Directed evolution of Alcaligenes faecalis nitrilase

Alcaligenes faecalis nitrilase (NITAf) was engineered in a directed evolution approach towards increased activity at its optimal pH as well as improved fitness at low pH values. Error prone PCR in combination with recombination of beneficial mutations resulted in a variant with increased specific activity for 2-phenylpropionitrile at pH 7.5. In addition, a new nitrilase variant (pHNIT45) was developed that is catalytically active at pH values as low as pH 4.5. Within 10 min this mutant fully hydrolyzes the base labile substrate (R)-2-Cl-mandelonitrile (10 mM) to give the product (R)-2-Cl-mandelic acid (conversion 100%, ee > 99%) with full retention of enantiopurity.