Engineering Nitrilase with Improved Enantioselectivity for Specific Hydrolysis and Hydration Reactions

Nitrilases are important biocatalysts that convert nitriles to carboxylic acids and ammonia, and recent studies have revealed their intrinsic hydration activity for the generation of amides. Notably, enantioselectivity is a pivotal prerequisite for their extensive application. Through the dual regulation of reaction specificity and enantioselectivity, novel enzymes with enantioselective hydrolysis/hydration specificity are expected to be achieved for the production of high-purity chiral carboxylic acids or amides. In this study, protein engineering was performed on the nitrilase from<i>Oryza sativa</i>(<i>Os</i>NIT), generating mutants with enhanced enantioselectivity for hydration and hydrolysis reactions. Using racemic mandelonitrile as the substrate, the mutant with enhanced enantioselective hydration efficiently produced (<i>R</i>)-mandelamide with the enantiomeric excess (<i>e</i>.<i>e</i>.) increased from 7.50% to 91.40%, while the enantioselective hydrolysis-improved mutant efficiently synthesized (<i>R</i>)-mandelamide with enantiomeric excess (<i>e</i>.<i>e</i>.) increased from 7.50% to 91.40%, while the enantioselective hydrolysis-improved mutant efficiently synthesized (<i>R</i>)-mandelic acid with <i>e</i>.<i>e</i>. increased from 46.70% to 96.45%. In-depth structural-functional analysis provided theoretical support for the alteration of catalytic performance and offered a valuable strategy for the regulation of multiple catalytic properties of nitrilases.