CATALITIC ACTIVITY OF IMMOBILIZED FUMARASE

In order to evaluate the influence of the immobilization techniques of fumarase on its immobilization yield and on its catalytic activity, it was covalently bonded to the surface of polymers (consisting of suitably functionalized ethylene–vinyl alcohol copolymers and functionalized poly(acrylamides), and physically entrapped into cross-linked poly(acrylamide) gels. The kinetic parameters of the hydration reaction of fumarate to l-malate were obtained by determining the UV absorbance variation of the fumarate double bond at 290 nm. When the enzyme is covalently bonded, both activity and stability of the enzymatic preparations are low; however, when fumarase is bonded to ethylene–vinyl alcohol copolymers by less denaturating and more spacing coupling agents (as glutaraldehyde), a better residual enzyme activity was obtained, and it was seen that this latter depended on the amount of bonded enzyme. Also the influence of the hydrophilicity of the polymer matrix on the amount of bonded enzyme and on its activity was evaluated. Satisfactory results were obtained by physical entrapment of the enzyme into poly(acrylamide) gel, with quantitative immobilization yields, a rather good enzyme activity (η = 38 ± 5%), and a constant catalytic activity, under operative conditions, for several days. The inhibiting effect of methanol concentrations up to 20% (v/v), in the reaction medium, was also evaluated for the different immobilized enzyme preparations