Design of Nitrilases with Superior Activity and Enantioselectivity towards Sterically Hindered Nitrile by Protein Engineering

Abstract The enantioselective hydrolysis of ortho ‐chloromandelonitrile with nitrilase is one of the most attractive approaches to prepare ( R )‐ ortho ‐chloromandelic acid. To date, efforts to develop this nitrilase‐mediated process were plagued by either insufficient ee p (enantiomeric excess of product) or low activity due to the steric hindrance from the ortho ‐substituted substrate. To improve the nitrilase potential for producing ( R )‐ ortho ‐chloromandelic acid, an enhancement of both activity and enantioselectivity towards sterically hindered nitriles would be highly desirable. Molecular docking of the ( R )‐ ortho ‐chloromandelonitrile into the active site of wild‐type 2A6 nitrilase (nitA) allowed the identification of proximal nitA active site residues. Several residues (52, 132, 189 and 190) were selected as targets for single and double point mutation to improve nitA activity and enantioselectivity towards ortho ‐chloromandelonitrile. Targeted mutagenesis yielded several nitA variants with superior activity and enantioselectivity. The best mutant T132A/F189T exhibited a 4.37‐fold higher specific activity (7.39 U/mg) towards ortho ‐chloromandelonitrile than the wild‐type nitA. More importantly, the enantioselectivity ( E ) was improved from 17.34 to >200, resulting in a highly enantiopure product. Molecular docking experiments further support the enhanced activity and enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of nitA are provided. The amino acids at sites 189 and 132 determine the activity and enantioselectivity towards ortho ‐chloromandelonitrile. With mutant T132A/F189T as a catalyst, a maximum of 450 mM of ( R )‐ ortho ‐chloromandelic acid was produced with a 90% conversion and >99% ee p within 3 h. This is the first time that a high productivity of ( R )‐ ortho ‐chloromandelic acid of up to 671.76 g L −1 d −1 using a nitrilase‐mediated approach is reported. The engineered T132A/F189T variant represents a promising and competitive biocatalyst for practical application in synthesizing ( R )‐ ortho ‐chloromandelic acid. magnified image