Improving Activity and Stability of <i>Candida boidinii</i> Formate Dehydrogenase Through Rational Active Site Engineering

Formate dehydrogenases represent a highly attractive tool for nicotinamide cofactor regeneration to drive biocatalytic reductions and oxygenations by NADH-dependent oxidoreductases with formate as a cheap, atom-economic terminal reductant. However, despite the clear benefits over other recycling systems, native FDHs remain rather unpopular, mainly due to their intrinsic, low specific activity. While the active site architecture is generally highly conserved across many FDHs from very diverse origins, smaller patterns of dissonance in the active site's vicinity can serve as hotspots for the rational design of more effective variants. In this study, we have created a series of Candida boidinii FDH variants incorporating nonconsensus amino acids found in Saccharomyces cerevisiae FDH. A double variant (C23S/F285D) was identified that exhibits a significantly higher specific activity, an overall more desirable pH and temperature profile, and an improved stability profile.