Thermostabilization of an esterase by alignment-guided focussed directed evolution

Protein Eng Des Sel. 2010 Dec;23(12):903-9. doi: 10.1093/protein/gzq071. Epub 2010 Oct 14.


Site-saturation libraries of the Pseudomonas fluorescens esterase were created targeting three surface positions to increase its thermostability on the basis of the B-factor iterative test principle. All three positions were saturated simultaneously using our recently developed protocol for the design of 'small, but smart' mutant libraries bearing only consensus-like mutations. Hence, the library size could be significantly reduced while ensuring a high hit rate. Variants could be identified that showed significantly improved stability (8° C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 9° C increased melting point, but unchanged catalytic properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Directed Molecular Evolution*
  • Enzyme Stability / genetics
  • Esterases / chemistry*
  • Esterases / genetics
  • Esterases / metabolism
  • Gene Library
  • Models, Molecular
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Protein Engineering
  • Pseudomonas fluorescens / enzymology
  • Sequence Alignment
  • Sequence Analysis, Protein
  • Temperature


  • Bacterial Proteins
  • Esterases