Genomic Data Mining Of Nitrilase And Its Application In Production Of(R)-o-Chloromandelic Acid

(R)-2-Chloromandelic acid is the key precursor for the synthesis of the platelet aggregation inhibitor Clopidogrel. Because of the racemization of2-chloromandelonitrile, the reaction carries out a dynamic kinetic resolution which provides theorethically100%of the product, and the starting material is very cheap. The hydrolysis of2-chloromandelonitrile by nitrilases becomes one of the promising approaches to produce (R)-2-chloromandelic acid.Because there was no nitrilase which could fulfill both the requirements for good eep and high substrate loading, a new nitrilase from Labrenzia aggregata was found from NCBI database based on data mining, which could hydrolyze2-chloromandelonitrile with high enantioselectivity, yielding (R)-2-chloromandelic acid with96.5%ee. The LaN was overexpressed in Escherichia coli BL21(DE3), purified and its catalytic properties were studied. The optimum temperature and pH of LaN was50℃and8.0, respectively. Besides, the enzyme was stable at30℃, pH7.0and8.0. A study of substrate spectrum showed that the enzyme exhibited the highest activity with phenylacetonitrile, and preferred to hydrolyze arylacetonitriles, but it showed no activity on aromatic nitriles and slight activities on aliphatic nitriles. When2-chloromandelonitrile was used as the substrate, the Km was0.39mM, Vmax was2.56μmol·min-1·mg-1, and kcat=1.60s-1.In order to relieve the substrate inhibition and improve the productivity of LaN, a fed-batch strategy and water-toluene system were used. In the water-toluene system,2-chloromandelonitrile of300mM (apparent concentration, based on total volume) could be transformed by LaN in8h, with an isolated yield of94.5%and96%ee. Afer recrystallization in toluene, the optical purity of the product was further improved to99%.