from 7.0 to 9.0 (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E). This finding suggests that these proteins are most likely intracellularly produced, consistent with the absence of signal peptides in their sequences. Even though the enzymes showed a slightly basic optimum pH, all retained 33–68% of their activity at pH 5.5. Interestingly, Est\u003Csub\u003E\u003Ci\u003EA6\u003C\u002Fi\u003E\u003C\u002Fsub\u003E shows two activity peaks, one at pH 5.5 and one at pH 9.0, while Est\u003Csub\u003E\u003Ci\u003EA5\u003C\u002Fi\u003E\u003C\u002Fsub\u003E, which only differs in two amino acids, has an optimum pH of 9.0 (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003ESequence analysis revealed that EstA\u003Csub\u003E5\u003C\u002Fsub\u003E and EstA\u003Csub\u003E6\u003C\u002Fsub\u003E which have their origins in a bacterium of the phylum \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E, differ in only 2 amino acids (99.4% identity). Positions 152 and 172 are occupied by Arg and Ala in EstA\u003Csub\u003E5\u003C\u002Fsub\u003E and by Cys and Thr in EstA\u003Csub\u003E6\u003C\u002Fsub\u003E, respectively. Notably, EstA\u003Csub\u003E5\u003C\u002Fsub\u003E was most active at 30°C, retaining more than 80% of the activity at temperatures from 20 to 45°C (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E). The optimum temperature for activity increased up to 45°C for EstA\u003Csub\u003E6\u003C\u002Fsub\u003E, which maintained more than 80% of its activity in the range from 30 to 60°C. Analysis by circular dichroism revealed that Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E showed a sigmoidal curve from which a temperature of denaturation of 60.4 ± 0.2°C could be obtained (\u003Ca href=\"#F3\"\u003EFigure 3\u003C\u002Fa\u003E). However, the curve for Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E shows two transitions, one with a denaturation temperature of 48.1 ± 0.8°C and a second at 75.7 ± 0.2°C. The presence of these two phases may therefore indicate that the presence of these two amino acids may contribute to protein stability and its denaturation under distinct conditions. This result may explain the higher optimum temperature for the activity of this enzyme compared to Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E, and the stabilization effect of Cys152 and Thr172. This difference in thermostability between Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E and Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E can probably be explained by the difference in amino acid 152, since Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E has a Cys that would allow it to make a possible disulfide bridge with Cys181, giving it greater thermostability than Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E (since Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E has an Arg at position 152 instead of a Cys), as shown by examination of the 3D models (\u003Ca href=\"#S9\"\u003ESupplementary Figure 2\u003C\u002Fa\u003E and \u003Ca href=\"#F4\"\u003EFigure 4\u003C\u002Fa\u003E). It is plausible that this difference may also be responsible for the different pH profiles of both enzymes (\u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E). If the disulfide bridge was present in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E, it could be removed by reduction or chemical modification. Activity tests revealed that both enzymes are resistant to the reducing agent beta-mercaptoethanol, with no activity lost even after 24 h of incubation at 30–45°C in the presence of a 1–10 mM inhibitor. By contrast, in the presence of the cysteine alkylating agent iodoacetamide, both enzymes were rapidly inactivated after 5 min. Thus, we could not verify the presumptive formation of the disulfide bridge in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E, and further studies are needed to test this assumption.\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003EFIGURE 3\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" name=\"figure3\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g003.jpg\" alt=\"www.frontiersin.org\" id=\"F3\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003EFigure 3.\u003C\u002Fstrong\u003E The thermal denaturation curve of Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E (filled circle) and Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E (open circle) at pH 7.0. The datasets were obtained by measuring the ellipticity changes at 220 nm obtained at different temperatures. For raw data see \u003Ca href=\"#S9\"\u003ESupplementary Table 2\u003C\u002Fa\u003E.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine mb15\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"Imageheaders\"\u003EFIGURE 4\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"FigureDesc\"\u003E\r\n\u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" name=\"figure4\" target=\"_blank\"\u003E\r\n\n \u003Cpicture\u003E\n \u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=480&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" media=\"(max-width: 563px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=370&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" media=\"(max-width: 1024px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=290&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" media=\"(max-width: 1441px)\"\u003E\u003Csource type=\"image\u002Fwebp\" srcset=\"https:\u002F\u002Fimages-provider.frontiersin.org\u002Fapi\u002Fipx\u002Fw=410&f=webp\u002Fhttps:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" media=\"\"\u003E\u003Csource type=\"image\u002Fjpg\" srcset=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" media=\"\"\u003E \u003Cimg src=\"https:\u002F\u002Fwww.frontiersin.org\u002Ffiles\u002FArticles\u002F868839\u002Ffmicb-13-868839-HTML\u002Fimage_m\u002Ffmicb-13-868839-g004.jpg\" alt=\"www.frontiersin.org\" id=\"F4\" loading=\"lazy\"\u003E\n \u003C\u002Fpicture\u003E\n\u003C\u002Fa\u003E\r\n\u003Cp\u003E\u003Cstrong\u003EFigure 4.\u003C\u002Fstrong\u003E Three-dimensional comparison of the Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E and Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E hydrolases \u003Cb\u003E(A)\u003C\u002Fb\u003E. Zoom into the region that is different between both proteins, where the possible interaction of Cys152 and Cys181 in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E can be seen in panel \u003Cb\u003E(B)\u003C\u002Fb\u003E. As shown, position 152 is occupied by Arg in Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E instead. Figure has been created using PMOL(TM) 2.2.3.\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"DottedLine\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Ca id=\"h5\" name=\"h5\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EDiscussion\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb15\"\u003EThe effects of environmental constraints as prime forces shaping AMD populations have only begun to be elucidated through omics studies (\u003Ca href=\"#B51\"\u003EMéndez-García et al., 2015\u003C\u002Fa\u003E). These effects are also of high interest in the context of the isolation and characterization of novel enzymes, for which limited data are available. However, the difficulty of cultivating organisms inhabiting AMD sites, which is due to their longer generation times, lower biomass yields, and cultivation conditions that are not yet fully understood, requires different strategies to overcome the problems associated not only with their cultivation, but also with the isolation of enzymes. Metagenomic approaches allow the screening of enzymes from such extreme environments. However, by using these tools, we have thus far explored only a small fraction of the enormous diversity on the planet, especially that of organisms inhabiting extremely acidic environments (\u003Ca href=\"#B21\"\u003EFerrer et al., 2015\u003C\u002Fa\u003E), again indicating the importance of establishing enzyme screening programs for AMD sites. The particular characteristics of the Los Rueldos AMD site (\u003Ca href=\"#B51\"\u003EMéndez-García et al., 2015\u003C\u002Fa\u003E) that make it an interesting study site include the following. First, it is populated by a larger diversity of \u003Ci\u003EBacteria\u003C\u002Fi\u003E and \u003Ci\u003EArchaea\u003C\u002Fi\u003E compared to other AMD sites, containing a total of 39 different species. Second, it has high microbial heterogeneity in local microniches defined by its O\u003Csub\u003E2\u003C\u002Fsub\u003E concentration gradients and spatial and biofilm architecture. As an example, only 1 of 18 species inhabiting the two distinct compartments in a stratified streamer investigated herein, namely, the oxic uppermost (B1A) and anoxic lowermost (B1B) sediment-attached strata, was shared. Therefore, it is plausible that Los Rueldos may also contain a greater diversity of microbial products such as enzymes.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EWe have sought to address this possibility by screening for esterases and lipases from the α\u002Fβ-hydrolase fold superfamily in microbial communities inhabiting the Los Rueldos AMD site. These enzymes are desired tools for biocatalysis in a variety of industrial sectors (\u003Ca href=\"#B16\"\u003EDaiha et al., 2015\u003C\u002Fa\u003E; \u003Ca href=\"#B21\"\u003EFerrer et al., 2015\u003C\u002Fa\u003E). Microorganisms that can survive under low pH values similar to those in Los Rueldos (pH ∼2) could be good sources of enzymes that can be used, for example, as additives in detergents, for the biobleaching of pulp and paper, in the clean-up of effluent streams from the textile processing industry, and in the degradation of plastics (\u003Ca href=\"#B35\"\u003EGomes et al., 2003\u003C\u002Fa\u003E; \u003Ca href=\"#B1\"\u003EAdrio and Demain, 2014\u003C\u002Fa\u003E; \u003Ca href=\"#B53\"\u003ENchedo Ariole and George-West, 2020\u003C\u002Fa\u003E) and other polymers (\u003Ca href=\"#B27\"\u003EFütterer et al., 2004\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EWe used two complementary approaches for enzyme mining. A sequence-based metagenomic approach that searched for homologous enzymes in the metagenomic sequence data and function-driven screens in which expression libraries were used to identify, by using specific colorimetric substrates (\u003Ca href=\"#B22\"\u003EFerrer et al., 2016\u003C\u002Fa\u003E; \u003Ca href=\"#B56\"\u003EPeña-García et al., 2016\u003C\u002Fa\u003E), clones containing enzymes of interest that could be missed in shallow metagenomics sequencing. By using both approaches, we identified 16 sequences that were potentially encoding esterases and lipases. The amino acid sequences were distantly related to sequences found in other AMD formations, which was in agreement with the distinct Los Rueldos-specific populations (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). Indeed, we only observed some degree of sequence identity (27–54%) to 3 homologs from the Carnoulès (lead-zinc) mine, France (\u003Ca href=\"#B10\"\u003EBertin et al., 