{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T19:46:10Z","timestamp":1773085570965,"version":"3.50.1"},"reference-count":54,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,4,5]],"date-time":"2022-04-05T00:00:00Z","timestamp":1649116800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Portugal 2020","award":["47033"],"award-info":[{"award-number":["47033"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p &lt; 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.<\/jats:p>","DOI":"10.3390\/ijms23074024","type":"journal-article","created":{"date-parts":[[2022,4,5]],"date-time":"2022-04-05T11:26:07Z","timestamp":1649157967000},"page":"4024","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8389-7543","authenticated-orcid":false,"given":"V\u00e2nia","family":"Cardoso","sequence":"first","affiliation":[{"name":"Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal\u2014Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, P\u00f3lo Universit\u00e1rio do Alto da Ajuda, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"},{"name":"NZYTech Ltd., Estrada do Pa\u00e7o do Lumiar, Campus do Lumiar, 1649-038 Lisboa, Portugal"}]},{"given":"Joana L. A.","family":"Br\u00e1s","sequence":"additional","affiliation":[{"name":"NZYTech Ltd., Estrada do Pa\u00e7o do Lumiar, Campus do Lumiar, 1649-038 Lisboa, Portugal"}]},{"given":"In\u00eas F.","family":"Costa","sequence":"additional","affiliation":[{"name":"NZYTech Ltd., Estrada do Pa\u00e7o do Lumiar, Campus do Lumiar, 1649-038 Lisboa, Portugal"}]},{"given":"Lu\u00eds M. A.","family":"Ferreira","sequence":"additional","affiliation":[{"name":"Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal\u2014Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, P\u00f3lo Universit\u00e1rio do Alto da Ajuda, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3894-3488","authenticated-orcid":false,"given":"Lu\u00eds T.","family":"Gama","sequence":"additional","affiliation":[{"name":"Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal\u2014Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, P\u00f3lo Universit\u00e1rio do Alto da Ajuda, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9667-0196","authenticated-orcid":false,"given":"Renaud","family":"Vincentelli","sequence":"additional","affiliation":[{"name":"Centre National de la Recherche Scientifique, Unit\u00e9 Mixte de Recherche 7257, Universit\u00e9 Aix-Marseille, 13288 Marseille, France"},{"name":"Institut National de la Recherche Agronomique, Unit\u00e9 sous Contrat 1408 Architecture et Fonction des Macromol\u00e9cules Biologiques, 13288 Marseille, France"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3434-8588","authenticated-orcid":false,"given":"Bernard","family":"Henrissat","sequence":"additional","affiliation":[{"name":"Centre National de la Recherche Scientifique, Unit\u00e9 Mixte de Recherche 7257, Universit\u00e9 Aix-Marseille, 13288 Marseille, France"},{"name":"Institut National de la Recherche Agronomique, Unit\u00e9 sous Contrat 1408 Architecture et Fonction des Macromol\u00e9cules Biologiques, 13288 Marseille, France"},{"name":"Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia"}]},{"given":"Carlos M. G. A.","family":"Fontes","sequence":"additional","affiliation":[{"name":"Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal\u2014Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, P\u00f3lo Universit\u00e1rio do Alto da Ajuda, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"},{"name":"NZYTech Ltd., Estrada do Pa\u00e7o do Lumiar, Campus do Lumiar, 1649-038 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1879","DOI":"10.1016\/j.carres.2009.05.021","article-title":"The structure, function, and biosynthesis of plant cell wall pectic polysaccharides","volume":"344","author":"Caffall","year":"2009","journal-title":"Carbohydr. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.cbpa.2006.02.035","article-title":"Bioethanol","volume":"10","author":"Gray","year":"2006","journal-title":"Curr. Opin. Chem. 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