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Using the previously evolved high-yield Thaumatin variant M-882 as the parental scaffold, we used an integrated rational design strategy combining HotSpot Wizard, FoldX, and PyRosetta. Candidate variants were evaluated by sfGFP-based fluorescence screening, SDS-PAGE, and absolute protein quantification. The variant R178H showed the most favorable overall balance of structural compatibility and thermal resilience. After heating at 80 \u00b0C for 2 h, R178H retained 63% of its soluble protein fraction, compared with 43% for M-882. Structural analysis suggested that replacing Arg178 with histidine reduced local steric hindrance and improved packing interactions. Molecular dynamics simulations further indicated that R178H adopted a more compact and stable conformation. These results clarify the structural basis for its improved heat resistance and provide a rational strategy for engineering thermostable sweet proteins for food-processing applications.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c06037","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T17:02:00Z","timestamp":1791306120000},"page":"32538-32548","source":"Crossref","is-referenced-by-count":0,"title":["Enhancing the Thermostability\nof Sweet Protein Thaumatin\nII by Integrated Rational Design"],"prefix":"10.1021","volume":"74","author":[{"given":"Lefei","family":"Wang","sequence":"first","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuxin","family":"Yang","sequence":"additional","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jintong","family":"Dong","sequence":"additional","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoqi","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4177-2416","authenticated-orcid":true,"given":"Zhengming","family":"Zhu","sequence":"additional","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6625-5557","authenticated-orcid":true,"given":"Ling","family":"Jiang","sequence":"additional","affiliation":[{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]},{"name":"Nanjing Tech University , , ,","place":["Nanjing, China, 211816"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,6]]},"reference":[{"key":"2026102109075877400_cit1","doi-asserted-by":"publisher","first-page":"414","DOI":"10.1016\/j.cmet.2023.02.003","article-title":"The global burden of metabolic disease:\ndata from 2000 to 2019","volume":"35","author":"Chew","year":"2023","journal-title":"Cell Metab."},{"key":"2026102109075877400_cit2","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1016\/j.fbp.2022.10.009","article-title":"Determination\nof recombinant thaumatin II secreted by Pichia pastoris using reversed-phase high-performance liquid chromatography","volume":"137","author":"Joseph","year":"2023","journal-title":"Food Bioprod. 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Biotechnol."},{"key":"2026102109075877400_cit6","doi-asserted-by":"publisher","first-page":"8693","DOI":"10.1021\/acs.jafc.4c00827","article-title":"Constructing an antibiotic-free protein\nexpression system for ovalbumin\nbiosynthesis in probiotic Escherichia coli Nissle 1917","volume":"72","author":"Liu","year":"2024","journal-title":"J. Agric. Food Chem."},{"key":"2026102109075877400_cit7","doi-asserted-by":"publisher","DOI":"10.3390\/nu16183162","article-title":"Chronic use\nof artificial sweeteners: pros and cons","volume":"16","author":"Kossiva","year":"2024","journal-title":"Nutrients"},{"key":"2026102109075877400_cit8","doi-asserted-by":"publisher","DOI":"10.1016\/j.foodres.2022.111853","article-title":"An updated\nmultifaceted overview of sweet proteins\nand dipeptides as sugar substitutes; the chemistry, health benefits,\ngut interactions, and safety","volume":"162","author":"Farag","year":"2022","journal-title":"Food Res. 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