Improving the Properties of Bacterial R-Selective Hydroxynitrile Lyases for Industrial Applications

• Published in: ChemCatChem 2015-01, Vol.7 (2), p.325-332
• Main Authors: Wiedner, Romana, Kothbauer, Bettina, Pavkov-Keller, Tea, Gruber-Khadjawi, Mandana, Gruber, Karl, Schwab, Helmut, Steiner, Kerstin
• Format: Article
• Language: English
• Subjects: Bacteriology; biocatalysis; cupins; cyanohydrin syntheses; lyases; protein engineering
• ISSN: 1867-3880; 1867-3899
• DOI: 10.1002/cctc.201402742

Hydroxynitrile lyases (HNLs) catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. The recent discovery of bacterial HNLs with a cupin fold gave rise to a new promising class of these enzymes. They are interesting candidates for the synthesis of cyanohydrins on an industrial scale owing to their high expression levels in Escherichia coli. The activity and enantioselectivity of the manganese‐dependent HNL from Granulicella tundricola (GtHNL) were significantly improved by site‐saturation mutagenesis of active site amino acids. The combination of beneficial mutations resulted in a variant with 490‐fold higher specific activity in comparison to the wild‐type enzyme. More importantly, GtHNL‐A40H/V42T/Q110H is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals. New lyase on the rise: A variant of the bacterial manganese‐dependent hydroxynitrile lyase from Granulicella tundricola is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.