Kinetic analysis of enzymatic sythesis of cephalexin

Abstract

Using Acetobacter turbidans enzyme, enzymatic synthesis of cephalexin from D-$\alpha$-phenylglycine methylester(PGM) and 7-animo-3-deacetoxycephalosporanic acid(7-ADCA) was attempted. This enzyme was partially purified through ammonium sulfate fractionation, DEAE, CM, and S-200 gel filtration. This enzyme showed its maximal synthetic activity at pH 6.0 and 40$^\circ$C. An unique kinetic pattern that the maximal conversion achieved was greatly affected by molar ratio of two substrates and the amount of enzyme loaded, was observed. In order to elucidate this phenomenon, the reaction mixture of all steps involved in enzymatic synthesis of cephalexin was analyzed by TLC and HPLC techniques. From this result, it was confirmed that this enzyme could catalyze simultaneously three reactions, which are the hydrolysis of PGM, transfer of phenylglycyl group to 7-ADCA to form cephalexin, and hydrolysis of cephalexin synthesized. It was also revealed that the hydrolysis of PGM and cephalexin, and synthetic reaction of cephalexin were not fully reversible. The optimal pH for hydrolysis was slightly higher than the other two reactions and the hydrolysis rate of PGM was decreased greatly by increasing 7-ADCA concentration. Considering all reaction steps, it was proposed that reaction mechanism of this enzyme was nearly identical with that of X. citri. From this proposed reaction mechanism, the rate equations derived in case of X. citri were used to evaluated the kinetic parameters. The unwanted product, phenylglycin, was found to act as a competitive inhibitor in the hydrolyzing reactions of PGM and cephalexin, and as a non-competitive inhibitor in the synthetic reaction. Using the rate equations and kinetic parameters determined, the reaction progress was simulated by computer and it was found that simulated result fitted well the experimental one.