By utilizing whole cell enzyme of the $\mbox{\underline{Xanthomonas}}$ $\mbox{\underline{citri}}$ IFO 3835, cephalexin is synthesized directly from 7-aminodeacetoxycephalosporanic acid (7-ADCA) and phenylglycine methylester (PGM). To date, cephalexin has been menufactured by chemical process involving fairly large number of steps to protect the amino group of phenylglycine and carboxyl group of 7-ADCA. However, the enzymatic process involves only a single step with 95% conversion within 2hr.
The studies on fermentation variables indicate that oxygen transfer is the limiting step both in the microbial growth and enzyme production. Penicillin acylase inducer failed to induce the enzyme production, and this enzyme is not considered as the penicillin acylase. Optimum conditions for the cephalexin synthesis was elucidated; 37℃, pH6.0, and the optimum mole ratio of PGM to 7-ADCA was 3.
8.63 Kcal per mole of activation energy, 4.0 mM of Km for 7-ADCA, and 12.0 mM of Km for PGM were estimated from experimental results. At a high concentration of substrates, 7-ADCA activates the synthesis but PGM inhibits; the inhibition constant of PGM, Ki, was 70 mM.
From the evaluation of enzyme kinetics, the mode of enzyme action appears to be the partial, rapid equilibrium ordered bi-bi reaction in which an activator, 7-ADCA, and a substrate, PGM, combine with the enzyme in an obligate order; PGM can add only to the enzyme 7-ADCA complex.
The particular decay pattern of whole cell enzyme shows the temporary increase of original activity up to 200% followed by rapid decrease. Enzyme loading effect was also evaluated. The more enzyme loading was introduced, the greater maximum velocity was observed. But the maximum conversion ratio was remarkably decreased, about two thirds of the maximum conversion.