{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T15:02:47Z","timestamp":1785337367120,"version":"3.55.0"},"reference-count":39,"publisher":"American Chemical Society (ACS)","issue":"29","license":[{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22308317"],"award-info":[{"award-number":["22308317"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Pioneer and Leading Goose R&D Program of Zhejiang","doi-asserted-by":"publisher","award":["2024C03013"],"award-info":[{"award-number":["2024C03013"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Pioneer and Leading Goose R&D Program of Zhejiang","doi-asserted-by":"publisher","award":["2025C01097"],"award-info":[{"award-number":["2025C01097"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,7,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Nicotinic acid is vital in pharmaceuticals, feed, food, and cosmetics. Nitrilase can catalyze the conversion of 3-cyanopyridine to nicotinic acid, but its industrial application is limited by poor thermostability and substrate tolerance. In this study, nitrilase from Betaproteobacteria bacterium was engineered via multistrategy synergistic evolution, including C-terminal loop truncation, consensus mutation, loop engineering and protein surface engineering. Through iterative saturation mutagenesis, we obtained mutant 4 M (L194F\/A201Q\/S208L\/M180Q-\u039447). Its activity reached 12.78 U\/mL. After 1 h at 50 \u00b0C, 4 M retained 10.46 U\/mL residual activity\u25003.25-fold higher than WT. Its melting and aggregation temperatures increased by 8.3 and 6.6 \u00b0C, respectively. Molecular dynamics revealed that enhanced thermostability resulted from reduced loop flexibility, strengthened hydrophobic interactions, and new hydrogen bonds. An engineered Vibrio natriegens strain produced 564.3 g\/L nicotinic acid, a 5.8-fold increase over WT. This work provides mechanistic insights into nitrilase thermostability.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c02804","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T14:42:29Z","timestamp":1784212949000},"page":"23046-23055","source":"Crossref","is-referenced-by-count":0,"title":["Enhancing the Robustness\nof Nitrilase from\n                    <i>Betaproteobacteria bacterium<\/i>\n                    by Multistrategy Synergistic\nEvolution"],"prefix":"10.1021","volume":"74","author":[{"given":"Qi","family":"Zhu","sequence":"first","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lanxin","family":"Xiao","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yijie","family":"Sun","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huifen","family":"Yu","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chennuo","family":"Zhu","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sai","family":"Fang","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Meng","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongliang","family":"Yan","sequence":"additional","affiliation":[{"name":"Zhejiang Jiuzhou Pharmaceutical Co., Ltd ., ,","place":["Hangzhou, China, 318000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6378-8451","authenticated-orcid":true,"given":"Lirong","family":"Yang","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]},{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 311215"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9700-7721","authenticated-orcid":true,"given":"Wenlong","family":"Zheng","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]},{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 311215"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9924-7307","authenticated-orcid":true,"given":"Jianping","family":"Wu","sequence":"additional","affiliation":[{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 310058"]},{"name":"Zhejiang University , , ,","place":["Hangzhou, China, 311215"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"2026072910063553500_cit1","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1016\/j.biortech.2018.03.109","article-title":"Efficient biocatalytic\nsynthesis of nicotinic acid by recombinant nitrilase via high density\nculture","volume":"260","author":"Gong","year":"2018","journal-title":"Bioresour. Technol."},{"key":"2026072910063553500_cit2","doi-asserted-by":"publisher","first-page":"9078","DOI":"10.1021\/acscatal.3c01469","article-title":"Structure-oriented\nengineering of amidase: modification\nof twisted access tunnel for efficient synthesis of 2-chloronicotinic\nacid","volume":"13","author":"Wu","year":"2023","journal-title":"ACS Catal."},{"key":"2026072910063553500_cit3","doi-asserted-by":"publisher","first-page":"175","DOI":"10.1016\/j.gresc.2023.07.002","article-title":"Iridium-catalyzed\nasymmetric\nreduction of \u03b1, \u03b2-unsaturated nitriles with water","volume":"5","author":"Lu","year":"2024","journal-title":"Green Synth. Catal."