Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution

Chembiochem. 2011 Jul 4;12(10):1529-35. doi: 10.1002/cbic.201100078. Epub 2011 May 12.


We describe here a strategy to improve the expression efficiency and enantioselectivity of Aspergillus niger epoxide hydrolase (ANEH) by directed evolution. Based on a blue-colony screening system using the LacZα (β-galactosidase α peptide) complementation solubility reporter, several ANEH variants out of 15 000 transformants from a random-mutagenesis library were identified that show improved recombinant expression in E. coli. Among them, Pro221Ser was subsequently used as a template for iterative saturation mutagenesis (ISM) at sites around the ANEH binding pocket. Following four rounds of ISM, a highly enantioselective mutant was identified that catalyzes the hydrolytic kinetic resolution of racemic glycidyl phenyl ether with a selectivity factor of E=160 in favor of the (S)-diol compared to WT ANEH characterized by E=4.6. Expression of this mutant is 50 times higher than that of WT ANEH. It also serves as an excellent stereoselective catalyst in the hydrolytic kinetic resolution and desymmetrization of several other structurally diverse epoxides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / enzymology*
  • Aspergillus niger / genetics
  • Directed Molecular Evolution / methods*
  • Epoxide Hydrolases / genetics*
  • Epoxide Hydrolases / isolation & purification
  • Epoxide Hydrolases / metabolism
  • Escherichia coli / genetics
  • Gene Expression
  • Models, Molecular
  • Mutation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Stereoisomerism
  • Substrate Specificity
  • beta-Galactosidase / metabolism


  • Recombinant Proteins
  • beta-Galactosidase
  • Epoxide Hydrolases