Objective: The intracellular alpha-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 degrees C. The enzyme was stable between pH 5.0 and 8.0, and at 35 degrees C. 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 k(cat)/K(m) were 123.7 +/-3.7, 2.9 +/- 0.6 and 101.3 +/- 2.1 mmol(-1) x s(-1) x L respectively. The k(cat)/K(m) 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 k(cat) 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.