2011\u003C\u002Fa\u003E) in 6 of 16 sequences. In addition, the large differences among the recovered enzymes may correspond to the high population diversity that characterizes the Los Rueldos site (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003ENotably, activity-based screens did not yield any active clones from the library created from the anoxic lowermost strata (B1B), while they yielded 10 active clones from the library created from the oxic uppermost strata (B1A). Thus, we searched for such enzymes in B1B by screening sequence data generated in a previous study (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E). It is plausible that the presence of low-O\u003Csub\u003E2\u003C\u002Fsub\u003E-adapted microbial species in B1B, in contrast to the aerobic species in B1A, may account for the low efficiency of heterologous gene expression after cloning of the genetic material in the \u003Ci\u003EE. coli\u003C\u002Fi\u003E host and, possibly, the lower efficiency of the screening tests in the B1B library compared to that obtained for B1A. However, the fact that similar proportions of identified proteins (by naïve and sequence screening) in B1A (7 of 12) and B1B (2 of 4) could be produced as soluble active proteins when expressed in \u003Ci\u003EE. coli\u003C\u002Fi\u003E suggests that this may not be the only reason explaining the absence of positive clones in the B1B library. We cannot rule out that the native promoters of the partial genes from microorganisms inhabiting B1B cloned in the pCCFOS1 fosmids were inactive in \u003Ci\u003EE. coli\u003C\u002Fi\u003E, resulting in failed active clones on the plates. The data provided in \u003Ca href=\"#T2\"\u003ETables 2\u003C\u002Fa\u003E, \u003Ca href=\"#T3\"\u003E3\u003C\u002Fa\u003E revealed that B1B enzymes were among the least-active enzymes among all hydrolases identified and characterized in the present study, and it is therefore also plausible that the low efficiency of the screening tests may have been due to the low activity level of enzymes from microorganisms inhabiting the anoxic B1B compartment. Additionally, it is plausible that different screening conditions (temperature, pH, inductor concentration, etc.) may be needed to detect other active proteins and that the enzymes from B1B would be more active under other assay conditions, the investigation of which is beyond the scope of the present study.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003ERegardless of the problems associated with the screening efficiency in different environments, including extreme AMD formations such as Los Rueldos, the analysis of the optimal pH profile of 10 out of the 16 hydrolases that could be produced in active form additionally revealed that their optimal pH was in the range from 7.0 to 9.0. This finding suggests that all hydrolases are presumptively produced intracellularly by acidophiles that thrive in the acidic Los Rueldos environment with a pH of 2.0. A similar phenotype has been found for other enzymes from AMD inhabitants, such as ATP-dependent DNA ligase from “\u003Ci\u003EF. acidarmanus\u003C\u002Fi\u003E” Fer1 (\u003Ca href=\"#B40\"\u003EJackson et al., 2007\u003C\u002Fa\u003E) and ene-reductase from “\u003Ci\u003EFerrovum\u003C\u002Fi\u003E” sp. JA12 (\u003Ca href=\"#B62\"\u003EScholtissek et al., 2016\u003C\u002Fa\u003E), with pH optima of 6.0–7.0. However, we observed that most of the enzymes showed a slightly acid-stable phenotype, retaining ∼33–68% of activity at pH 5.5. It is plausible that the identification of enzymes with neutral-like pH optima is a consequence of screening tests performed at neutral pH, using a vector and host that allow mostly intracellular proteins to be produced and that presumably acid-stable enzymes could not be detected. In the future, performing naïve screens at such low pH values may help obtain additional active clones. However, while specific adaptations need to be explored in great detail, the retention of a high activity level at a slightly acidic pH might be attributed to the prevalence of acidic amino acids (negatively charged at a neutral pH) on the surfaces of these enzymes (\u003Ca href=\"#S9\"\u003ESupplementary Figure 2\u003C\u002Fa\u003E), as reported for other proteins from acidophiles (\u003Ca href=\"#B66\"\u003EWu et al., 2020\u003C\u002Fa\u003E). Indeed, the relative frequencies of acidic residues in proteins in this study ranged from 67 to 54%, except for Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E (37%). As no major differences in pH profiles were observed when comparing the enzymes with the highest (Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E: 67%) and lowest (Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E: 37%) percentages of acidic residues, it is possible that other factors affect the activity and stability of the studied proteins from the Los Rueldos site. An example of this is the differences in stability against pH and temperature of Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E and Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E, which show very different features and have only a two amino acid difference despite having the same percentage of acidic residues.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EFurthermore, the biochemical properties of the esterases reported in this study revealed that all enzymes showed an activity–stability trade-off characteristic of mesophilic-adapted enzymes (from 30 to 65°C), which is a phenotype that has also been found for enzymes from other AMD inhabitants (\u003Ca href=\"#B31\"\u003EGolyshina et al., 2006\u003C\u002Fa\u003E; \u003Ca href=\"#B40\"\u003EJackson et al., 2007\u003C\u002Fa\u003E; \u003Ca href=\"#B54\"\u003EOhara et al., 2014\u003C\u002Fa\u003E; \u003Ca href=\"#B62\"\u003EScholtissek et al., 2016\u003C\u002Fa\u003E). It is noticeable, however, that 5 of 10 characterized enzymes retained at least 50% of their maximal activity at temperatures as low as 12°C. The fact that the Los Rueldos site is characterized by a relatively low temperature compared to other AMD sites (\u003Ca href=\"#B50\"\u003EMéndez-García et al., 2014\u003C\u002Fa\u003E) may account for this low-temperature-active phenotype. However, the lack of biochemical information on enzymes from other AMD sites does not allow us to validate this assumption.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EFinally, it should be emphasized that the activity levels of the characterized enzymes (maximum for the best \u003Ci\u003Ep\u003C\u002Fi\u003ENP substrates: approximately 680 to 3 U\u002Fmg, depending on the hydrolase) were in the range of other reported enzymes of different origins with esterase and lipase activities (\u003Ca href=\"#B21\"\u003EFerrer et al., 2015\u003C\u002Fa\u003E; \u003Ca href=\"#B48\"\u003EMartínez-Martínez et al., 2018\u003C\u002Fa\u003E). The data suggest that the low-O\u003Csub\u003E2\u003C\u002Fsub\u003E-adapted microbial species developed in the anoxic lowermost (B1B) sediment-attached strata do contain less-active enzymes than those developed in the oxic uppermost (B1A) strata under the conditions used herein. Whether this is typical \u003Ci\u003Ein vivo\u003C\u002Fi\u003E or is a result of bias due to the assay conditions would require the characterization of a larger number of enzymes from both microenvironments in the Los Rueldos AMD system. The capacity of six of the enzymes from the Los Rueldos AMD formation, and thus the bacteria that contain them, for degrading acrylic- and terephthalate-like esters is noticeable. These enzymes could potentially be of use in developing plastic degradation strategies that have yet to be explored. In this context, the taxonomic distribution of top protein hits and the results of genome linguistics analysis suggested that the metagenomic fragments containing the six characterized enzymes that can potentially degrade plastic substrates most likely belong to \u003Ci\u003EActinobacteria\u003C\u002Fi\u003E (genera related to \u003Ci\u003EAcidithrix\u003C\u002Fi\u003E and \u003Ci\u003EAcidimicrobium\u003C\u002Fi\u003E\u002F\u003Ci\u003EFerrimicrobium\u003C\u002Fi\u003E), \u003Ci\u003EAcidobacteria\u003C\u002Fi\u003E, and \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E (some are related to \u003Ci\u003EAcidiphilium\u003C\u002Fi\u003E). These are groups of acidophiles that have been largely neglected with respect to enzyme discovery.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb15\"\u003EPolyethylene terephthalate-degrading bioprospecting has shown that only a tiny fraction of carboxylic ester hydrolases can degrade PET and its intermediates BHET and MHET, including Actinomyces (i.e., \u003Ci\u003EThermobifida\u003C\u002Fi\u003E, \u003Ci\u003EThermomonospora\u003C\u002Fi\u003E, and \u003Ci\u003ESaccharomonospora\u003C\u002Fi\u003E), \u003Ci\u003EBacillus\u003C\u002Fi\u003E, \u003Ci\u003EFirmicutes\u003C\u002Fi\u003E (e.g., \u003Ci\u003EClostridium\u003C\u002Fi\u003E), \u003Ci\u003EBacteroidetes\u003C\u002Fi\u003E, \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E (e.g., \u003Ci\u003EPseudomonas\u003C\u002Fi\u003E, \u003Ci\u003EEnterobacteria\u003C\u002Fi\u003E, and \u003Ci\u003EIdeonella sakaiensis\u003C\u002Fi\u003E), and fungi [\u003Ci\u003EFusarium\u003C\u002Fi\u003E and \u003Ci\u003EThermomyces\u003C\u002Fi\u003E (\u003Ci\u003EHumicola\u003C\u002Fi\u003E)] (for a recent example, see \u003Ca href=\"#B68\"\u003EYoshida et al., 2016\u003C\u002Fa\u003E; \u003Ca href=\"#B17\"\u003EDanso et al., 2018\u003C\u002Fa\u003E; \u003Ca href=\"#B11\"\u003EBollinger et al., 2020\u003C\u002Fa\u003E; \u003Ca href=\"#B67\"\u003EYan et al., 2021\u003C\u002Fa\u003E). In this study, we found that bacteria from the genera \u003Ci\u003EAcidithrix\u003C\u002Fi\u003E (the host of Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E1\u003C\u002Fsub\u003E), \u003Ci\u003EAcidimicrobium\u003C\u002Fi\u003E\u002F\u003Ci\u003EFerrimicrobium\u003C\u002Fi\u003E (the host of Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E), and unknown genera from the phylum \u003Ci\u003EProteobacteria\u003C\u002Fi\u003E (the hosts of Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E5\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E6\u003C\u002Fsub\u003E, Est\u003Csub\u003E\u003Ci\u003EA\u003C\u002Fi\u003E8\u003C\u002Fsub\u003E, and Est\u003Csub\u003E\u003Ci\u003EB\u003C\u002Fi\u003E2\u003C\u002Fsub\u003E) could degrade BHET and could potentially degrade PET or PET oligomers under conditions yet to be explored, as no PET hydrolysis was detected under the assay conditions employed herein. Thus, the metagenomics approach applied herein expands the range of microorganisms containing enzymes supporting BHET hydrolysis and, possibly, PET depolymerization. For effective PET hydrolysis, in addition to a high degradation rate at 40–70°C, a broad range of pH stability (toward both the alkaline and acidic ranges) is one of the prerequisites of applied enzymes (\u003Ca href=\"#B49\"\u003EMaurya et al., 2020\u003C\u002Fa\u003E), and these features were characteristic of some of the esterases from Los Rueldos reported herein. Further studies will reveal the catalytic efficiency and stability of hydrolases from Los Rueldos AMD systems to establish PET degradation systems or to support these systems in combination with other known PET-degrading enzymes.