{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T10:18:00Z","timestamp":1788257880381,"version":"build-2803163510"},"reference-count":37,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:00:00Z","timestamp":1788048000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32272267"],"award-info":[{"award-number":["32272267"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32272267"],"award-info":[{"award-number":["32272267"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Jiangsu Provincial Frontier Technology Research and Development Program","award":["BF2024073"],"award-info":[{"award-number":["BF2024073"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3\u2013DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L\/T508Y\/Y603F (M1) was obtained. The half-life of M1 at 50 \u00b0C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 \u00b0C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application.<\/jats:p>","DOI":"10.3390\/ijms27177773","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T09:48:36Z","timestamp":1788256116000},"page":"7773","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evolutionary Adaptability Coupled with Computation-Driven Engineering for Thermostability Enhancement of a Deoxynivalenol-Detoxifying Fusion Enzyme"],"prefix":"10.3390","volume":"27","author":[{"given":"Yiting","family":"Pan","sequence":"first","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Hao","family":"Zhu","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Qingwei","family":"Jiang","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Bin","family":"Ma","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Changhe","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Fengxia","family":"Lu","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"supervision","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"given":"Huibing","family":"Chi","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4393-7608","authenticated-orcid":false,"given":"Ping","family":"Zhu","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1007\/s12393-020-09241-0","article-title":"Degradation of Deoxynivalenol by Atmospheric-Pressure Cold Plasma and Sequential Treatments with Heat and UV Light","volume":"13","author":"Feizollahi","year":"2021","journal-title":"Food Eng. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.watres.2019.04.049","article-title":"Effect of Microbial Activity and Microbial Community Structure on the Formation of Dissolved Organic Nitrogen (DON) and Bioavailable DON Driven by Low Temperatures","volume":"159","author":"Liao","year":"2019","journal-title":"Water Res."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Li, X.-Z., Hassan, Y.I., Lepp, D., Zhu, Y., and Zhou, T. (2022). 3-Keto-DON, but Not 3-Epi-DON, Retains the in Planta Toxicological Potential after the Enzymatic Biotransformation of Deoxynivalenol. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23137230"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1007\/s12550-017-0279-9","article-title":"Chronic DON Exposure and Acute LPS Challenge: Effects on Porcine Liver Morphology and Function","volume":"33","author":"Renner","year":"2017","journal-title":"Mycotoxin. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29105","DOI":"10.1038\/srep29105","article-title":"Microbial Biotransformation of DON: Molecular Basis for Reduced Toxicity","volume":"6","author":"Pierron","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"7350","DOI":"10.1021\/acs.jafc.5c14433","article-title":"Advances in Microbial and Enzymatic Degradation of Deoxynivalenol (DON)","volume":"74","author":"Sun","year":"2026","journal-title":"J. Agric. Food Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129512","DOI":"10.1016\/j.ijbiomac.2024.129512","article-title":"Self-Cascade Deoxynivalenol Detoxification by an Artificial Enzyme with Bifunctions of Dehydrogenase and Aldo\/Keto Reductase from Genome Mining","volume":"261","author":"Niu","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9539","DOI":"10.1021\/acs.jafc.5c15071","article-title":"Reshaping the Binding Pocket of Aldo-Keto Reductase for Enhanced Stereoselectivity and Activity","volume":"74","author":"Niu","year":"2026","journal-title":"J. Agric. Food Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6929","DOI":"10.1038\/s41598-017-07319-0","article-title":"The Enzymatic Epimerization of Deoxynivalenol by Devosia Mutans Proceeds through the Formation of 3-Keto-DON Intermediate","volume":"7","author":"Hassan","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.crstbi.2022.07.002","article-title":"Engineering Enhanced Thermostability into the Geobacillus Pallidus Nitrile Hydratase","volume":"4","author":"Sewell","year":"2022","journal-title":"Curr. Res. Struct. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"343","DOI":"10.3724\/abbs.2023033","article-title":"Data-Driven Strategies for the Computational Design of Enzyme Thermal Stability: Trends, Perspectives, and Prospects","volume":"55","author":"Dou","year":"2023","journal-title":"ABBS"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Mendes, G.d.R.L., Pazdiora, P.C., Viana, V.E., Dallagnol, L.J., Calgaro, L.C., da Silva, G.J., Del Ponte, E.M., and Oliveira, A.C. (2025). de Comparative Enzymatic and Gene Expression Responses in Wheat to DON- and NIV-Producing Fusarium Species. Biology, 14.","DOI":"10.3390\/biology14081063"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1021\/acs.jcim.2c01083","article-title":"Accurate Prediction of Enzyme Thermostabilization with Rosetta Using AlphaFold Ensembles","volume":"63","author":"Peccati","year":"2023","journal-title":"J. Chem. Inf. Model."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Qian, H., Zhang, C., Lu, Z., Xia, B., Bie, X., Zhao, H., Lu, F., and Yang, G.