Characteristics of GL-7-ACA acylase immobilized on silica gel through silanization

Abstract

Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme which converts GL-7-ACA to 7-aminocephalosporanic acid (7-ACA), a starting material for semisynthetic cephalosporin antibiotics. In this study, GL-7-ACA acylase was immobilized on silica gel modified with 3-aminopropyltriethoxysilane, followed by glutaraldehyde for the production of 7-ACA. The Michaelis–Menten kinetic parameters, K m and V max, for free GL-7-ACA acylase were estimated to be 9.9 mM and 171.36 μg/min, and K m and V max for GL-7-ACA acylase immobilized on silica gel were estimated to be 15.4 mM and 114.94 μg/min, respectively. The pH values for optimum activity of the free and immobilized GL-7-ACA acylase were almost the same. However, the pH-dependent activity profile for the immobilized GL-7-ACA acylase is considerably expanded. The stability of the immobilized GL-7-ACA acylase was significantly improved over its free form at lower pH values. The effect of temperature was also investigated. Both the free and immobilized enzymes have the highest activity at 50 °C. The temperature dependence of the reaction rate obeyed the Arrhenius law. The activation energies ( E a) of the reactions catalyzed by the free and immobilized GL-7-ACA acylase were calculated to be 7.2 and 5.0 kcal/mol, respectively. In order to investigate the thermal stability of the immobilized GL-7-ACA acylase, experiments were carried out at various temperatures (20–60 °C). The thermal inactivation constant rate ( k i) values of the immobilized GL-7-ACA acylase at all temperatures tested in this study were lower than those of the free GL-7-ACA acylase, and the half-lives for inactivation of the immobilized GL-7-ACA acylase at all temperatures tested were higher than those of the free GL-7-ACA acylase. Consequently, the thermal stability of the immobilized GL-7-ACA acylase was increased significantly compared to that of the free GL-7-ACA acylase.