Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis combined with site-directed mutagenesis was performed to identify the key residues responsible for nitrilase enantioselectivity. Five enzyme mutants exhibiting distinct selectivity were generated and four "hot spots" (M113, R128, A136, and 1168) responsible for enantioselectivity toward mandelonitrile were identified and characterized. Furthermore, through saturation mutagenesis, positions 113 and 128 were confirmed to substantially influence the enantioselectivity of PpL19, and certain replacements of the methionine at position 113, in particular, were found to reverse the enantioselectivity of PpL19 from S- to R-selectivity. Two other single mutants of the enzyme, PpL19-A136Y and -I168Y, also showed reversed selectivity and preferentially produced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively). By combining the beneficial mutations, two enantiocomplementary nitrilase mutants, PpL19-LH and PpL19-GYY, were created, which exhibited high 5- and R-selectivity toward mandelonitrile, respectively: PpL19-LH showed the highest S-selectivity toward mandelonitrile ever reported (91.1% ee), and, notably, the PpL19-GYY mutant was identified to be highly R-selective (90.1% ee) and thus an unexpected enantiocomplementary mutant for mandelonitrile. (C) 2015 Elsevier Inc. All rights reserved.