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Whereas classical approaches relied on rational design and experimental screening, advances in high-throughput sequencing, modeling, and machine learning have enabled predictive exploration of sequence\u2013structure\u2013function relationships in enzymes. Importantly, the latest protein language models and deep learning approaches enable accurate prediction of mutational outcomes, stability engineering, and functional annotation at an unprecedented scale. Generative AI models also enable the design of novel enzymes by predicting protein sequences with tailored catalytic functions and broadened substrate specificity. AI combined with design\u2013build\u2013test\u2013learn (DBTL) automation and synthetic biology has enabled the creation of closed-loop engineering workflows for rapid, iterative optimization. This review examines enzyme engineering from classical methods to AI-assisted biocatalyst development, highlighting key advances, challenges, and emerging trends in autonomous laboratories, sustainable biocatalysis, and computational protein design.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c04272","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T11:01:33Z","timestamp":1786100493000},"page":"24761-24780","source":"Crossref","is-referenced-by-count":1,"title":["Enzyme Engineering:\nFrom Classical Strategies to AI-Driven\nBiocatalyst Design"],"prefix":"10.1021","volume":"74","author":[{"given":"Mati","family":"Ullah","sequence":"first","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad","family":"Rizwan","sequence":"additional","affiliation":[{"name":"Huazhong Agriculture University , , ,","place":["Wuhan, Hubei, China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vivian","family":"Andoh","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Raza","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, Anhui, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2502-4482","authenticated-orcid":true,"given":"Muhammad","family":"Naeem","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad Ihtesham","family":"Waheed","sequence":"additional","affiliation":[{"name":"University of Agriculture , , , ,","place":["Peshawar, Khyber Pakhtunkhwa, Pakistan, 25000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0202-1043","authenticated-orcid":true,"given":"Yong","family":"Chen","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"issue":"9","key":"2026081910154177500_cit1","doi-asserted-by":"publisher","first-page":"5347","DOI":"10.1021\/acs.chemrev.2c00879","article-title":"Bioinspired framework catalysts: from enzyme immobilization\nto biomimetic catalysis","volume":"123","author":"Wang","year":"2023","journal-title":"Chem. 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