Enhancement of the thermostability and catalytic activity of d-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution

d-Amino-acid amidases, which catalyze the stereospecific hydrolysis of d-amino-acid amide to yield d-amino acid and ammonia, have attracted increasing attention as catalysts for stereospecific production of d-amino acids. We screened for the enzyme variants with improved thermostability generated by a directed evolution method with the goal of the application of evolved enzyme to the production of d-amino acids. Random mutagenesis by error-prone PCR and a filter-based screening was repeated twice, and as a result the most thermostable mutant BFB40 was obtained. Gene analysis of the BFB40 mutant indicated that the mutant enzyme had K278 M and E303 V mutations. To compare the enzyme characteristics with the wild-type enzyme, the mutant enzyme, BFB40, was purified from the Escherichia coli (E. coli) transformant. Both the thermostability and apparent optimum temperature of the BFB40 were shifted upward by 5 °C compared with those of the wild-type enzyme. The apparent Km value for d-phenylalaninamide of BFB40 enzyme was almost the same with that of the wild-type enzyme, whereas Vmax value was enhanced about three-fold. Almost complete hydrolysis of d-phenylalaninamide was achieved in 2 h from 1.0 M of racemic phenylalaninamide–HCl using the cells of E. coli transformant expressing BFB40 enzyme, the conversion of which was 1.7-fold higher than the case using cells expressing wild-type enzyme after the same reaction time.