\u003C\u002Fp\u003E\r\n\u003Cp class=\"mb0\"\u003EIt is plausible that the capacity to degrade plastic substrates comes from the adaptation of the active sites of enzymes to metabolize microbial polymeric substances that are naturally occurring in AMDs. Thus, in Los Rueldos, as in other AMDs such as the Richmond Mine at Iron Mountain (\u003Ca href=\"#B41\"\u003EJiao et al., 2010\u003C\u002Fa\u003E, \u003Ca href=\"#B42\"\u003E2011\u003C\u002Fa\u003E; \u003Ca href=\"#B47\"\u003EMartínez-Martínez et al., 2013\u003C\u002Fa\u003E), the organisms present might contribute to the use\u002Fdegradation of extracellular polymeric substances (EPS), which requires a broad range of enzymes (\u003Ca href=\"#B26\"\u003EFlemming and Wingender, 2010\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Ca id=\"h6\" name=\"h6\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EData Availability Statement\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EThe datasets presented in this study can be found in online repositories. The names of the repository\u002Frepositories and accession number(s) can be found in the article\u002F\u003Ca href=\"#S9\"\u003ESupplementary Material\u003C\u002Fa\u003E.\u003C\u002Fp\u003E\r\n\u003Ca id=\"h7\" name=\"h7\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EAuthor Contributions\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EMF and AP conceived this study. MM-M contributed to gene cloning and expression. PV, MM-M, LF-L, and MF performed biochemical data and interpreted the data. SR and VG performed the 3D modeling. CM-G and MF contributed sample processing and library construction. OG contributed the phylogenetic analysis. MF drafted and revised the manuscript. All authors discussed, read, approved the manuscript, and authorized its submission for publication.\u003C\u002Fp\u003E\r\n\u003Ca id=\"fun1\" name=\"fun1\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EFunding\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EThis study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We acknowledge financial support under Grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET, GA No. 604814) (MF), BIO2017-85522-R (MF), PID2020-112758RB-I00 (MF), PID2019-106370RB-I00 (VG), and PDC2021-121534-I00 (MF) from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.13039\u002F501100011033\"\u003EDoi: 10.13039\u002F501100011033\u003C\u002Fa\u003E), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU\u002FPRTR”), and Grant 2020AEP061 (MF) from the Agencia Estatal CSIC. OG acknowledges the support of the Centre for Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) \u003Ci\u003Evia\u003C\u002Fi\u003E the Welsh Government. SR thanks the Spanish Ministry of Science and Innovation for a Ph.D. fellowship (FPU19\u002F00608).\u003C\u002Fp\u003E\r\n\u003Ca id=\"conf1\" name=\"conf1\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EConflict of Interest\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003C\u002Fp\u003E\r\n\u003Ca id=\"disclaimer1\" name=\"disclaimer1\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EPublisher’s Note\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\u003C\u002Fp\u003E\r\n\u003Ca id=\"ack1\" name=\"ack1\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EAcknowledgments\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EWe acknowledge David Almendral for supporting the circular dichroism analysis and Rafael Bargiela for preparation of \u003Ca href=\"#F2\"\u003EFigure 2\u003C\u002Fa\u003E. We also acknowledge the support of Cristina Coscolín for supporting the BHET degradation tests and Jesús Sánchez for the support in the study of acid mine drainage (AMD) systems and the evaluation and review of this work.\u003C\u002Fp\u003E\r\n\u003Ca id=\"S9\" name=\"S9\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003ESupplementary Material\u003C\u002Fh2\u003E\r\n\u003Cp class=\"mb0\"\u003EThe Supplementary Material for this article can be found online at: \u003Ca href=\"https:\u002F\u002Fwww.frontiersin.org\u002Farticles\u002F10.3389\u002Ffmicb.2022.868839\u002Ffull#supplementary-material\"\u003Ehttps:\u002F\u002Fwww.frontiersin.org\u002Farticles\u002F10.3389\u002Ffmicb.2022.868839\u002Ffull#supplementary-material\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003Ca id=\"refer1\" name=\"refer1\"\u003E\u003C\u002Fa\u003E\u003Ch2\u003EReferences\u003C\u002Fh2\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B1\" id=\"B1\"\u003E\u003C\u002Fa\u003EAdrio, J. L., and Demain, A. L. (2014). Microbial enzymes: tools for biotechnological processes. \u003Ci\u003EBiomolecules\u003C\u002Fi\u003E 4, 117–139. doi: 10.3390\u002Fbiom4010117\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F24970208\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3390\u002Fbiom4010117\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+enzymes%3A+tools+for+biotechnological+processes%2E&journal=Biomolecules&author=Adrio+J.+L.&author=Demain+A.+L.&publication_year=2014&volume=4&pages=117–139\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B2\" id=\"B2\"\u003E\u003C\u002Fa\u003EAlcaide, M., Tchigvintsev, A., Martinez-Martinez, M., Popovic, A., Reva, O. N., Lafraya, A., et al. (2015). Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp \u003Ci\u003ERimicaris exoculata\u003C\u002Fi\u003E by using functional metagenomics. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 81, 2125–2136. doi: 10.1128\u002FAEM.03387-14\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F25595762\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.03387-14\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Identification+and+characterization+of+carboxyl+esterases+of+gill+chamber-associated+microbiota+in+the+deep-sea+shrimp+Rimicaris+exoculata+by+using+functional+metagenomics%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Alcaide+M.&author=Tchigvintsev+A.&author=Martinez-Martinez+M.&author=Popovic+A.&author=Reva+O.+N.&author=Lafraya+A.&publication_year=2015&volume=81&pages=2125–2136\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B3\" id=\"B3\"\u003E\u003C\u002Fa\u003EAltschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. \u003Ci\u003ENucleic Acids Res\u003C\u002Fi\u003E. 25, 3389–3402. doi: 10.1093\u002Fnar\u002F25.17.3389\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F9254694\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1093\u002Fnar\u002F25.17.3389\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Gapped+BLAST+and+PSI-BLAST%3A+a+new+generation+of+protein+database+search+programs%2E&journal=Nucleic+Acids+Res%2E&author=Altschul+S.+F.&author=Madden+T.+L.&author=Schäffer+A.+A.&author=Zhang+J.&author=Zhang+Z.&author=Miller+W.&publication_year=1997&volume=25&pages=3389–3402\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B4\" id=\"B4\"\u003E\u003C\u002Fa\u003EArpigny, J. L., and Jaeger, K. E. (1999). Bacterial lipolytic enzymes: classification and properties. \u003Ci\u003EBiochem. J.\u003C\u002Fi\u003E 343, 177–183. doi: 10.1042\u002Fbj3430177\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1042\u002Fbj3430177\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Bacterial+lipolytic+enzymes%3A+classification+and+properties%2E&journal=Biochem%2E+J%2E&author=Arpigny+J.+L.&author=Jaeger+K.+E.&publication_year=1999&volume=343&pages=177–183\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B5\" id=\"B5\"\u003E\u003C\u002Fa\u003EBaker, B. J., Comolli, L. R., Dick, G. J., Hauser, L. J., Hyatt, D., Dill, B. D., et al. (2010). Enigmatic, ultrasmall, uncultivated Archaea. \u003Ci\u003EProc. Natl. Acad. Sci. U.S.A.\u003C\u002Fi\u003E 107, 8806–8811. doi: 10.1073\u002Fpnas.0914470107\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F20421484\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1073\u002Fpnas.0914470107\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Enigmatic%2C+ultrasmall%2C+uncultivated+Archaea%2E&journal=Proc%2E+Natl%2E+Acad%2E+Sci%2E+U%2ES%2EA%2E&author=Baker+B.+J.&author=Comolli+L.+R.&author=Dick+G.+J.&author=Hauser+L.+J.&author=Hyatt+D.&author=Dill+B.+D.&publication_year=2010&volume=107&pages=8806–8811\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B6\" id=\"B6\"\u003E\u003C\u002Fa\u003EBaker, B. J., Tyson, G. W., Webb, R. I., Flanagan, J., Hugenholtz, P., Allen, E. E., et al. (2006). Lineages of acidophilic archaea revealed by community genomic analysis. \u003Ci\u003EScience\u003C\u002Fi\u003E 314, 1933–1935. doi: 10.1126\u002Fscience.1132690\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F17185602\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1126\u002Fscience.1132690\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Lineages+of+acidophilic+archaea+revealed+by+community+genomic+analysis%2E&journal=Science&author=Baker+B.+J.&author=Tyson+G.+W.&author=Webb+R.+I.&author=Flanagan+J.&author=Hugenholtz+P.&author=Allen+E.+E.&publication_year=2006&volume=314&pages=1933–1935\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B7\" id=\"B7\"\u003E\u003C\u002Fa\u003EBarth, S., Fischer, M., Schmid, R. D., and Pleiss, J. (2004). The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions. \u003Ci\u003EBioinformatics\u003C\u002Fi\u003E 20, 2845–2847. doi: 10.1093\u002Fbioinformatics\u002Fbth284\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F15117755\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1093\u002Fbioinformatics\u002Fbth284\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+database+of+epoxide+hydrolases+and+haloalkane+dehalogenases%3A+one+structure%2C+many+functions%2E&journal=Bioinformatics&author=Barth+S.&author=Fischer+M.&author=Schmid+R.+D.&author=Pleiss+J.&publication_year=2004&volume=20&pages=2845–2847\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B8\" id=\"B8\"\u003E\u003C\u002Fa\u003EBauer, T. L., Buchholz, P. C. F., and Pleiss, J. (2020). The modular structure of α\u002Fβ-hydrolases. \u003Ci\u003EFEBS J.\u003C\u002Fi\u003E 287, 1035–1053. doi: 10.1111\u002Ffebs.15071\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F31545554\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1111\u002Ffebs.15071\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+modular+structure+of+α%2Fβ-hydrolases%2E&journal=FEBS+J%2E&author=Bauer+T.+L.&author=Buchholz+P.+C.+F.&author=Pleiss+J.&publication_year=2020&volume=287&pages=1035–1053\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B9\" id=\"B9\"\u003E\u003C\u002Fa\u003EBaweja, M., Nain, L., Kawarabayasi, Y., and Shukla, P. (2016). Current technological improvements in enzymes toward their biotechnological applications. \u003Ci\u003EFront. Microbiol.\u003C\u002Fi\u003E 7:965. doi: 10.3389\u002Ffmicb.2016.00965\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F27379087\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffmicb.2016.00965\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Current+technological+improvements+in+enzymes+toward+their+biotechnological+applications%2E&journal=Front%2E+Microbiol%2E&author=Baweja+M.