-Y. (2018). Consensus Design for Improved Thermostability of Lipoxygenase from Anabaena Sp. PCC 7120. BMC Biotechnol., 18.","DOI":"10.1186\/s12896-018-0468-4"},{"key":"ref_15","first-page":"246","article-title":"Contamination and Biotransformation of Deoxynivalenol (DON) in Common Commercial Foods: Current Status, Challenges, and Future Perspectives","volume":"7","author":"Zhao","year":"2025","journal-title":"Green Synth. Catal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"140468","DOI":"10.1016\/j.ijbiomac.2025.140468","article-title":"Improving the Thermostability of Ulvan Lyase from Polysaccharide Lyase Family 25 Based on Multiple Computational Rational Design Strategies","volume":"302","author":"Du","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"134530","DOI":"10.1016\/j.ijbiomac.2024.134530","article-title":"Machine Learning-Guided Multi-Site Combinatorial Mutagenesis Enhances the Thermostability of Pectin Lyase","volume":"277","author":"Zhang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1002\/mlf2.12152","article-title":"GRAPE-WEB: An Automated Computational Redesign Web Server for Improving Protein Thermostability","volume":"3","author":"Sun","year":"2024","journal-title":"mLife"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1582","DOI":"10.1093\/bioinformatics\/bty862","article-title":"The EVcouplings Python Framework for Coevolutionary Sequence Analysis","volume":"35","author":"Hopf","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"139067","DOI":"10.1016\/j.ijbiomac.2024.139067","article-title":"Engineering Thermostability of Industrial Enzymes for Enhanced Application Performance","volume":"291","author":"Xu","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3833","DOI":"10.1021\/acs.jafc.3c07554","article-title":"High-Throughput Screening Techniques for the Selection of Thermostable Enzymes","volume":"72","author":"Li","year":"2024","journal-title":"J. Agric. Food Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3383","DOI":"10.1021\/acs.jcim.6c00102","article-title":"Hydroxylase Thermostability Prediction Based on Self-Trained Semisupervised Iteration and Bayesian Dynamic Tuning","volume":"66","author":"Liu","year":"2026","journal-title":"J. Chem. Inf. Model."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1080\/07388551.2019.1682963","article-title":"Recent Advances in the Improvement of Enzyme Thermostability by Structure Modification","volume":"40","author":"Xu","year":"2020","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"108385","DOI":"10.1016\/j.foodhyd.2022.108385","article-title":"Improved Enzyme Thermal Stability, Loading and Bioavailability Using Alginate Encapsulation","volume":"137","author":"Weng","year":"2023","journal-title":"Food Hydrocoll."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"107063","DOI":"10.1016\/j.fbio.2025.107063","article-title":"Identification of a Novel DON Dehydrogenase and Computational Insights into the Decisive Role of Cofactor Affinity in DON Detoxification","volume":"71","author":"Ouyang","year":"2025","journal-title":"Food Biosci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3857","DOI":"10.1007\/s00204-017-2012-z","article-title":"Glucuronidation of Deoxynivalenol (DON) by Different Animal Species: Identification of Iso-DON Glucuronides and Iso-Deepoxy-DON Glucuronides as Novel DON Metabolites in Pigs, Rats, Mice, and Cows","volume":"91","author":"Hametner","year":"2017","journal-title":"Arch. Toxicol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.bbrc.2023.02.064","article-title":"Improving Thermostability of Bacillus Amyloliquefaciens Alpha-Amylase by Multipoint Mutations","volume":"653","author":"Yuan","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"102161","DOI":"10.1016\/j.biteb.2025.102161","article-title":"Increased Activity under Acidic Conditions and Improved Thermostability of Aspergillus Niger Phytase A","volume":"30","author":"Dotsenko","year":"2025","journal-title":"Bioresour. Technol. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"142646","DOI":"10.1016\/j.ijbiomac.2025.142646","article-title":"Modifying Loop Regions in Lipase from Caldibacillus Thermoamylovorans for Enhancing Thermostability","volume":"309","author":"Pan","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2057","DOI":"10.1080\/10408398.2021.1970508","article-title":"Overview of Strategies for Developing High Thermostability Industrial Enzymes: Discovery, Mechanism, Modification and Challenges","volume":"63","author":"Wu","year":"2023","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Tiwari, A., Krisnawati, D.I., Cheng, T.-M., and Kuo, T.-R. (2024). Precision Thermostability Predictions: Leveraging Machine Learning for Examining Laccases and Their Associated Genes. IJMS, 25.","DOI":"10.3390\/ijms252313035"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"110123","DOI":"10.1016\/j.enzmictec.2022.110123","article-title":"Rational Engineering of a Metalloprotease to Enhance Thermostability and Activity","volume":"162","author":"Zhu","year":"2023","journal-title":"Enzym. Microb. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5141","DOI":"10.1038\/s41467-024-49119-x","article-title":"Simultaneous Enhancement of Multiple Functional Properties Using Evolution-Informed Protein Design","volume":"15","author":"Fram","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.1111\/1751-7915.12874","article-title":"The Enzymatic Detoxification of the Mycotoxin Deoxynivalenol: Identification of DepA from the DON Epimerization Pathway","volume":"11","author":"Carere","year":"2017","journal-title":"Microb. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2489","DOI":"10.1021\/acs.jctc.6b00120","article-title":"Thermostability of Enzymes from Molecular Dynamics Simulations","volume":"12","author":"Zeiske","year":"2016","journal-title":"J. Chem. Theory Comput."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5903","DOI":"10.1080\/10408398.2021.1895056","article-title":"Mechanisms of Deoxynivalenol (DON) Degradation during Different Treatments: A Review","volume":"62","author":"Feizollahi","year":"2021","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.tifs.2022.04.002","article-title":"Elimination of Fusarium Mycotoxin Deoxynivalenol (DON) via Microbial and Enzymatic Strategies: Current Status and Future Perspectives","volume":"124","author":"Tian","year":"2022","journal-title":"Trends Food Sci. Technol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/17\/7773\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T09:55:37Z","timestamp":1788256537000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/17\/7773"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,30]]},"references-count":37,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ijms27177773"],"URL":"https:\/\/doi.org\/10.3390\/ijms27177773","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,30]]}}}