Improvement of stability of immobilized GL-7-ACA acylase through modification with glutaraldehyde

Abstract

Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-ACA (7-aminocephalosporanic acid), a starting material for semisynthetic cephalosporin antibiotics. Alleviation of the adverse effect of glutaraldehyde is very important for the enhancement of activity and activity recovery of the immobilized GL-7-ACA acylase. Optimal conditions for the optimization of glutaraldehyde were deduced using statistical analysis. Analysis of variance (ANOVA) was introduced for the determination of significant factors that affect the modification of glutaraldehyde. ANOVA results suggest the most important factors are the interaction between heating temperature and heating time, and the interaction between pH and glutaraldehyde concentration. Both factors are significant for activity and activity recovery at a 1% level of significance. In order to determine the optimal conditions for significant factors tested by ANOVA, response surface methodology (RSM) was employed. Through RSM analysis, it was found that the optimal conditions for pH, heating temperature, heating time and concentrations of glutaraldehyde for the modification of glutaraldehyde were found to be: pH 8.0, 62 °C, 25 min and 2% concentration of glutaraldehyde, respectively. The values of activity and activity recovery at the optimal points were 18.72 U/g-matrix and 36.52%, respectively. These values for activity and activity recovery were increased up to 2.0- and 2.2-fold, respectively, when compared to those obtained using untreated glutaraldehyde. These increased values were obtained by improvement of immobilization techniques through the modification of glutaraldehyde without the modification of enzyme. As a consequence, this method would be considerably useful in the reuse of immobilized GL-7-ACA acylase for the enhancement of productivity of 7-ACA. More superior effects might be obtained if reuse of immobilized GL-7-ACA acylase should be introduced in scale-up for the mass production of 7-ACA.