Purification and characterization of alpha-amino acid ester hydrolase from Xanthomonas rubrillineans
红纹黄单胞菌α-氨基酸酯水解酶的分离纯化及酶学性质

Objective] The intracellular α-amino acid ester hydrolase(AEH) from Xanthomonas rubrillineans was purified and characterized.Methods] AEH was extracted by butyl acetate,and then purified to electrophoretic homogeneity by calcium phosphate gel precipitation,ammonium sulfate fraction precipitation,anion exchange with DEAE Sephadex A-50,cation exchange with CM cellulose 52,and Sephadex G 200 column chromatography.Results] The subunit molecular mass of AEH was 70 kDa by SDS-PAGE.The optimal reaction pH for cefaclor synthesis was 6.8,and optimal temperature was 42 ℃.The enzyme was stable between pH 5.0 and 8.0,and at 35 ℃.The enzyme activity was enhanced by Mn2+ and Ca2+,however was strongly inhabited by Cu2+,Fe2+ and high concentration of acetone.The kinetic parameters that the enzyme hydrolyzed D-Phenylglycine methyl ester,D-Hydroxyphenylglycine methyl ester and cefaclor were determined,and the values of kcat/Km were 123.7±3.7,2.9±0.6 and 101.3±2.1 mmol-1·s-1·L respectively.The kcat/Km values indicated that the enzyme hydrolyzed D-Phenylglycine methyl ester more efficiently than other substrates.The mechanism of enzymatic reaction with bi-substrates by AEH is Ping-Pong kinetics,and the kcat value that the enzyme catalyzed the synthesis of cefaclor is 547.3±38.2 s-1.Conclusion] The studies of AEH from Xanthomonas rubrillineans were rare,and our research may provide an important basis for industrial application of AEH for beta-lactam antibiotics synthesis.