},{"key":"2026072910063553500_cit4","doi-asserted-by":"publisher","first-page":"1104","DOI":"10.1016\/j.biortech.2017.08.039","article-title":"Significantly\nenhanced substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma and cell immobilization","volume":"244","author":"Dong","year":"2017","journal-title":"Bioresour. Technol."},{"key":"2026072910063553500_cit5","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2024.108093","article-title":"Cryo-EM structure\nof bacterial nitrilase reveals insight into oligomerization, substrate\nrecognition, and catalysis","volume":"216","author":"Aguirre-Sampieri","year":"2024","journal-title":"J. Struct. Biol."},{"key":"2026072910063553500_cit6","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1080\/07388551.2020.1827367","article-title":"Nitrilase: a promising biocatalyst\nin industrial applications for green chemistry","volume":"41","author":"Shen","year":"2021","journal-title":"Crit. 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Macromol."},{"key":"2026072910063553500_cit18","doi-asserted-by":"publisher","first-page":"1235","DOI":"10.1007\/s10295-010-0902-7","article-title":"An improved\nnitrilase-mediated bioprocess\nfor synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2","volume":"38","author":"Sharma","year":"2011","journal-title":"J.\nInd. Microbiol. Biotechnol."},{"key":"2026072910063553500_cit19","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1007\/s11274-012-1195-y","article-title":"Biosynthesis\nof\nnicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150","volume":"29","author":"Jin","year":"2013","journal-title":"World J. Microbiol. 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Bull. Life\nSci."},{"key":"2026072910063553500_cit25","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-43631-2","article-title":"Discovery\nand remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery","volume":"14","author":"Tian","year":"2023","journal-title":"Nat. Commun."},{"key":"2026072910063553500_cit26","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1021\/cbe.5c00069","article-title":"Semirational protein engineering yields archaerhodopsin-3-based\nfluorescent genetically encoded voltage indicators with enhanced brightness\nand red shifted absorption bands","volume":"2","author":"Nikolaev","year":"2025","journal-title":"Chem. Bio\nEng."},{"key":"2026072910063553500_cit27","doi-asserted-by":"crossref","unstructured":"Dao, T. M.; Ghent, S.; Uversky, V. 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Microbiol."},{"key":"2026072910063553500_cit30","doi-asserted-by":"publisher","DOI":"10.1126\/science.adr6006","article-title":"Rapid in\nsilico directed evolution by a protein language model with EVOLVEpro","volume":"387","author":"Jiang","year":"2025","journal-title":"Science"},{"key":"2026072910063553500_cit31","doi-asserted-by":"publisher","DOI":"10.1002\/aic.18211","article-title":"Engineering residues on C interface\nto improve thermostability of nitrilase for biosynthesis of Pregabalin precursor","volume":"69","author":"Lu","year":"2023","journal-title":"AIChE J."},{"key":"2026072910063553500_cit32","doi-asserted-by":"publisher","DOI":"10.1016\/j.bioorg.2023.107055","article-title":"Identification and evolution of non-traditional nitrilase\nfrom Spirosoma linguale DSM 74 with\nhigh hydration activity","volume":"143","author":"Sun","year":"2024","journal-title":"Bioorg. 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Biol.\nMacromol."},{"key":"2026072910063553500_cit39","doi-asserted-by":"publisher","first-page":"4422","DOI":"10.1021\/acs.biochem.6b00370","article-title":"Structural basis of the substrate\nrange and enantioselectivity of two (S)-selective \u03c9-transaminases","volume":"55","author":"van Oosterwijk","year":"2016","journal-title":"Biochemistry"}],"container-title":["Journal of Agricultural\nand Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c02804","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/29\/23046\/65979858\/acs.jafc.6c02804.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/29\/23046\/65979858\/acs.jafc.6c02804.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T14:15:46Z","timestamp":1785334546000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/29\/23046\/5212184\/Enhancing-the-Robustness-of-Nitrilase-from"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,16]]},"references-count":39,"journal-issue":{"issue":"29","published-online":{"date-parts":[[2026,7,16]]},"published-print":{"date-parts":[[2026,7,29]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c02804","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,16]]}}}