Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: Crystal structures and phage display selection

  • Dröge M
  • Boersma Y
  • Van Pouderoyen G
 et al. 
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Abstract

Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.

Author-supplied keywords

  • Bacillus subtilis
  • Crystal structure
  • Directed evolution
  • Enantiomeric phosphonate inhibitors
  • Lipases
  • Phage display

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Authors

  • Melloney J. Dröge

  • Ykelien L. Boersma

  • Gertie Van Pouderoyen

  • Titia E. Vrenken

  • Carsten J. Rüggeberg

  • Manfred T. Reetz

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