Rational Design\nof Formate Dehydrogenase for Enhanced\nThermal Stability and Catalytic Activity in Bioelectrocatalysis

Formate dehydrogenase can be utilized as a biocatalyst\nin the bioelectrocatalysis\nof converting CO<sub>2</sub> into formic acid. However, its industrial\napplication has been hindered by limited thermal stability. This study\nsuccessfully obtained a mutant (D533S/E684I) with enhanced thermal\nstability and catalytic activity through the rational design of flexible\nregions. The mutant exhibited a half-life (<i>t</i><sub>1/2</sub>) 1.5 times longer than the wild type (WT) at 35 °C,\nalong with a specific enzyme activity 7.46 times higher than that\nof the WT. Additionally, the catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value) of the mutant\ntoward the substrate was 2.72 s<sup>–1</sup>·mM<sup>–1</sup>, representing a 19.4-fold increase compared to the WT (0.14 s<sup>–1</sup>·mM<sup>–1</sup>). Formic acid production\nreached 53.4 mM through bioelectrocatalysis after 10 h, utilizing\nthe mutant as the biocatalyst. Molecular dynamics simulations and\nstructural analysis were employed to investigate the molecular mechanisms\nbehind the enhanced thermal stability and activity. The displacement\nof a highly flexible region in the mutant may counteract the stability-activity\ntrade-off. This study proposed a method for improving both thermal\nstability and activity in enzyme evolution.