SYNTHESIS OF BETA-LACTAM ANTIBIOTICS CONTAINING ALPHA-AMINOPHENYLACETYL GROUP IN THE ACYL MOIETY CATALYZED BY D-(-)-PHENYLGLYCYL-BETA-LACTAMIDE AMIDOHYDROLASE

D-(-)-Phenylglycyl-beta-lactamide amidohydrolase was isolated from Xanthomonas sp., purified, and characterized. A characteristic feature of the enzyme is its high specificity for substrates containing an alpha-aminophenylacetic group in the acyl moiety. Cephalexin and D-C-(-)-phenylglycine methyl ester (MEPG), being nonspecific penicillin acylase (EC 3.5.1.11) substrates, have the highest values of bimolecular constant k(cat)/K(m) (2.8 . 10(5) and 2.0 . 10(5) M-1 . s-1, respectively) in the case of amidohydrolase. On the contrary, benzylpenicillin is not hydrolyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase. The other peculiarity of the enzyme is its affinity for the charged forms of substrates. Using the amidohydrolase, it was found that the values of DELTAG(pH7.0)-degrees' for hydrolysis of the amide bond in cephalexin and ampicillin are -3.3 and -2.3 kJ mol-1, respectively. They are less by a minimum of 2.7 kJ mol-1 than those for other beta-lactam antibiotics. Detailed thermodynamic and kinetic studies of the synthesis of cephalexin from MEPG and 7-aminodeacetoxycephalosporanic acid (7-ADCA) catalyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase were undertaken. A kinetic scheme is proposed which describes well the experimental curves. The value of conversion of ''nucleus'' was found to be 76% when the synthesis was carried out from a 31.5 mm solution of 7-ADCA and an 88.5 mm solution of MEPG at pH 6.2 (optimum conditions). A 75% conversion of 7-aminocephalosporanic acid (7-ACA) was achieved in the synthesis of cephaloglycine catalyzed by D-(-)-phenylglycyl-beta-lactamide amidohydrolase.