&author=Nain+L.&author=Kawarabayasi+Y.&author=Shukla+P.&publication_year=2016&volume=7\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B10\" id=\"B10\"\u003E\u003C\u002Fa\u003EBertin, P. N., Heinrich-Salmeron, A., Pelletier, E., Goulhen-Chollet, F., Arsène-Ploetze, F., Gallien, S., et al. (2011). Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics. \u003Ci\u003EISME J\u003C\u002Fi\u003E. 5, 1735–1747. doi: 10.1038\u002Fismej.2011.51\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F21562598\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002Fismej.2011.51\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Metabolic+diversity+among+main+microorganisms+inside+an+arsenic-rich+ecosystem+revealed+by+meta-+and+proteo-genomics%2E&journal=ISME+J%2E&author=Bertin+P.+N.&author=Heinrich-Salmeron+A.&author=Pelletier+E.&author=Goulhen-Chollet+F.&author=Arsène-Ploetze+F.&author=Gallien+S.&publication_year=2011&volume=5&pages=1735–1747\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B11\" id=\"B11\"\u003E\u003C\u002Fa\u003EBollinger, A., Thies, S., Knieps-Grünhagen, E., Gertzen, C., Kobus, S., Höppner, A., et al. (2020). A Novel polyester hydrolase from the marine bacterium \u003Ci\u003EPseudomonas aestusnigri\u003C\u002Fi\u003E Structural and functional insights. \u003Ci\u003EFront. Microbiol.\u003C\u002Fi\u003E 11:114. doi: 10.3389\u002Ffmicb.2020.00114\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F32117139\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffmicb.2020.00114\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+Novel+polyester+hydrolase+from+the+marine+bacterium+Pseudomonas+aestusnigri+Structural+and+functional+insights%2E&journal=Front%2E+Microbiol%2E&author=Bollinger+A.&author=Thies+S.&author=Knieps-Grünhagen+E.&author=Gertzen+C.&author=Kobus+S.&author=Höppner+A.&publication_year=2020&volume=11\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B12\" id=\"B12\"\u003E\u003C\u002Fa\u003EBradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. \u003Ci\u003EAnal. Biochem\u003C\u002Fi\u003E. 72, 248–254. doi: 10.1006\u002Fabio.1976.9999\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F942051\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1006\u002Fabio.1976.9999\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+rapid+and+sensitive+method+for+the+quantification+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%2E&journal=Anal%2E+Biochem%2E&author=Bradford+M.+M.&publication_year=1976&volume=72&pages=248–254\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B13\" id=\"B13\"\u003E\u003C\u002Fa\u003ECai, K., Delaviz, Y., Banh, M., Guo, Y., and Santerre, J. P. (2014). Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process. \u003Ci\u003EDent Mater\u003C\u002Fi\u003E. 30, 848–860. doi: 10.1016\u002Fj.dental.2014.05.031\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F24950808\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.dental.2014.05.031\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Biodegradation+of+composite+resin+with+ester+linkages%3A+identifying+human+salivary+enzyme+activity+with+a+potential+role+in+the+esterolytic+process%2E&journal=Dent+Mater%2E&author=Cai+K.&author=Delaviz+Y.&author=Banh+M.&author=Guo+Y.&author=Santerre+J.+P.&publication_year=2014&volume=30&pages=848–860\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B14\" id=\"B14\"\u003E\u003C\u002Fa\u003EChen, L. X., Huang, L. N., Méndez-García, C., Kuang, J. L., Hua, Z. S., Liu, J., et al. (2016). Microbial communities, processes and functions in acid mine drainage ecosystems. \u003Ci\u003ECurr. Opin. Biotechnol\u003C\u002Fi\u003E. 38, 150–158. doi: 10.1016\u002Fj.copbio.2016.01.013\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26921733\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.copbio.2016.01.013\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+communities%2C+processes+and+functions+in+acid+mine+drainage+ecosystems%2E&journal=Curr%2E+Opin%2E+Biotechnol%2E&author=Chen+L.+X.&author=Huang+L.+N.&author=Méndez-García+C.&author=Kuang+J.+L.&author=Hua+Z.+S.&author=Liu+J.&publication_year=2016&volume=38&pages=150–158\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B15\" id=\"B15\"\u003E\u003C\u002Fa\u003EChen, L. X., Méndez-García, C., Dombrowski, N., Servín-Garcidueñas, L. E., Eloe-Fadrosh, E. A., Fang, B. Z., et al. (2018). Metabolic versatility of small archaea Micrarchaeota and Parvarchaeota. \u003Ci\u003EISME J.\u003C\u002Fi\u003E 12, 756–775. doi: 10.1038\u002Fs41396-017-0002-z\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F29222443\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002Fs41396-017-0002-z\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Metabolic+versatility+of+small+archaea+Micrarchaeota+and+Parvarchaeota%2E&journal=ISME+J%2E&author=Chen+L.+X.&author=Méndez-García+C.&author=Dombrowski+N.&author=Servín-Garcidueñas+L.+E.&author=Eloe-Fadrosh+E.+A.&author=Fang+B.+Z.&publication_year=2018&volume=12&pages=756–775\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B16\" id=\"B16\"\u003E\u003C\u002Fa\u003EDaiha Kde, G., Angeli, R., de Oliveira, S. D., and Almeida, R. V. (2015). Are lipases still important biocatalysts? a study of scientific publications and patents for technological forecasting. \u003Ci\u003EPLoS One\u003C\u002Fi\u003E 10:e0131624. doi: 10.1371\u002Fjournal.pone.0131624\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26111144\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1371\u002Fjournal.pone.0131624\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Are+lipases+still+important+biocatalysts%B4+a+study+of+scientific+publications+and+patents+for+technological+forecasting%2E&journal=PLoS+One&author=Daiha&author=Kde+G.&author=Angeli+R.&author=de+Oliveira+S.+D.&author=Almeida+R.+V.&publication_year=2015&volume=10\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B17\" id=\"B17\"\u003E\u003C\u002Fa\u003EDanso, D., Schmeisser, C., Chow, J., Zimmermann, W., Wei, R., Leggewie, C., et al. (2018). New insights into the function and global distribution of polyethylene terephthalate (PET)-degrading bacteria and enzymes in marine and terrestrial metagenomes. \u003Ci\u003EAppl. Environ. Microbiol.\u003C\u002Fi\u003E 84, e2773–e2717. doi: 10.1128\u002FAEM.02773-17\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F29427431\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.02773-17\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=New+insights+into+the+function+and+global+distribution+of+polyethylene+terephthalate+%28PET%29-degrading+bacteria+and+enzymes+in+marine+and+terrestrial+metagenomes%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Danso+D.&author=Schmeisser+C.&author=Chow+J.&author=Zimmermann+W.&author=Wei+R.&author=Leggewie+C.&publication_year=2018&volume=84&pages=e2773–e2717\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B18\" id=\"B18\"\u003E\u003C\u002Fa\u003EDelavat, F., Phalip, V., Forster, A., Plewniak, F., Lett, M. C., and Lièvremont, D. (2012). Amylases without known homologues discovered in an acid mine drainage: significance and impact. \u003Ci\u003ESci. Rep\u003C\u002Fi\u003E. 2:354. doi: 10.1038\u002Fsrep00354\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F22482035\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002Fsrep00354\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Amylases+without+known+homologues+discovered+in+an+acid+mine+drainage%3A+significance+and+impact%2E&journal=Sci%2E+Rep%2E&author=Delavat+F.&author=Phalip+V.&author=Forster+A.&author=Plewniak+F.&author=Lett+M.+C.&author=Lièvremont+D.&publication_year=2012&volume=2\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B19\" id=\"B19\"\u003E\u003C\u002Fa\u003EDonahue, Craig, J., Exline, Jennifer, A., and Warner Cynthia. (2003). Chemical recycling of pop bottles: the synthesis of dibenzyl terephthalate from the plastic polyethylene terephthalate. \u003Ci\u003EJ. Chem. Educ.\u003C\u002Fi\u003E 80:79. doi: 10.1021\u002Fed080p79\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1021\u002Fed080p79\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Chemical+recycling+of+pop+bottles%3A+the+synthesis+of+dibenzyl+terephthalate+from+the+plastic+polyethylene+terephthalate%2E&journal=J%2E+Chem%2E+Educ%2E&author=Donahue&author=Craig+J.&author=Exline&author=Jennifer+A.&publication_year=2003&volume=80\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B20\" id=\"B20\"\u003E\u003C\u002Fa\u003EDopson, M., Baker-Austin, C., Hind, A., Bowman, J. P., and Bond, P. L. (2004). Characterization of \u003Ci\u003EFerroplasma\u003C\u002Fi\u003E isolates and \u003Ci\u003EFerroplasma acidarmanus\u003C\u002Fi\u003E sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 70, 2079–2088. doi: 10.1128\u002FAEM.70.4.2079-2088.2004\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F15066799\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.70.4.2079-2088.2004\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Characterization+of+Ferroplasma+isolates+and+Ferroplasma+acidarmanus+sp%2E+nov%2E%2C+extreme+acidophiles+from+acid+mine+drainage+and+industrial+bioleaching+environments%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Dopson+M.&author=Baker-Austin+C.&author=Hind+A.&author=Bowman+J.+P.&author=Bond+P.+L.&publication_year=2004&volume=70&pages=2079–2088\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B21\" id=\"B21\"\u003E\u003C\u002Fa\u003EFerrer, M., Bargiela, R., Martínez-Martínez, M., Mir, J., Koch, R., Golyshina, O. V., et al. (2015). Biodiversity for biocatalysis: a review of the α\u002Fβ-hydrolase fold superfamily of esterases-lipases discovered in metagenomes. \u003Ci\u003EBiocatal. Biotransform\u003C\u002Fi\u003E. 33, 235–249. doi: 10.3109\u002F10242422.2016.1151416\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3109\u002F10242422.2016.1151416\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Biodiversity+for+biocatalysis%3A+a+review+of+the+α%2Fβ-hydrolase+fold+superfamily+of+esterases-lipases+discovered+in+metagenomes%2E&journal=Biocatal%2E+Biotransform%2E&author=Ferrer+M.&author=Bargiela+R.&author=Martínez-Martínez+M.&author=Mir+J.&author=Koch+R.&author=Golyshina+O.+V.&publication_year=2015&volume=33&pages=235–249\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B22\" id=\"B22\"\u003E\u003C\u002Fa\u003EFerrer, M., Martínez-Martínez, M., Bargiela, R., Streit, W. R., Golyshina, O. V., and Golyshin, P. N. (2016). Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. \u003Ci\u003EMicrob Biotechnol\u003C\u002Fi\u003E. 9, 22–34. doi: 10.1111\u002F1751-7915.12309\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26275154\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1111\u002F1751-7915.12309\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Estimating+the+success+of+enzyme+bioprospecting+through+metagenomics%3A+current+status+and+future+trends%2E&journal=Microb+Biotechnol%2E&author=Ferrer+M.&author=Martínez-Martínez+M.&author=Bargiela+R.&author=Streit+W.