Screening for enantioselective nitrilases:: kinetic resolution of racemic mandelonitrile to (R)-(-)-mandelic acid by new bacterial isolates

Several new microorganisms have been isolated with high nitrilase activity against (RS)-mandelonitrile using the enrichment culture technique. The organisms were cultivated in liquid culture and the enzyme activity was determined at different phases of growth. The organisms having high enzyme titre were further grown and used as catalysts for the transformation of mandelonitrile to mandelic acid. The percentage conversion was checked with RP-HPLC and the enantiomeric excess was determined on a chiral column. Three isolates gave the desired product, (R)-(-)-mandelic acid with high ee (%) and were identified as Pseudomonas putida, Microbacterium paraoxydans and Microbacterium liquefaciens. All three isolates showed good specific activity (0.33-0.50 U/mg min) with high ee (>93%) and E values. The conversion of racemic mandelonitrile to mandelic acid by these isolates was compared: P. putida was found to be the most suitable biocatalyst for further studies as it showed higher reaction rate (k(Rxn)), lower K-m, better growth rate (mu), good yield and ee values and higher stability compared to the other two microorganisms. (C) 2003 Elsevier Ltd. All rights reserved.