{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:55:57Z","timestamp":1791273357679,"version":"4.1.0"},"reference-count":43,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Basic Research Program of China","doi-asserted-by":"publisher","award":["2023YFA0914000"],"award-info":[{"award-number":["2023YFA0914000"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    The stability\u2010activity trade\u2010off remains a major bottleneck in engineering robust biocatalysts for industrial applications. Here, we developed an integrated computational strategy to simultaneously enhance the catalytic activity and thermostability of 7\u03b1\u2010hydroxysteroid dehydrogenase (7\u03b1\u2010HSDH) by engineering highly flexible regions, with a particular focus on \u03b1\u2010helices. Evolutionary conservation analysis combined with multi\u2010temperature molecular dynamics (MD) simulations was first applied to identify weakly conserved and flexible \u03b1\u2010helices. Subsequently, computational tools (PROSS and Pythia) were utilized to generate mutations within these flexible regions. Through modular assembly of synergistic mutations, the optimal mutant M9 (D12K\/V15I\/V50E\/I56R\/D66A\/T76R\/A126I\/A132H\/S158A\/S161A\/A203P\/H208Q) was obtained. Compared with the WT, M9 exhibited a 10.5\u00b0C increase in melting temperature (\n                    <jats:italic>T<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>m<\/jats:italic>\n                    <\/jats:sub>\n                    ), a 2.3\u2010fold prolongation of half\u2010life (\n                    <jats:italic>t<\/jats:italic>\n                    <jats:sub>1\/2<\/jats:sub>\n                    ) at 40\u00b0C (reaching 75.3\u2009h), a 3.14\u2010fold improvement in catalytic efficiency (\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>cat<\/jats:sub>\n                    <jats:italic>\/K<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>m<\/jats:italic>\n                    <\/jats:sub>\n                    ) to 29,032.73\u2009mM\n                    <jats:sup>\u22121<\/jats:sup>\n                    s\n                    <jats:sup>\u22121<\/jats:sup>\n                    , and enhanced substrate tolerance. Structural and molecular dynamics analyses indicated that the enhanced performance of M9 was associated with increased rigidity of key \u03b1\u2010helix regions, redistribution of surface electrostatic potential, and remodeling of the substrate\u2010binding pocket. This study thus offers a novel design framework for improving the catalytic activity, thermostability, and substrate tolerance, balancing the stability\u2010activity trade\u2010off.\n                  <\/jats:p>","DOI":"10.1002\/bit.70399","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:49:48Z","timestamp":1791272988000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering the \u03b1\u2010Helix Dynamics of Hydroxysteroid Dehydrogenase by Computational Design to Balance Catalytic Activity and Stability"],"prefix":"10.1002","author":[{"given":"Shu\u2010Fang","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ting","family":"Zou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory of Bioorganic 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and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ya\u2010Ru","family":"Shen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou 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