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

摘要

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 sterreospecific 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 K278M and E303V 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 deg C compared with those of the wild-type enzyme. The apparent K_m value for D-phenylalaninamide of BFB40 enzyme was almost the same with that of the wild-type enzyme, whereas V_max value was enhanced about three-fold. Almost complete hydrolysis of D-phenylaninamide was ahcieved 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.