An Improved Racemase/Acylase Biotransformation for the Preparation of Enantiomerically Pure Amino Acids

Using directed evolution, a variant N-acetyl amino acid racemase (NAAAR G291D/F323Y) has been developed with up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids. The variant has been coupled with an enantiospecific acylase to give a preparative scale dynamic kinetic resolution which allows 98% conversion of N-acetyl-DL-allylglycine into D-allylglycine in 18 h at high substrate concentrations (50 g L(-1)). This is the first example of NAAAR operating under conditions which would allow it to be successfully used on an industrial scale for the production of enantiomerically pure α-amino acids. X-ray crystal analysis of the improved NAAAR variant allowed a comparison with the wild-type enzyme. We postulate that a network of novel interactions that result from the introduction of the two side chains is the source of improved catalytic performance.