+R.&author=Golyshina+O.+V.&author=Golyshin+P.+N.&publication_year=2016&volume=9&pages=22–34\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B23\" id=\"B23\"\u003E\u003C\u002Fa\u003EFerrer, M., Méndez-García, C., Bargiela, R., Chow, J., Alonso, S., García-Moyano, A., et al. (2019). Decoding the ocean’s microbiological secrets for marine enzyme biodiscovery. \u003Ci\u003EFEMS Microbiol. Lett\u003C\u002Fi\u003E. 366, 0378–1097. doi: 10.1093\u002Ffemsle\u002Ffny285\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F30534987\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1093\u002Ffemsle\u002Ffny285\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Decoding+the+ocean’s+microbiological+secrets+for+marine+enzyme+biodiscovery%2E&journal=FEMS+Microbiol%2E+Lett%2E&author=Ferrer+M.&author=Méndez-García+C.&author=Bargiela+R.&author=Chow+J.&author=Alonso+S.&author=García-Moyano+A.&publication_year=2019&volume=366&pages=0378–1097\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B24\" id=\"B24\"\u003E\u003C\u002Fa\u003EFiner, Y., and Santerre, J. P. (2004). Salivary esterase activity and its association with the biodegradation of dental composites. \u003Ci\u003EJ. Dent Res\u003C\u002Fi\u003E. 83, 22–26. doi: 10.1177\u002F154405910408300105\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F14691108\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1177\u002F154405910408300105\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Salivary+esterase+activity+and+its+association+with+the+biodegradation+of+dental+composites%2E&journal=J%2E+Dent+Res%2E&author=Finer+Y.&author=Santerre+J.+P.&publication_year=2004&volume=83&pages=22–26\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B25\" id=\"B25\"\u003E\u003C\u002Fa\u003EFischer, M., and Pleiss, J. (2003). The Lipase Engineering Database: a navigation and analysis tool for protein families. \u003Ci\u003ENucleic Acids Res\u003C\u002Fi\u003E. 31, 319–321. doi: 10.1093\u002Fnar\u002Fgkg015\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F12520012\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1093\u002Fnar\u002Fgkg015\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+Lipase+Engineering+Database%3A+a+navigation+and+analysis+tool+for+protein+families%2E&journal=Nucleic+Acids+Res%2E&author=Fischer+M.&author=Pleiss+J.&publication_year=2003&volume=31&pages=319–321\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B26\" id=\"B26\"\u003E\u003C\u002Fa\u003EFlemming, H. C., and Wingender, J. (2010). The biofilm matrix. \u003Ci\u003ENat. Rev. Microbiol.\u003C\u002Fi\u003E 8, 623–633. doi: 10.3390\u002Fnano10081527\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F32759748\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3390\u002Fnano10081527\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+biofilm+matrix%2E&journal=Nat%2E+Rev%2E+Microbiol%2E&author=Flemming+H.+C.&author=Wingender+J.&publication_year=2010&volume=8&pages=623–633\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B27\" id=\"B27\"\u003E\u003C\u002Fa\u003EFütterer, O., Angelov, A., Liesegang, H., Gottschalk, G., Schleper, C., Schepers, B., et al. (2004). Genome sequence of \u003Ci\u003EPicrophilus torridus\u003C\u002Fi\u003E and its implications for life around pH 0. \u003Ci\u003EProc. Natl. Acad. Sci. U.S.A\u003C\u002Fi\u003E. 101, 9091–9096. doi: 10.1073\u002Fpnas.0401356101\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F15184674\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1073\u002Fpnas.0401356101\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Genome+sequence+of+Picrophilus+torridus+and+its+implications+for+life+around+pH+0%2E&journal=Proc%2E+Natl%2E+Acad%2E+Sci%2E+U%2ES%2EA%2E&author=Fütterer+O.&author=Angelov+A.&author=Liesegang+H.&author=Gottschalk+G.&author=Schleper+C.&author=Schepers+B.&publication_year=2004&volume=101&pages=9091–9096\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B28\" id=\"B28\"\u003E\u003C\u002Fa\u003EGavrilov, S. N., Korzhenkov, A. A., Kublanov, I. V., Bargiela, R., Zamana, L. V., Popova, A. A., et al. (2019). Microbial communities of polymetallic deposits’ acidic ecosystems of continental climatic zone with high temperature contrasts. \u003Ci\u003EFront. Microbiol\u003C\u002Fi\u003E. 10:1573. doi: 10.3389\u002Ffmicb.2019.01573\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F31379766\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffmicb.2019.01573\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+communities+of+polymetallic+deposits’+acidic+ecosystems+of+continental+climatic+zone+with+high+temperature+contrasts%2E&journal=Front%2E+Microbiol%2E&author=Gavrilov+S.+N.&author=Korzhenkov+A.+A.&author=Kublanov+I.+V.&author=Bargiela+R.&author=Zamana+L.+V.&author=Popova+A.+A.&publication_year=2019&volume=10\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B29\" id=\"B29\"\u003E\u003C\u002Fa\u003EGiunta, C. I., Cea-Rama, I., Alonso, S., Briand, M. L., Bargiela, R., Coscolín, C., et al. (2020). Tuning the properties of natural promiscuous enzymes by engineering their nano-environment. \u003Ci\u003EACS Nano\u003C\u002Fi\u003E 14, 17652–17664. doi: 10.1021\u002Facsnano.0c08716\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F33306346\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1021\u002Facsnano.0c08716\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Tuning+the+properties+of+natural+promiscuous+enzymes+by+engineering+their+nano-environment%2E&journal=ACS+Nano&author=Giunta+C.+I.&author=Cea-Rama+I.&author=Alonso+S.&author=Briand+M.+L.&author=Bargiela+R.&author=Coscolín+C.&publication_year=2020&volume=14&pages=17652–17664\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B30\" id=\"B30\"\u003E\u003C\u002Fa\u003EGolyshina, O. V. (2011). Environmental, biogeographic, and biochemical patterns of archaea of the family \u003Ci\u003EFerroplasmaceae\u003C\u002Fi\u003E. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 77, 5071–5078. doi: 10.1128\u002FAEM.00726-11\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F21685165\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.00726-11\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Environmental%2C+biogeographic%2C+and+biochemical+patterns+of+archaea+of+the+family+Ferroplasmaceae%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Golyshina+O.+V.&publication_year=2011&volume=77&pages=5071–5078\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B31\" id=\"B31\"\u003E\u003C\u002Fa\u003EGolyshina, O. V., Golyshin, P. N., Timmis, K. N., and Ferrer, M. (2006). The ‘pH optimum anomaly’ of intracellular enzymes of \u003Ci\u003EFerroplasma acidiphilum\u003C\u002Fi\u003E. \u003Ci\u003EEnviron. Microbiol\u003C\u002Fi\u003E. 8, 416–425. doi: 10.1111\u002Fj.1462-2920.2005.00907.x\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F16478448\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1111\u002Fj.1462-2920.2005.00907.x\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+‘pH+optimum+anomaly’+of+intracellular+enzymes+of+Ferroplasma+acidiphilum%2E&journal=Environ%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Golyshin+P.+N.&author=Timmis+K.+N.&author=Ferrer+M.&publication_year=2006&volume=8&pages=416–425\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B32\" id=\"B32\"\u003E\u003C\u002Fa\u003EGolyshina, O. V., Lünsdorf, H., Kublanov, I. V., Goldenstein, N. I., Hinrichs, K. U., and Golyshin, P. N. (2016). The novel extremely acidophilic, cell-wall-deficient archaeon \u003Ci\u003ECuniculiplasma divulgatum\u003C\u002Fi\u003E gen.Nov.sp nov.represents a new family, \u003Ci\u003ECuniculiplasmataceae\u003C\u002Fi\u003E fam. nov.of the order Thermoplasmatales. \u003Ci\u003EInt. J. Syst. Evol. Microbiol\u003C\u002Fi\u003E. 66, 332–340. doi: 10.1099\u002Fijsem.0.000725\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26518885\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1099\u002Fijsem.0.000725\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+novel+extremely+acidophilic%2C+cell-wall-deficient+archaeon+Cuniculiplasma+divulgatum+gen%2ENov%2Esp+nov%2Erepresents+a+new+family%2C+Cuniculiplasmataceae+fam%2E+nov%2Eof+the+order+Thermoplasmatales%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Lünsdorf+H.&author=Kublanov+I.+V.&author=Goldenstein+N.+I.&author=Hinrichs+K.+U.&author=Golyshin+P.+N.&publication_year=2016&volume=66&pages=332–340\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B33\" id=\"B33\"\u003E\u003C\u002Fa\u003EGolyshina, O. V., Pivovarova, T. A., Karavaiko, G. I., Kondratéva, T. F., Moore, E. R., Abraham, W. R., et al. (2000). \u003Ci\u003EFerroplasma acidiphilum\u003C\u002Fi\u003E gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the \u003Ci\u003EFerroplasmaceae\u003C\u002Fi\u003E fam. nov., comprising a distinct lineage of the Archaea. \u003Ci\u003EInt. J. Syst. Evol. Microbiol\u003C\u002Fi\u003E. 3, 997–1006. doi: 10.1099\u002F00207713-50-3-997\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F10843038\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1099\u002F00207713-50-3-997\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Ferroplasma+acidiphilum+gen%2E+nov%2E%2C+sp%2E+nov%2E%2C+an+acidophilic%2C+autotrophic%2C+ferrous-iron-oxidizing%2C+cell-wall-lacking%2C+mesophilic+member+of+the+Ferroplasmaceae+fam%2E+nov%2E%2C+comprising+a+distinct+lineage+of+the+Archaea%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Pivovarova+T.+A.&author=Karavaiko+G.+I.&author=Kondratéva+T.+F.&author=Moore+E.+R.&author=Abraham+W.+R.&publication_year=2000&volume=3&pages=997–1006\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B34\" id=\"B34\"\u003E\u003C\u002Fa\u003EGolyshina, O. V., Yakimov, M. M., Lünsdorf, H., Ferrer, M., Nimtz, M., Timmis, K. N., et al. (2009). \u003Ci\u003EAcidiplasma aeolicum\u003C\u002Fi\u003E gen. nov., sp. nov., a euryarchaeon of the family Ferroplasmaceae isolated from a hydrothermal pool, and transfer of \u003Ci\u003EFerroplasma cupricumulans\u003C\u002Fi\u003E to \u003Ci\u003EAcidiplasma cupricumulans\u003C\u002Fi\u003E comb. nov. \u003Ci\u003EInt. J. Syst. Evol. Microbiol\u003C\u002Fi\u003E. 59, 2815–2823. doi: 10.1099\u002Fijs.0.009639-0\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F19628615\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1099\u002Fijs.0.009639-0\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Acidiplasma+aeolicum+gen%2E+nov%2E%2C+sp%2E+nov%2E%2C+a+euryarchaeon+of+the+family+Ferroplasmaceae+isolated+from+a+hydrothermal+pool%2C+and+transfer+of+Ferroplasma+cupricumulans+to+Acidiplasma+cupricumulans+comb%2E+nov%2E&journal=Int%2E+J%2E+Syst%2E+Evol%2E+Microbiol%2E&author=Golyshina+O.+V.&author=Yakimov+M.+M.&author=Lünsdorf+H.&author=Ferrer+M.&author=Nimtz+M.&author=Timmis+K.+N.&publication_year=2009&volume=59&pages=2815–2823\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B35\" id=\"B35\"\u003E\u003C\u002Fa\u003EGomes, I., Gomes, J., and Steiner, W. (2003). Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium \u003Ci\u003ERhodothermus marinus\u003C\u002Fi\u003E: production and partial characterization. \u003Ci\u003EBioresour. Technol\u003C\u002Fi\u003E. 90, 207–214. doi: 10.1016\u002Fs0960-8524(03)00110-x\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fs0960-8524(03)00110-x\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Highly+thermostable+amylase+and+pullulanase+of+the+extreme+thermophilic+eubacterium+Rhodothermus+marinus%3A+production+and+partial+characterization%2E&journal=Bioresour%2E+Technol%2E&author=Gomes+I.&author=Gomes+J.&author=Steiner+W.&publication_year=2003&volume=90&pages=207–214\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B36\" id=\"B36\"\u003E\u003C\u002Fa\u003EGuazzaroni, M. E., Morgante, V., Mirete, S., and González-Pastor, J. E. (2013). Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment. \u003Ci\u003EEnviron. Microbiol\u003C\u002Fi\u003E. 15, 1088–1102. doi: 10.1111\u002F1462-2920.12021\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F23145860\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1111\u002F1462-2920.12021\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Novel+acid+resistance+genes+from+the+metagenome+of+the+Tinto+River%2C+an+extremely+acidic+environment%2E&journal=Environ%2E+Microbiol%2E&author=Guazzaroni+M.+E.&author=Morgante+V.&author=Mirete+S.&author=González-Pastor+J.+E.&publication_year=2013&volume=15&pages=1088–1102\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B37\" id=\"B37\"\u003E\u003C\u002Fa\u003EHajighasemi, M., Tchigvintsev, A., Nocek, B., Flick, R., Popovic, A., Hai, T., et al. (2018). Screening and characterization of novel polyesterases from environmental metagenomes with high hydrolytic activity against synthetic polyesters. \u003Ci\u003EEnviron. Sci. Technol\u003C\u002Fi\u003E. 52, 12388–12401. doi: 10.1021\u002Facs.est.8b04252\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F30284819\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1021\u002Facs.est.8b04252\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Screening+and+characterization+of+novel+polyesterases+from+environmental+metagenomes+with+high+hydrolytic+activity+against+synthetic+polyesters%2E&journal=Environ%2E+Sci%2E+Technol%2E&author=Hajighasemi+M.&author=Tchigvintsev+A.&author=Nocek+B.&author=Flick+R.&author=Popovic+A.&author=Hai+T.&publication_year=2018&volume=52&pages=12388–12401\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B38\" id=\"B38\"\u003E\u003C\u002Fa\u003EHe, Y., Zhang, F., Saleh, E., Vaithilingam, J., Aboulkhair, N., Begines, B., et al. (2017). A tripropylene glycol diacrylate-based polymeric support ink for material jetting. \u003Ci\u003EAddit. Manuf\u003C\u002Fi\u003E. 16, 153–161. doi: 10.1016\u002Fj.addma.2017.06.001\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.addma.2017.06.001\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+tripropylene+glycol+diacrylate-based+polymeric+support+ink+for+material+jetting%2E&journal=Addit%2E+Manuf%2E&author=He+Y.&author=Zhang+F.&author=Saleh+E.&author=Vaithilingam+J.&author=Aboulkhair+N.&author=Begines+B.&publication_year=2017&volume=16&pages=153–161\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B39\" id=\"B39\"\u003E\u003C\u002Fa\u003EHuang, L.-N., Kuang, J.-L., and Shu, W.-S. (2016). Microbial ecology and evolution in the acid mine drainage model system. \u003Ci\u003ETrends Microbiol.\u003C\u002Fi\u003E 24, 581–593. doi: 10.1016\u002Fj.tim.2016.03.004\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F27050827\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.tim.2016.03.004\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+ecology+and+evolution+in+the+acid+mine+drainage+model+system%2E&journal=Trends+Microbiol%2E&author=Huang+L.-N.&author=Kuang+J.-L.&author=Shu+W.-S.&publication_year=2016&volume=24&pages=581–593\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B40\" id=\"B40\"\u003E\u003C\u002Fa\u003EJackson, B. R., Noble, C., Lavesa-Curto, M., Bond, P. L., and Bowater, R. P. (2007). Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “\u003Ci\u003EFerroplasma acidarmanus\u003C\u002Fi\u003E” Fer1. \u003Ci\u003EExtremophiles\u003C\u002Fi\u003E 11, 315–327. doi: 10.1007\u002Fs00792-006-0041-2\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F17136487\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs00792-006-0041-2\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Characterization+of+an+ATP-dependent+DNA+ligase+from+the+acidophilic+archaeon+“Ferroplasma+acidarmanus”+Fer1%2E&journal=Extremophiles&author=Jackson+B.+R.&author=Noble+C.&author=Lavesa-Curto+M.&author=Bond+P.+L.&author=Bowater+R.+P.&publication_year=2007&volume=11&pages=315–327\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B41\" id=\"B41\"\u003E\u003C\u002Fa\u003EJiao, Y., Cody, G. D., Harding, A. K., Wilmes, P., Schrenk, M., Wheeler, K. E., et al. (2010). Characterization of extracellular polymeric substances from acidophilic microbial biofilms. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 76, 2916–2922. doi: 10.1128\u002FAEM.02289-09\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F20228116\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.02289-09\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Characterization+of+extracellular+polymeric+substances+from+acidophilic+microbial+biofilms%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Jiao+Y.&author=Cody+G.+D.&author=Harding+A.+K.&author=Wilmes+P.&author=Schrenk+M.&author=Wheeler+K.+E.&publication_year=2010&volume=76&pages=2916–2922\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B42\" id=\"B42\"\u003E\u003C\u002Fa\u003EJiao, Y., D’haeseleer, P., Dill, B. D., Shah, M., Verberkmoes, N. C., Hettich, R. L., et al. (2011). Identification of biofilm matrix associated proteins from an acid mine drainage microbial community. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 77, 5230–5237. doi: 10.1128\u002FAEM.03005-10\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F21685158\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.03005-10\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Identification+of+biofilm+matrix+associated+proteins+from+an+acid+mine+drainage+microbial+community%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Jiao+Y.&author=D’haeseleer+P.&author=Dill+B.+D.&author=Shah+M.&author=Verberkmoes+N.+C.&author=Hettich+R.+L.&publication_year=2011&volume=77&pages=5230–5237\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B43\" id=\"B43\"\u003E\u003C\u002Fa\u003EJohnson, D. B., and Quatrini, R. (2020). Acidophile microbiology in space and time. \u003Ci\u003ECurr. Issues Mol. Biol.\u003C\u002Fi\u003E 39, 63–76. doi: 10.21775\u002Fcimb.039.063\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F32083998\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.21775\u002Fcimb.039.063\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Acidophile+microbiology+in+space+and+time%2E&journal=Curr%2E+Issues+Mol%2E+Biol%2E&author=Johnson+D.+B.&author=Quatrini+R.&publication_year=2020&volume=39&pages=63–76\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B44\" id=\"B44\"\u003E\u003C\u002Fa\u003EJumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021). Highly accurate protein structure prediction with AlphaFold. \u003Ci\u003ENature\u003C\u002Fi\u003E 596, 583–589. doi: 10.1038\u002Fs41586-021-03819-2\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F34265844\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002Fs41586-021-03819-2\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Highly+accurate+protein+structure+prediction+with+AlphaFold%2E&journal=Nature&author=Jumper+J.&author=Evans+R.&author=Pritzel+A.&author=Green+T.&author=Figurnov+M.&author=Ronneberger+O.&publication_year=2021&volume=596&pages=583–589\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B45\" id=\"B45\"\u003E\u003C\u002Fa\u003EKorzhenkov, A. A., Toshchakov, S. V., Bargiela, R., Gibbard, H., Ferrer, M., Teplyuk, A. V., et al. (2019). Archaea dominate the microbial community in an ecosystem with low-to-moderate temperature and extreme acidity. \u003Ci\u003EMicrobiome\u003C\u002Fi\u003E 7:11. doi: 10.1186\u002Fs40168-019-0623-8\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F30691532\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1186\u002Fs40168-019-0623-8\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Archaea+dominate+the+microbial+community+in+an+ecosystem+with+low-to-moderate+temperature+and+extreme+acidity%2E&journal=Microbiome&author=Korzhenkov+A.+A.&author=Toshchakov+S.+V.&author=Bargiela+R.&author=Gibbard+H.&author=Ferrer+M.&author=Teplyuk+A.+V.&publication_year=2019&volume=7\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B46\" id=\"B46\"\u003E\u003C\u002Fa\u003ELaemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. \u003Ci\u003ENature\u003C\u002Fi\u003E 227, 680–685. doi: 10.1038\u002F227680a0\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F5432063\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002F227680a0\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4%2E&journal=Nature&author=Laemmli+U.+K.&publication_year=1970&volume=227&pages=680–685\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B47\" id=\"B47\"\u003E\u003C\u002Fa\u003EMartínez-Martínez, M., Alcaide, M., Tchigvintsev, A., Reva, O., Polaina, J., and Bargiela, R. (2013). Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach. \u003Ci\u003EAppl. Environ. Microbiol\u003C\u002Fi\u003E. 79, 3553–3562. doi: 10.1128\u002FAEM.00240-13\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F23542620\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1128\u002FAEM.00240-13\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Biochemical+diversity+of+carboxyl+esterases+and+lipases+from+Lake+Arreo+%28Spain%29%3A+a+metagenomic+approach%2E&journal=Appl%2E+Environ%2E+Microbiol%2E&author=Martínez-Martínez+M.&author=Alcaide+M.&author=Tchigvintsev+A.&author=Reva+O.&author=Polaina+J.&author=Bargiela+R.&publication_year=2013&volume=79&pages=3553–3562\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B48\" id=\"B48\"\u003E\u003C\u002Fa\u003EMartínez-Martínez, M., Coscolín, C., Santiago, G., Chow, J., Stogios, P. J., and Bargiela, R. (2018). The Inmare Consortium.Determinants and prediction of esterase substrate promiscuity patterns. \u003Ci\u003EACS Chem. Biol.\u003C\u002Fi\u003E 13, 225–234. doi: 10.1021\u002Facschembio.7b00996\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F29182315\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1021\u002Facschembio.7b00996\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=The+Inmare+Consortium%2EDeterminants+and+prediction+of+esterase+substrate+promiscuity+patterns%2E&journal=ACS+Chem%2E+Biol%2E&author=Martínez-Martínez+M.&author=Coscolín+C.&author=Santiago+G.&author=Chow+J.&author=Stogios+P.+J.&author=Bargiela+R.