A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.
Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.
Krammer
B
B
Research Centre Applied Biocatalysis, Graz, Austria.
Rumbold
K
K
Tschemmernegg
M
M
Pöchlauer
P
P
Schwab
H
H
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D002999:Clone Cells; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli; D028482:Hevea / Q000201:enzymology*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020539:Sequence Analysis, Protein
D050505:Mutant Proteins; D009570:Nitriles; D000446:Aldehyde-Lyases