Catalytic activity of immobilized fumarase

In order to evaluate the influence of the immobilization techniques of fuma rase on its immobilization yield and on its catalytic activity, it was cova lently bonded to the surface of polymers (consisting of suitably functional ized 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 we re obtained by determining the UV absorbance variation of the fumarate doub le bond at 290 nm. When the enzyme is covalently bonded, both activity and stability of the en zymatic preparations are low; however, when fumarase is bonded to ethylene- vinyl alcohol copolymers by less denaturating and more spacing coupling age nts (as glutaraldehyde), a better residual enzyme activity was obtained, an d it was seen that this latter depended on the amount of bonded enzyme. Als o the influence of the hydrophilicity of the polymer matrix on the amount o f bonded enzyme and on its activity was evaluated. Satisfactory results were obtained by physical entrapment of the enzyme int o poly(acrylamide) gel, with quantitative immobilization yields, a rather g ood enzyme activity (eta = 38 +/- 5%), and a constant catalytic activity, u nder operative conditions, for several days. The inhibiting effect of methanol concentrations up to 20% (v/v), in the re action medium, was also evaluated for the different immobilized enzyme prep arations. (C) 2001 Elsevier Science B.V. All rights reserved.