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

Chembiochem. 2014 Dec 15;15(18):2710-8. doi: 10.1002/cbic.201402501. Epub 2014 Oct 24.


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][Me2 PO4 ]). 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][Me2 PO4 ], through an enhancement of the kcat 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][Me2 PO4 ] 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 pKa of residue E163 from 4.8 to 5.5. Calculation of the pKa of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (pKa 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.

Keywords: biocatalysis; directed evolution; ionic liquids; pKa; protein stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Candida / chemistry
  • Candida / enzymology*
  • Candida / metabolism
  • Directed Molecular Evolution*
  • Enzyme Stability
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / genetics*
  • Formate Dehydrogenases / metabolism*
  • Imidazoles / chemistry*
  • Imidazoles / metabolism
  • Ionic Liquids / chemistry*
  • Ionic Liquids / metabolism
  • Models, Molecular
  • Mutation
  • Protein Unfolding


  • 1,3-dimethylimidazolium
  • Imidazoles
  • Ionic Liquids
  • Formate Dehydrogenases