&publication_year=2018&volume=13&pages=225–234\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B49\" id=\"B49\"\u003E\u003C\u002Fa\u003EMaurya, A., Bhattacharya, A., and Khare, S. K. (2020). Enzymatic remediation of polyethylene terephthalate (PET)-based polymers for effective management of plastic wastes: an Overview. \u003Ci\u003EFront. Bioeng. Biotechnol\u003C\u002Fi\u003E. 8:602325. doi: 10.3389\u002Ffbioe.2020.602325\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F33330434\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffbioe.2020.602325\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Enzymatic+remediation+of+polyethylene+terephthalate+%28PET%29-based+polymers+for+effective+management+of+plastic+wastes%3A+an+Overview%2E&journal=Front%2E+Bioeng%2E+Biotechnol%2E&author=Maurya+A.&author=Bhattacharya+A.&author=Khare+S.+K.&publication_year=2020&volume=8\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B50\" id=\"B50\"\u003E\u003C\u002Fa\u003EMéndez-García, C., Mesa, V., Sprenger, R. R., Richter, M., Diez, M. S., and Solano, J. (2014). Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage. \u003Ci\u003EISME J\u003C\u002Fi\u003E. 8, 1259–1274. doi: 10.1038\u002Fismej.2013.242\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F24430486\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1038\u002Fismej.2013.242\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+stratification+in+low+pH+oxic+and+suboxic+macroscopic+growths+along+an+acid+mine+drainage%2E&journal=ISME+J%2E&author=Méndez-García+C.&author=Mesa+V.&author=Sprenger+R.+R.&author=Richter+M.&author=Diez+M.+S.&author=Solano+J.&publication_year=2014&volume=8&pages=1259–1274\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B51\" id=\"B51\"\u003E\u003C\u002Fa\u003EMéndez-García, C., Peláez, A. I., Mesa, V., Sánchez, J., Golyshina, O. V., and Ferrer, M. (2015). Microbial diversity and metabolic networks in acid mine drainage habitats. \u003Ci\u003EFront. Microbiol.\u003C\u002Fi\u003E 6:475. doi: 10.3389\u002Ffmicb.2015.00475\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26074887\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffmicb.2015.00475\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+diversity+and+metabolic+networks+in+acid+mine+drainage+habitats%2E&journal=Front%2E+Microbiol%2E&author=Méndez-García+C.&author=Peláez+A.+I.&author=Mesa+V.&author=Sánchez+J.&author=Golyshina+O.+V.&author=Ferrer+M.&publication_year=2015&volume=6\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B52\" id=\"B52\"\u003E\u003C\u002Fa\u003EMénigaud, S., Mallet, L., Picord, G., Churlaud, C., Borrel, A., and Deschavanne, P. (2012). GOHTAM: a website for ’Genomic Origin of Horizontal Transfers. \u003Ci\u003EAlign. Metagenom. Bioinform\u003C\u002Fi\u003E. 28, 1270–1271. doi: 10.1093\u002Fbioinformatics\u002Fbts118\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F22426345\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1093\u002Fbioinformatics\u002Fbts118\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=GOHTAM%3A+a+website+for+’Genomic+Origin+of+Horizontal+Transfers%2E&journal=Align%2E+Metagenom%2E+Bioinform%2E&author=Ménigaud+S.&author=Mallet+L.&author=Picord+G.&author=Churlaud+C.&author=Borrel+A.&author=Deschavanne+P.&publication_year=2012&volume=28&pages=1270–1271\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B53\" id=\"B53\"\u003E\u003C\u002Fa\u003ENchedo Ariole, C., and George-West, O. (2020). Bioplastic degradation potential of microorganisms isolated from the soil. \u003Ci\u003EAm. J. Chem. Eng\u003C\u002Fi\u003E. 4:1. doi: 10.11648\u002Fj.ajcbe.20200401.11\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.11648\u002Fj.ajcbe.20200401.11\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Bioplastic+degradation+potential+of+microorganisms+isolated+from+the+soil%2E&journal=Am%2E+J%2E+Chem%2E+Eng%2E&author=Nchedo+Ariole+C.&author=George-West+O.&publication_year=2020&volume=4\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B54\" id=\"B54\"\u003E\u003C\u002Fa\u003EOhara, K., Unno, H., Oshima, Y., Hosoya, M., Fujino, N., Hirooka, K., et al. (2014). Structural insights into the low pH adaptation of a unique carboxylesterase from \u003Ci\u003EFerroplasma\u003C\u002Fi\u003E: altering the pH optima of two carboxylesterases. \u003Ci\u003EJ. Biol. Chem\u003C\u002Fi\u003E. 289, 24499–24510. doi: 10.1074\u002Fjbc.M113.521856\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F25043762\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1074\u002Fjbc.M113.521856\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Structural+insights+into+the+low+pH+adaptation+of+a+unique+carboxylesterase+from+Ferroplasma%3A+altering+the+pH+optima+of+two+carboxylesterases%2E&journal=J%2E+Biol%2E+Chem%2E&author=Ohara+K.&author=Unno+H.&author=Oshima+Y.&author=Hosoya+M.&author=Fujino+N.&author=Hirooka+K.&publication_year=2014&volume=289&pages=24499–24510\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B55\" id=\"B55\"\u003E\u003C\u002Fa\u003EPellis, A., Cantone, S., Ebert, C., and Gardossi, L. (2017). Evolving biocatalysis to meet bioeconomy challenges and opportunities. \u003Ci\u003ENew Biotechnol\u003C\u002Fi\u003E. 40, 154–169. doi: 10.1016\u002Fj.nbt.2017.07.005\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F28743564\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.nbt.2017.07.005\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Evolving+biocatalysis+to+meet+bioeconomy+challenges+and+opportunities%2E&journal=New+Biotechnol%2E&author=Pellis+A.&author=Cantone+S.&author=Ebert+C.&author=Gardossi+L.&publication_year=2017&volume=40&pages=154–169\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B56\" id=\"B56\"\u003E\u003C\u002Fa\u003EPeña-García, C., Martínez-Martínez, M., Reyes-Duarte, D., and Ferrer, M. (2016). High throughput screening of esterases, lipases and phospholipases in mutant and metagenomic libraries: a review. \u003Ci\u003EComb. Chem. High Throughput Screen\u003C\u002Fi\u003E 19, 605–615. doi: 10.2174\u002F1386207319666151110123927\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26552433\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.2174\u002F1386207319666151110123927\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=High+throughput+screening+of+esterases%2C+lipases+and+phospholipases+in+mutant+and+metagenomic+libraries%3A+a+review%2E&journal=Comb%2E+Chem%2E+High+Throughput+Screen&author=Peña-García+C.&author=Martínez-Martínez+M.&author=Reyes-Duarte+D.&author=Ferrer+M.&publication_year=2016&volume=19&pages=605–615\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B57\" id=\"B57\"\u003E\u003C\u002Fa\u003EPlacido, A., Hai, T., Ferrer, M., Chernikova, T. N., Distaso, M., and Armstrong, D. (2015). Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase. \u003Ci\u003EAppl. Microbiol. Biotechnol.\u003C\u002Fi\u003E 99, 10031–10046. doi: 10.1007\u002Fs00253-015-6873-x\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26266751\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs00253-015-6873-x\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Diversity+of+hydrolases+from+hydrothermal+vent+sediments+of+the+Levante+Bay%2C+Vulcano+Island+%28Aeolian+archipelago%29+identified+by+activity-based+metagenomics+and+biochemical+characterization+of+new+esterases+and+an+arabinopyranosidase%2E&journal=Appl%2E+Microbiol%2E+Biotechnol%2E&author=Placido+A.&author=Hai+T.&author=Ferrer+M.&author=Chernikova+T.+N.&author=Distaso+M.&author=Armstrong+D.&publication_year=2015&volume=99&pages=10031–10046\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B58\" id=\"B58\"\u003E\u003C\u002Fa\u003EPütz, A. (2006). \u003Ci\u003EIsolierung, Identifizierung und Biochemische Charakterisierung Dialkylphthalat Spaltender Esterasen.\u003C\u002Fi\u003E [Ph D thesis]. Düsseldorf: HeinrichHeine-Universität, 39–40.\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&journal=Isolierung%2C+Identifizierung+und+Biochemische+Charakterisierung+Dialkylphthalat+Spaltender+Esterasen%2E&author=Pütz+A.&publication_year=2006&pages=39–40\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B59\" id=\"B59\"\u003E\u003C\u002Fa\u003ERao, L., Xue, Y., Zheng, Y., Lu, J. R., and Ma, Y. A. (2013). A novel alkaliphilic bacillus esterase belongs to the 13(th) bacterial lipolytic enzyme family. \u003Ci\u003EPLoS One\u003C\u002Fi\u003E 8:e60645. doi: 10.1371\u002Fjournal.pone.0060645\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F23577139\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1371\u002Fjournal.pone.0060645\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+novel+alkaliphilic+bacillus+esterase+belongs+to+the+13%28th%29+bacterial+lipolytic+enzyme+family%2E&journal=PLoS+One&author=Rao+L.&author=Xue+Y.&author=Zheng+Y.&author=Lu+J.+R.&author=Ma+Y.+A.&publication_year=2013&volume=8\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B60\" id=\"B60\"\u003E\u003C\u002Fa\u003EReyes-Duarte, D., Ferrer, M., and García-Arellano, H. (2012). Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries. \u003Ci\u003EMethods Mol. Biol\u003C\u002Fi\u003E. 861, 101–113. doi: 10.1007\u002F978-1-61779-600-5_6\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002F978-1-61779-600-5_6\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Functional-based+screening+methods+for+lipases%2C+esterases%2C+and+phospholipases+in+metagenomic+libraries%2E&journal=Methods+Mol%2E+Biol%2E&author=Reyes-Duarte+D.&author=Ferrer+M.&author=García-Arellano+H.&publication_year=2012&volume=861&pages=101–113\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B61\" id=\"B61\"\u003E\u003C\u002Fa\u003ESantiago, G., Martínez-Martínez, M., Alonso, S., Bargiela, R., Coscolín, C., Golyshin, P. N., et al. (2018). Rational engineering of multiple active sites in an ester hydrolase. \u003Ci\u003EBiochemistry\u003C\u002Fi\u003E 57, 2245–2255. doi: 10.1021\u002Facs.biochem.8b00274\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F29600855\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1021\u002Facs.biochem.8b00274\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Rational+engineering+of+multiple+active+sites+in+an+ester+hydrolase%2E&journal=Biochemistry&author=Santiago+G.&author=Martínez-Martínez+M.&author=Alonso+S.&author=Bargiela+R.&author=Coscolín+C.&author=Golyshin+P.+N.&publication_year=2018&volume=57&pages=2245–2255\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B62\" id=\"B62\"\u003E\u003C\u002Fa\u003EScholtissek, A., Ullrich, S. R., Mühling, M., Schlömann, M., Paul, C. E., and Tischler, D. (2016). A thermophilic-like ene-reductase originating from an acidophilic iron oxidizer. \u003Ci\u003EAppl. Microbiol. Biotechnol\u003C\u002Fi\u003E. 101:2. doi: 10.1007\u002Fs00253-016-7782-3\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F27542380\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs00253-016-7782-3\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+thermophilic-like+ene-reductase+originating+from+an+acidophilic+iron+oxidizer%2E&journal=Appl%2E+Microbiol%2E+Biotechnol%2E&author=Scholtissek+A.&author=Ullrich+S.+R.&author=Mühling+M.&author=Schlömann+M.&author=Paul+C.+E.&author=Tischler+D.&publication_year=2016&volume=101\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B63\" id=\"B63\"\u003E\u003C\u002Fa\u003ESharma, A., Kawarabayasi, Y., and Satyanarayana, T. (2012). Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications. \u003Ci\u003EExtremophiles\u003C\u002Fi\u003E 16, 1–19. doi: 10.1007\u002Fs00792-011-0402-3\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F22080280\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs00792-011-0402-3\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Acidophilic+bacteria+and+archaea%3A+acid+stable+biocatalysts+and+their+potential+applications%2E&journal=Extremophiles&author=Sharma+A.&author=Kawarabayasi+Y.&author=Satyanarayana+T.&publication_year=2012&volume=16&pages=1–19\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B64\" id=\"B64\"\u003E\u003C\u002Fa\u003EThapa, S., Mishra, J., Arora, N., Mishra, P., Li, H., O’Hair, J., et al. (2020). Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation. \u003Ci\u003ERev. Environ. Sci. Biotechnol.\u003C\u002Fi\u003E 19, 621–648. doi: 10.1007\u002Fs11157-020-09536-y\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs11157-020-09536-y\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Microbial+cellulolytic+enzymes%3A+diversity+and+biotechnology+with+reference+to+lignocellulosic+biomass+degradation%2E&journal=Rev%2E+Environ%2E+Sci%2E+Biotechnol%2E&author=Thapa+S.&author=Mishra+J.&author=Arora+N.&author=Mishra+P.&author=Li+H.&author=O’Hair+J.&publication_year=2020&volume=19&pages=621–648\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B65\" id=\"B65\"\u003E\u003C\u002Fa\u003EVan den Berg, B. (2010). Crystal structure of a full-length autotransporter. \u003Ci\u003EJ. Mol. Biol\u003C\u002Fi\u003E. 396, 627–633. doi: 10.1016\u002Fj.jmb.2009.12.061\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F20060837\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.jmb.2009.12.061\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Crystal+structure+of+a+full-length+autotransporter%2E&journal=J%2E+Mol%2E+Biol%2E&author=Van+den+Berg+B.&publication_year=2010&volume=396&pages=627–633\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B66\" id=\"B66\"\u003E\u003C\u002Fa\u003EWu, X., Zhang, Q., Zhang, L., Liu, S., Chen, G., Zhang, H., et al. (2020). Insights into the role of exposed surface charged residues in the alkali-tolerance of GH11 xylanase. \u003Ci\u003EFront. Microbiol.\u003C\u002Fi\u003E 8:872. doi: 10.3389\u002Ffmicb.2020.00872\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F32457729\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.3389\u002Ffmicb.2020.00872\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Insights+into+the+role+of+exposed+surface+charged+residues+in+the+alkali-tolerance+of+GH11+xylanase%2E&journal=Front%2E+Microbiol%2E&author=Wu+X.&author=Zhang+Q.&author=Zhang+L.&author=Liu+S.&author=Chen+G.&author=Zhang+H.&publication_year=2020&volume=8\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B67\" id=\"B67\"\u003E\u003C\u002Fa\u003EYan, F., Wei, R., Cui, Q., Bornscheuer, U. T., and Liu, Y. J. (2021). Thermophilic whole-cell degradation of polyethylene terephthalate using engineered \u003Ci\u003EClostridium thermocellum\u003C\u002Fi\u003E. \u003Ci\u003EMicrob Biotechnol.\u003C\u002Fi\u003E 14, 374–385. doi: 10.1111\u002F1751-7915.13580\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F32343496\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1111\u002F1751-7915.13580\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=Thermophilic+whole-cell+degradation+of+polyethylene+terephthalate+using+engineered+Clostridium+thermocellum%2E&journal=Microb+Biotechnol%2E&author=Yan+F.&author=Wei+R.&author=Cui+Q.&author=Bornscheuer+U.+T.&author=Liu+Y.+J.&publication_year=2021&volume=14&pages=374–385\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"References\" style=\"margin-bottom:0.5em;\"\u003E\r\n\u003Cp class=\"ReferencesCopy1\"\u003E\u003Ca name=\"B68\" id=\"B68\"\u003E\u003C\u002Fa\u003EYoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., et al. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). \u003Ci\u003EScience\u003C\u002Fi\u003E 351, 1196–1199. doi: 10.1126\u002Fscience.aad6359\u003C\u002Fp\u003E\r\n\u003Cp class=\"ReferencesCopy2\"\u003E\u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F26965627\" target=\"_blank\"\u003EPubMed Abstract\u003C\u002Fa\u003E | \u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.1126\u002Fscience.aad6359\" target=\"_blank\"\u003ECrossRef Full Text\u003C\u002Fa\u003E | \u003Ca href=\"http:\u002F\u002Fscholar.google.com\u002Fscholar_lookup?&title=A+bacterium+that+degrades+and+assimilates+poly%28ethylene+terephthalate%29%2E&journal=Science&author=Yoshida+S.&author=Hiraga+K.&author=Takehana+T.&author=Taniguchi+I.&author=Yamaji+H.&author=Maeda+Y.&publication_year=2016&volume=351&pages=1196–1199\" target=\"_blank\"\u003EGoogle Scholar\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"thinLineM20\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cdiv class=\"AbstractSummary\"\u003E\r\n\u003Cp\u003E\u003Cspan\u003EKeywords\u003C\u002Fspan\u003E: acidophiles, acidophilic bacteria, acid mine drainage, biodiversity, extremozymes, esterase, metagenomics, plastic\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003Cspan\u003ECitation:\u003C\u002Fspan\u003E Vidal P, Martínez-Martínez M, Fernandez-Lopez L, Roda S, Méndez-García C, Golyshina OV, Guallar V, Peláez AI and Ferrer M (2022) Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation. \u003Ci\u003EFront. Microbiol.\u003C\u002Fi\u003E 13:868839. doi: 10.3389\u002Ffmicb.2022.868839\u003C\u002Fp\u003E\r\n\u003Cp id=\"timestamps\"\u003E\r\n\u003Cspan\u003EReceived:\u003C\u002Fspan\u003E 03 February 2022; \u003Cspan\u003EAccepted:\u003C\u002Fspan\u003E 14 March 2022;\u003Cbr\u003E\u003Cspan\u003EPublished:\u003C\u002Fspan\u003E 19 May 2022.\u003C\u002Fp\u003E\r\n\u003Cdiv\u003E\r\n\u003Cp\u003EEdited by:\u003C\u002Fp\u003E\r\n\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F111623\u002Foverview\"\u003ERolf Daniel\u003C\u002Fa\u003E, University of Göttingen, Germany\u003C\u002Fdiv\u003E\r\n\u003Cdiv\u003E\r\n\u003Cp\u003EReviewed by:\u003C\u002Fp\u003E\r\n\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F231858\u002Foverview\"\u003EWolfgang R. Streit\u003C\u002Fa\u003E, University of Hamburg, Germany\u003Cbr\u003E\r\n\u003Ca href=\"https:\u002F\u002Floop.frontiersin.org\u002Fpeople\u002F1576541\u002Foverview\"\u003EJiujun Cheng\u003C\u002Fa\u003E, University of Waterloo, Canada\u003C\u002Fdiv\u003E\r\n\u003Cp\u003E\u003Cspan\u003ECopyright\u003C\u002Fspan\u003E © 2022 Vidal, Martínez-Martínez, Fernandez-Lopez, Roda, Méndez-García, Golyshina, Guallar, Peláez and Ferrer. This is an open-access article distributed under the terms of the \u003Ca rel=\"license\" href=\"http:\u002F\u002Fcreativecommons.org\u002Flicenses\u002Fby\u002F4.0\u002F\" target=\"_blank\"\u003ECreative Commons Attribution License (CC BY)\u003C\u002Fa\u003E. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003Cspan\u003E*Correspondence:\u003C\u002Fspan\u003E Manuel Ferrer, \u003Ca id=\"encmail\"\u003EbWZlcnJlckBpY3AuY3NpYy5lcw==\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003Cspan\u003E\u003Csup\u003E†\u003C\u002Fsup\u003EPresent address:\u003C\u002Fspan\u003E Mónica Martínez-Martínez, Departamento de Ciencias de la Salud, Facultad de Ciencias Biomédicas, Universidad Europea de Madrid, Villaviciosa de Odón, Spain\u003C\u002Fp\u003E\r\n\u003Cdiv class=\"clear\"\u003E\u003C\u002Fdiv\u003E\r\n\u003C\u002Fdiv\u003E",menuHtml:"\u003Cul class=\"flyoutJournal\"\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h1\"\u003EAbstract\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h2\"\u003EIntroduction\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h3\"\u003EMaterials and Methods\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h4\"\u003EResults\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h5\"\u003EDiscussion\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h6\"\u003EData Availability Statement\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#h7\"\u003EAuthor Contributions\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#fun1\"\u003EFunding\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#conf1\"\u003EConflict of Interest\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#disclaimer1\"\u003EPublisher’s Note\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#ack1\"\u003EAcknowledgments\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#S9\"\u003ESupplementary Material\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca href=\"#refer1\"\u003EReferences\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n"},files:[{name:"EPUB.epub",fileServerPackageEntryId:f,type:{code:aF,name:aF}},{name:aG,fileServerPackageEntryId:"fmicb-13-868839\u002Ffmicb-13-868839.pdf",type:{code:r,name:r}},{name:aG,fileServerPackageEntryId:f,type:{code:r,name:r}},{name:"fmicb-13-868839.xml",fileServerPackageEntryId:"fmicb-13-868839\u002Ffmicb-13-868839.xml",type:{code:"NLM_XML",name:"XML"}},{name:"Provisional PDF.pdf",fileServerPackageEntryId:f,type:{code:r,name:r}}]},currentArticlePageMetaInfo:{title:aH,link:[{rel:"canonical",href:aI}],meta:[{hid:z,property:z,name:z,content:aJ},{hid:aK,property:aK,name:"title",content:aH},{hid:aL,property:aL,name:z,content:aJ},{hid:aM,name:aM,content:"acidophiles,Acidophilic bacteria,acid mine 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Microbiol.","1664-302X",void 0,"Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation",6,18,11,13,1920,"por-journal.com",7,"escubed.org",1918,"fipp","https:\u002F\u002Fd2csxpduxe849s.cloudfront.net\u002Fmedia\u002FE32629C6-9347-4F84-81FEAEF7BFA342B3\u002FD7C8E8F5-EB21-4B26-BE7D25D7B2089254\u002Fwebimage-9F239CC8-42C5-479C-ADDCDB2931B331FF.png","22C10171-81B3-4DA6-99342F272A32E8BB","jpg","2022-06-27T10:00:07Z",1440,"fmicb",51,"journal_field","10.3389\u002Ffmicb.2022.868839","\u003Cp\u003EAcid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and \u003Citalic\u003Ein silico\u003C\u002Fitalic\u003E metagenomic approaches, we retrieved 16 esterases from the α\u002Fβ-hydrolase fold superfamily with the closest match from uncultured acidophilic \u003Citalic\u003EAcidobacteria, Actinobacteria (Acidithrix, Acidimicrobium\u003C\u002Fitalic\u003E, and \u003Citalic\u003EFerrimicrobium), Acidiphilium\u003C\u002Fitalic\u003E, and other \u003Citalic\u003EProteobacteria\u003C\u002Fitalic\u003E inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in \u003Citalic\u003EEscherichia coli\u003C\u002Fitalic\u003E. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U\u002Fg at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.\u003C\u002Fp\u003E","Paula","Mónica",1685724,"Laura",1685246,"Sergi","Department of Life Sciences, Barcelona Supercomputing Center",217882,"Celia","Área de Microbiología, Departamento Biología Funcional e Instituto de Biotecnología de Asturias, Universidad de Oviedo",224799,"Olga V.",517366,"Víctor",93877,"Ana I.",236735,"Manuel",111623,"Rolf","Germany",231858,1576541,"Jiujun",{},2940,"EPUB","fmicb-13-868839.pdf","Frontiers | Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation","https:\u002F\u002Fwww.frontiersin.org\u002Fjournals\u002Fmicrobiology\u002Farticles\u002F10.3389\u002Ffmicb.2022.868839\u002Ffull","Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose 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