Synthesis of β-lactam antibiotics containing α-aminophenylacetyl group in the acyl moiety catalyzed by d-(—)-Phenylglycyl-β-lactamide amidohydrolase

d-(—)-Phenylglycyl-β-lactamide amidohydrolase was isolated from Xanthomonas sp., purified, and characterized. A characteristic feature of the enzyme is its high specificity for substrates containing an α-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-β-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 ΔG°' pH7.0 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 β-lactam antibiotics. Detailed thermodynamic and kinetic studies of the synthesis of cephalexin from MEPG and 7-aminodeacetoxycephalosporanic acid (7-ADCA) catalyzed by d-(—)-phenylglycyl-β-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 m m solution of 7-ADCA and an 88.5 m m 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-β-lactamide amidohydrolase.