Characterization of an industrial biocatalyst: Immobilized glutaryl-7-ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (G A), one of the two enzymes involved in the biotransformation of cephalospor in C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K- m value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optima l at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were o btained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Imm obilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of dif ferent amounts of H2O2, a side product that might be present in the plant-s cale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H2O2 concentration that might be rea ched under operative conditions. Finally, biocatalyst performance in the co mplete two-step enzymatic conversion process from CefC to 7-ACA was determi ned on a laboratory scale. Following the complete conversion of a 75 mM sol ution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this inte rmediate into the final product 7-ACA. This reaction was repeated for 42 cy cles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cyc les, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7- ACA. (C) 2000 John Wiley & Sons, Inc.