Formate dehydrogenase (FDH) catalyzes the reversible formate oxidation using NAD + to yield carbon dioxide and NADH. FDH is widely utilized for cofactor regeneration, enabling the continuous supplying of NADH for chiral chemical biosynthesis. Herein, we employed semi‐rational engineering to improve the thermostability of highly catalytic FDH from Bacillus simplex (BsFDH). Through two rounds of mutational screening, BsFDH Q125F variant was identified to exhibit significantly improved thermostability with a 25‐fold increase in half‐life at 60°C. Additionally, the BsFDH Q125F variant revealed significant stability against chemo‐inactivation. The 100‐ns molecular dynamics stimulation demonstrated a reduced overall root‐mean‐square deviation for the BsFDH Q125F , with enhanced local packing primarily driven by intra‐ and inter‐subunit π–π interaction among four tandem histidine and phenylalanine residues, thereby restricting the movement of flexible loop130−160. The implementation of the BsFDH Q125F variant for cofactor regeneration in 4‐nitrophenol detoxification demonstrated its superior stability and efficiency under bioconversion, rendering it suitable for various biocatalytic industrial applications.
Boonkumkrong et al. (Thu,) studied this question.