A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis

Chembiochem. 2021 Mar 16;22(6):996-1000. doi: 10.1002/cbic.202000658. Epub 2021 Jan 12.


Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.

Keywords: amino-benzamidoxime; cyanohydrins; directed evolution; high-throughput screening; hydrocyanation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / metabolism*
  • Aldehydes / chemistry
  • Aldehydes / metabolism
  • Biocatalysis
  • Directed Molecular Evolution
  • Escherichia coli / enzymology
  • High-Throughput Screening Assays / methods*
  • Nitriles / chemistry
  • Nitriles / metabolism*
  • Stereoisomerism
  • Substrate Specificity


  • Aldehydes
  • Nitriles
  • cyanohydrin
  • Aldehyde-Lyases
  • mandelonitrile lyase