{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T22:06:54Z","timestamp":1788991614107,"version":"build-2803163510"},"reference-count":46,"publisher":"American Chemical Society (ACS)","issue":"35","license":[{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"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":["32200061"],"award-info":[{"award-number":["32200061"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Innovation & Entrepreneurship Team Project of Guangzhou, China","doi-asserted-by":"publisher","award":["2024D03J0012"],"award-info":[{"award-number":["2024D03J0012"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>d-Allulose is a promising low-calorie rare sweetener; however, its industrial production is limited by the insufficient catalytic activity and thermostability of d-allulose 3-epimerases (DAEases). In this study, a combinatorial engineering strategy integrating lid-domain modulation and computational design was developed to improve enzyme performance. Lid engineering identified a key mutation (N120D) associated with enhanced catalytic turnover, while computational screening identified beneficial mutations (T69S, D71E, and S128A). The best mutant, T69S\/D71E\/N120D\/S128A, exhibited a 1.9-fold increase in specific activity, a 2.2-fold improvement in catalytic efficiency, and a 34.1% longer half-life at 60 \u00b0C. It produced 152 g\/L d-allulose from 500 g\/L d-fructose within 3 h, achieving a 30.4% conversion yield under mildly acidic and high-temperature conditions. Mechanistic analyses suggested that improved performance resulted from the combinatorial effects of enhanced structural rigidity and altered lid-domain dynamics, consistent with improved catalytic turnover while maintaining thermostability. This work provides an efficient biocatalyst and a generalizable protein engineering strategy.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c08333","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T21:41:01Z","timestamp":1788298861000},"page":"27767-27778","source":"Crossref","is-referenced-by-count":0,"title":["Combinatorial Lid-Domain\nEngineering and Computational\nDesign Enable Simultaneous Enhancement of Activity and Thermostability\nof D-Allulose 3-Epimerase"],"prefix":"10.1021","volume":"74","author":[{"given":"Qichen","family":"Liu","sequence":"first","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinrui","family":"Tang","sequence":"additional","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjing","family":"Sun","sequence":"additional","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cunsheng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2241-4813","authenticated-orcid":true,"given":"Mei","family":"Zhao","sequence":"additional","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5817-1609","authenticated-orcid":true,"given":"Xianghui","family":"Qi","sequence":"additional","affiliation":[{"name":"Jiangsu University , , 301 Xuefu Road , ,","place":["Zhenjiang, China, 212013"]},{"name":"Guangzhou University , , 230 Wai Huan Xi Road , ,","place":["Guangzhou, China, 510006"]},{"name":"Guangzhou University , , 230 Wai Huan Xi Road , ,","place":["Guangzhou, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,27]]},"reference":[{"issue":"3","key":"2026090917080003100_cit1","doi-asserted-by":"publisher","first-page":"510","DOI":"10.1271\/bbb.90707","article-title":"Study on the postprandial\nblood glucose suppression effect of D-psicose in borderline diabetes\nand the safety of long-term ingestion by normal human subjects","volume":"74","author":"Hayashi","year":"2010","journal-title":"Biosci. 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Agric.\nFood Chem."},{"key":"2026090917080003100_cit43","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2024.1365814","article-title":"Biochemical characterization, structure-guided mutagenesis,\nand application\nof a recombinant D-allulose 3-epimerase from Christensenellaceae bacterium for the biocatalytic production of D-allulose","volume":"12","author":"Guan","year":"2024","journal-title":"Front. Bioeng. Biotechnol."},{"issue":"40","key":"2026090917080003100_cit44","doi-asserted-by":"publisher","first-page":"25562","DOI":"10.1021\/acs.jafc.5c09894","article-title":"Integrated\nprotein engineering and expression balancing enable efficient D-allulose\nproduction","volume":"73","author":"Cai","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"2026090917080003100_cit45","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2023.108094","article-title":"Role of distal sites in enzyme engineering","volume":"63","author":"Gu","year":"2023","journal-title":"Biotechnol. 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Food Chem."}],"container-title":["Journal of Agricultural\nand Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c08333","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/35\/27767\/67530675\/acs.jafc.6c08333.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/35\/27767\/67530675\/acs.jafc.6c08333.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T21:12:06Z","timestamp":1788988326000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/35\/27767\/5341456\/Combinatorial-Lid-Domain-Engineering-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,27]]},"references-count":46,"journal-issue":{"issue":"35","published-online":{"date-parts":[[2026,8,27]]},"published-print":{"date-parts":[[2026,9,9]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c08333","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,27]]}}}