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Keywords:

  • asymmetric synthesis;
  • directed evolution;
  • enzyme catalysis;
  • hydroxynitrile lyase;
  • Pichia pastoris
Thumbnail image of graphical abstract

Random mutagenesis and recombination of PaHNL5 was carried out in Pichia pastoris by employment of an overlap extension PCR-based strategy. New fast and stereoselective enzyme variants were obtained, and these led to improved turnover rates with the nonnatural substrate 2-chlorobenzaldehyde. A new catalyst combining all favorable mutations was generated, and even very small amounts of this variant enabled complete conversion of 2-chlorobenzaldehyde into the (R)-hydroxynitrile with >99 % ee in only 4 h.