Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability

The formate dehydrogenase (FDH) from Candida boidinii is a well-known enzyme in biocatalysis for NADH regeneration. Nevertheless, it has low activity in a water-miscible ionic liquid (1,3-dimethylimidazolium dimethyl phosphate, [MMIm]-[Me2PO4]). In this work, this enzyme was subjected to directed evolution by using error-prone PCR, and a mutant (N187S/T321S) displaying higher activity was obtained following selection based on the formazan-based colorimetric assay. The mutation N187S is responsible for improved activity both in aqueous solution and in [MMIm][Me2PO4], through an enhancement of the k(cat) value by a factor of 5.8. Fluorescence experiments performed in the presence of a quenching agent revealed that the mutant does not unfold in the presence of 50% (v/v) [MMIm][Me2PO4] whereas the wild-type enzyme does. Molecular modelling revealed that the mutation is located at the monomer-monomer interface and causes an increase in the pK(a) of residue E163 from 4.8 to 5.5. Calculation of the pK(a) of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pK(a) up to 6.2). We have identified a new site for improving FDH thermostability and tolerance to ionic liquids, and it is linked to the local charge of the enzymes in this class.