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Directed Evolution of a Formate Dehydrogenase for Increased Tolerance to Ionic Liquids Reveals a New Site for Increasing the Stability
Author(s) -
Carter Julie L. L.,
Bekhouche Mourad,
Noiriel Alexandre,
Blum Loïc J.,
Doumèche Bastien
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402501
Subject(s) - ionic liquid , formate dehydrogenase , formate , directed evolution , chemistry , ionic bonding , biochemistry , organic chemistry , catalysis , ion , mutant , gene
Abstract 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][Me 2 PO 4 ]). 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][Me 2 PO 4 ], 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][Me 2 PO 4 ] 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 p K a of residue E163 from 4.8 to 5.5. Calculation of the p K a of this residue in other microbial FDHs showed that thermostable FDHs have a highly basic glutamate at this position (p K 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.