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Here, we developed a multidimensional consensus computational framework integrating sequence conservation, structural dynamics, and thermodynamic prediction to identify functional mutation hotspots in PcPel1834. Iterative saturation mutagenesis yielded M5 (S212E\/Q225L\/F241L\/L260A\/G296T), which showed markedly enhanced thermostability and moderately improved apparent catalytic efficiency. M5 increased the melting temperature from 52.1 to 65.0 \u00b0C and retained nearly full activity after 24 h at 50 \u00b0C. Its specific activity and kcat\/Km reached 4259 U\/mg and 1427 mL\/s\/mg, respectively, both higher than WT. Epistatic analysis and MD simulations indicated that M5 enhanced thermostability through hydrogen-bond remodeling, backbone rigidification, and strengthened long-range communication, while distal mutations allosterically preorganized the catalytic pocket to improve substrate binding and catalytic geometry. In ramie degumming, M5 showed improved pectin removal and fiber quality. This study provides a practical strategy for engineering thermostable pectate lyases with improved catalytic performance.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c06845","type":"journal-article","created":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T14:01:38Z","timestamp":1786888898000},"page":"26057-26070","source":"Crossref","is-referenced-by-count":0,"title":["Multidimensional\nComputational Engineering of Pectate\nLyase for Enhanced Thermostability and Moderately Improved Catalytic\nEfficiency"],"prefix":"10.1021","volume":"74","author":[{"given":"Ziqi","family":"Hou","sequence":"first","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gen","family":"Lu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tong","family":"Shu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simin","family":"Liu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1652-8689","authenticated-orcid":true,"given":"Ya","family":"Wu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaofeng","family":"Hu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pandeng","family":"Li","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianyi","family":"Yu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunhua","family":"Fu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2517-2266","authenticated-orcid":true,"given":"Longjiang","family":"Yu","sequence":"additional","affiliation":[{"name":"Huazhong University of Science and Technology , , ,","place":["Wuhan, China, 430070"]},{"name":"Ministry of Education , , ,","place":["Wuhan, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,16]]},"reference":[{"issue":"1","key":"2026090104482369700_cit1","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1038\/s44160-023-00417-0","article-title":"Engineered Enzymes for the Synthesis of Pharmaceuticals\nand Other High-Value Products","volume":"3","author":"Reetz","year":"2024","journal-title":"Nat. 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