Immobilization of cephalexin semisynthesizing enzyme

Abstract

To immobilize partially purified cephalosporin acylase from Xanthomonas citri IFO 3835, various immobilization methods were tested. From these experiments, adsorption onto bentonite was selected as cephalosporin acylase immobilization method. From optimization experiments of adsorption conditions, 5 mg betonite/ml and 6-7 mg protein/ml of enzyme concentration were selected as optimum conditions of bentonite adsorption. And adsorption was performed at 20℃ by intermittent stirring at intervals of 5 minutes. Enzyme immobilized by the above method was investigated for its characteristics. The optimum pH and temperature of immobilized enzyme reaction were identical with free enzyme as pH 6.5 and 35℃, respectively. But immobilized enzyme was more stable than free enzyme in pH change and at higher temperature. Thus the pH profile of immobilized enzyme was broader than free enzyme, and the half lives of immobilized enzyme and free enzyme at 35℃ were 360 hours and 8 hours, respectively. The substrate inhibition by 7-ADCA was reduced by immobilization Namely, the maximum initial reaction rates of immobilized enzyme and free enzyme were obtained at 7-ADCA concentration of 50 mM and 40 mM, respectively. In enzyme reactor operation, the immobilized enzyme was repeatedly used in a batch type enzyme reactor of stirred tank. By continuous feeding of PGM into enzyme reactor, the conversion ratio was significantly improved when the flow rate of PGM was 1 mM/min at the enzyme loading of 0.5 uit/ml.