Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block

Chembiochem. 2006 May;7(5):805-9. doi: 10.1002/cbic.200500546.

Abstract

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry*
  • Alkynes / pharmacology
  • Binding Sites / genetics
  • Butanols / chemistry*
  • Butanols / pharmacology
  • Directed Molecular Evolution
  • Esterases / chemistry*
  • Esterases / drug effects
  • Esterases / genetics
  • Kinetics
  • Models, Molecular
  • Molecular Conformation
  • Mutation
  • Protein Conformation
  • Protein Structure, Tertiary
  • Pseudomonas fluorescens / enzymology*
  • Pseudomonas fluorescens / genetics
  • Stereoisomerism
  • Structure-Activity Relationship
  • Substrate Specificity / genetics
  • Time Factors

Substances

  • Alkynes
  • Butanols
  • Esterases