Substrate Specificity of α-Amino Acid Ester Hydrolase from Xanthomonas citri

Enzymological properties of the purified α-amino acid ester hydrolase from Xanthomonas citri IFO 3835 were studied. The enzyme catalyzed both hydrolysis of α-amino acid esters (e.g. D-α-phenylglycine methyl ester (D-PG-OMe)) and transfer of the acyl group from the amino acid esters to amine nucleophiles (e.g. 7-aminocephalosporanic acid, 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and 6-aminopenicillanic acid). The substrate specificity of the enzyme in hydrolysis was parallel with that in transfer. The enzyme required a free amino group on the α-carbon of the donor ester, but did not exhibit absolute stereospecificity. α-Amino acid derivatives having an acid-amide bond were hydrolyzed at much lower rates than the corresponding ester derivatives. The optimum pH for both hydrolysis and transfer was 6.4, and the optimum temperature, 35°C. The enzyme was inactivated completely within 15 min at pH 6.0 and 50°C. FeSO4, CuSO4, and HgCl2 inhibited the enzyme. At pH 6.4 and 30&C, the Michaelis constants were estimated to be 8.26mM for D-PG-OMe and 2.99mM for cephalexin. The apparent Michaelis constant for 7-ADCA was estimated to be 5.08 mm, With D-PG-OMe as an acyl donor, the molecular activity (ko) for hydrolysis was calculated to be 1.11×104 sec-1.