{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:14:01Z","timestamp":1785428041743,"version":"3.56.0"},"reference-count":88,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T00:00:00Z","timestamp":1781481600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union\u2019s Horizon Europe Research and Innovation Program","award":["101079425"],"award-info":[{"award-number":["101079425"]}]},{"DOI":"10.13039\/100012623","name":"Federation of European Biochemical Societies","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100012623","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/0100zbm77","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Excellence Scholarship and the High-Level Scientific Stay (SSHN) fellowship"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Bioprospecting for extremozymes from unique ecological niches is crucial for developing robust biocatalysts for green chemistry. Here, we report the de novo hybrid genome assembly of Penicillium crustosum CTM10622, isolated from the humid montane forest of El Fe\u00efdja National Park, Tunisia. Using Illumina NextSeq\u2122 500 and Nanopore PromethION 2 Solo, a highly contiguous 31.38 Mb assembly (N50 = 1.94 Mb; 98.3% BUSCOs) was achieved. This robust genomic foundation enabled the identification of an extensive hydrolase repertoire, leading to the discovery of a novel alkaline serine lipase, PCLIP, subsequently heterologously expressed in Pichia pastoris. Recombinant rPCLIP exhibited a high specific activity (15,000 U\/mg at pH 10, 65 \u00b0C) and exceptional thermostability, with half-lives of 14 and 8 h at 80 and 90 \u00b0C, respectively. The enzyme\u2019s identity as a serine lipase was confirmed by its complete inhibition by Orlistat or tetrahydrolipstatin (THL) (51 \u00b5M), PMSF (5 mM), and diisopropylfluorophosphate (DIFP) (2 mM). To determine its substrate specificity, advanced computational approaches, including convolutional neural network-based docking and explicitly solvated molecular dynamics, were employed to compare rPCLIP with its homologue PCrL, a recombinant serine alkaline lipase from Penicillium crustosum Thom P22. While rPCLIP showed optimal experimental activity toward short-chain glyceryl tributyrate, simulations revealed that long-chain trioctanoin acts as a \u2018thermodynamic trap\u2019 due to over-stabilization. Conversely, the rigid rPCrL favors tricaprylin, driven by a \u2018hydrophobic engine\u2019 effect where the solvated environment forces chain burial with minimal entropic penalty. The findings demonstrate that rPCLIP specificity is driven by a delicate interplay of geometric complementarity, Van der Waals enthalpy, and conformational entropy.<\/jats:p>","DOI":"10.3390\/ijms27125389","type":"journal-article","created":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T00:49:01Z","timestamp":1781570941000},"page":"5389","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hybrid Whole-Genome Sequencing of Penicillium crustosum CTM10622 Uncovers a Highly Thermostable Alkaline Serine Lipase with Biotechnological Relevance"],"prefix":"10.3390","volume":"27","author":[{"given":"Sondes","family":"Mechri","sequence":"first","affiliation":[{"name":"Laboratoire des Biotechnologies Microbiennes et Enzymatiques et Biomol\u00e9cules (LBMEB, code LR15CBS06), Centre de Biotechnologie de Sfax (CBS), Universit\u00e9 de Sfax (USF), Route Sidi Mansour Km 6, Sfax BP 1177, Tunisia"},{"name":"Universit\u00e9 Lyon 1, CNRS, INSERM, Plateforme de G\u00e9nomique et Microg\u00e9nomique ProfileXpert, SFR Sant\u00e9 Lyon Est, UMR-S3453, US7, Facult\u00e9 de M\u00e9decine Rockefeller, 69373 Lyon Cedex, France"},{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, Villeurbanne, France"},{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, G\u00e9nie Enzymatique, Membranes Biomim\u00e9tiques et Assemblages Supramol\u00e9culaires (GEMBAS), F-69622 Villeurbanne Cedex, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Afef","family":"Najjari","sequence":"additional","affiliation":[{"name":"Laboratoire de Microorganismes et Biomol\u00e9cules Actives (LMBA, LR03ES03), D\u00e9partement de Biologie, Facult\u00e9 des Sciences de Tunis (FST), Universit\u00e9 