Abstract:Formate dehydrogenase(FDH) is one of the most effective enzymes for NADH regeneration and is extensively utilized in the food, pharmaceutical and chemical industries. However, wild-type FDH often suffers from low enzyme activity and poor catalytic efficiency, resulting in lower product conversion and hindering industrial production. In order to obtain FDH with improved catalytic performance, FDH from Candida boidinii was used as a template and HOTSPOT WIZARD v3.1 was employed for three-dimensional structure simulation and prediction. And 2 mutants, P68G and Q197K, were constructed. In comparison to the wild type, they exhibited 11% and 33% higher enzyme activity, respectively. The improved activity was attributed to the replacement of the phenyl ring on the side chain of residue P68G by hydrogen, reducing steric hindrance for formate substrate entry, while the mutation of the side chain amide group in Q197K to aminobutyl enhanced the flexibility of the enzyme. However, these 2 mutations negatively affected the thermal stability of FDH. Hence, a cysteine mutation was introduced at position I239 to form a disulfide bond with C262, thereby enhancing the thermal stability. Ultimately, the mutant strain CbFDH Q197K/I239C was obtained, which exhibited remarkable improvement in thermal stability, displaying a 31% increase in enzyme activity compared to the wild-type and a 45% increase compared to I239C. This study demonstrates that the activity and thermal stability of FDH could be enhanced through semi-rational protein structure prediction, providing a reliable theoretical foundation for the efficient construction of FDH with stable performance and strong reducing power.
