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FDH is widely utilized for cofactor regeneration, enabling the continuous supplying of NADH for chiral chemical biosynthesis. Herein, we employed semi\u2010rational engineering to improve the thermostability of highly catalytic FDH from\n                    <jats:italic>Bacillus simplex<\/jats:italic>\n                    (BsFDH). Through two rounds of mutational screening, BsFDH\n                    <jats:sup>Q125F<\/jats:sup>\n                    variant was identified to exhibit significantly improved thermostability with a 25\u2010fold increase in half\u2010life at 60\u00b0C. Additionally, the BsFDH\n                    <jats:sup>Q125F<\/jats:sup>\n                    variant revealed significant stability against chemo\u2010inactivation. The 100\u2010ns molecular dynamics stimulation demonstrated a reduced overall root\u2010mean\u2010square deviation for the BsFDH\n                    <jats:sup>Q125F<\/jats:sup>\n                    , with enhanced local packing primarily driven by intra\u2010 and inter\u2010subunit \u03c0\u2013\u03c0 interaction among four tandem histidine and phenylalanine residues, thereby restricting the movement of flexible loop130\u2212160. The implementation of the BsFDH\n                    <jats:sup>Q125F<\/jats:sup>\n                    variant for cofactor regeneration in 4\u2010nitrophenol detoxification demonstrated its superior stability and efficiency under bioconversion, rendering it suitable for various biocatalytic industrial applications.\n                  <\/jats:p>","DOI":"10.1002\/cbic.70426","type":"journal-article","created":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T06:46:22Z","timestamp":1781246782000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Semi\u2010Rational Engineering of Formate Dehydrogenase Reveals Loop Dynamics as a Key Determinant of Thermostability"],"prefix":"10.1002","volume":"27","author":[{"given":"Rattima","family":"Boonkumkrong","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology Faculty of Science Mahidol University  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