de Tunis El Manar (UTM), El Manar, Tunis 2092, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4542-5088","authenticated-orcid":false,"given":"S\u00e9verine","family":"Croze","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Lyon 1, CNRS, INSERM, Plateforme de G\u00e9nomique et Microg\u00e9nomique ProfileXpert, SFR Sant\u00e9 Lyon Est, UMR-S3453, US7, Facult\u00e9 de M\u00e9decine Rockefeller, 69373 Lyon Cedex, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3261-7884","authenticated-orcid":false,"given":"Fakher","family":"Frikha","sequence":"additional","affiliation":[{"name":"Laboratoire de Proc\u00e9d\u00e9s de Criblage Mol\u00e9culaire et Cellulaire (LPCMC, code LR15CBS07), Centre de Biotechnologie de Sfax (CBS), Universit\u00e9 de Sfax (USF), Route Sidi Mansour Km 6, Sfax BP 1177, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadia","family":"Zarai","sequence":"additional","affiliation":[{"name":"Laboratoire des Biotechnologies Microbiennes et Enzymatiques et Biomol\u00e9cules (LBMEB, code LR15CBS06), Centre de Biotechnologie de Sfax (CBS), Universit\u00e9 de Sfax (USF), Route Sidi Mansour Km 6, Sfax BP 1177, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadda-Imene","family":"Ouzari","sequence":"additional","affiliation":[{"name":"Laboratoire de Microorganismes et Biomol\u00e9cules Actives (LMBA, LR03ES03), D\u00e9partement de Biologie, Facult\u00e9 des Sciences de Tunis (FST), Universit\u00e9 de Tunis El Manar (UTM), El Manar, Tunis 2092, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0979-1614","authenticated-orcid":false,"given":"Alexandre","family":"Noiriel","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, Villeurbanne, France"},{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, G\u00e9nie Enzymatique, Membranes Biomim\u00e9tiques et Assemblages Supramol\u00e9culaires (GEMBAS), F-69622 Villeurbanne Cedex, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6054-8842","authenticated-orcid":false,"given":"Ebru Toksoy","family":"\u00d6ner","sequence":"additional","affiliation":[{"name":"Industrial Biotechnology and Systems Biology Research Group (IBSB), Department of Bioengineering, Marmara University, 34854 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8020-4294","authenticated-orcid":false,"given":"Abdelkarim","family":"Abousalham","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, Villeurbanne, France"},{"name":"Universit\u00e9 Lyon 1, CNRS, ICBMS, UMR 5246, G\u00e9nie Enzymatique, Membranes Biomim\u00e9tiques et Assemblages Supramol\u00e9culaires (GEMBAS), F-69622 Villeurbanne Cedex, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1930-2637","authenticated-orcid":false,"given":"Marilize Le","family":"Roes-Hill","sequence":"additional","affiliation":[{"name":"Applied Microbial and Health Biotechnology Institute (AMHBI), Cape Peninsula University of Technology (CPUT), Bellville P.O. Box 1906, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Slim","family":"Tounsi","sequence":"additional","affiliation":[{"name":"Laboratoire des Biopesticides (LB, code LR15CBS04), Centre de Biotechnologie de Sfax (CBS), Universit\u00e9 de Sfax (USF), Route Sidi Mansour Km 6, Sfax BP 1177, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel","family":"Lachuer","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Lyon 1, CNRS, INSERM, Plateforme de G\u00e9nomique et Microg\u00e9nomique ProfileXpert, SFR Sant\u00e9 Lyon Est, UMR-S3453, US7, Facult\u00e9 de M\u00e9decine Rockefeller, 69373 Lyon Cedex, France"},{"name":"Universit\u00e9 Lyon 1, CNRS, INSERM, Centre de Recherche en Canc\u00e9rologie de Lyon, UMR 1052, UMR 5286, 28 Rue Laennec, 69008 Lyon Cedex, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5182-7353","authenticated-orcid":false,"given":"Bassem","family":"Jaouadi","sequence":"additional","affiliation":[{"name":"Laboratoire des Biotechnologies Microbiennes et Enzymatiques et Biomol\u00e9cules (LBMEB, code LR15CBS06), Centre de Biotechnologie de Sfax (CBS), Universit\u00e9 de Sfax (USF), Route Sidi Mansour Km 6, Sfax BP 1177, Tunisia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Schell, T., Greve, C., and Podsiadlowski, L. 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