Efficient CO₂-Reducing Activity of NAD-Dependent Formate Dehydrogenase from <i>Thiobacillus</i> sp. KNK65MA for Formate Production from CO₂ Gas

<div><p>NAD-dependent formate dehydrogenase (FDH) from <i>Candida boidinii</i> (CbFDH) has been widely used in various CO₂-reduction systems but its practical applications are often impeded due to low CO₂-reducing activity. In this study, we demonstrated superior CO₂-reducing properties of FDH from <i>Thiobacillus</i> sp. KNK65MA (TsFDH) for production of formate from CO₂ gas. To discover more efficient CO₂-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO₂-reducing activities were evaluated. All FDHs including CbFDH showed better CO₂-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO₂ reduction reaction. In particular, the FDH from <i>Thiobacillus</i> sp. KNK65MA (TsFDH) exhibited the highest CO₂-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO₂ reduction to formate oxidation in catalytic efficiency (<i>k</i>_cat/<i>K</i>_B) compared to CbFDH. Formate was produced from CO₂ gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO₂-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO₂ reduction systems.</p>