Determination of the intrinsic kinetic constants of immobilized glucose oxidase and catalase

Biotechnol Bioeng. 1987 Apr;29(6):696-704. doi: 10.1002/bit.260290606.

Abstract

Models of membrane systems containing immobilized glucose oxidase and catalase operating together or independently have been developed. A rotated disk electrode apparatus was employed with novel electrochemical operating conditions to experimentally determine mass transfer and intrinsic kinetic parameters of enzyme-containing membranes. The value of a mass transfer parameter that describes internal and external diffusion was first determined under conditions that do not permit the enzyme reactions. In a subsequent experiment with the reaction allowed, kinetic parameters corresponding to the intrinsic maximal velocity and Michaelis constants of the immobilized enzymes were estimated by regression analysis of data based on an appropriate two- or three- parameter model. It was found that immobilization reduced the maximal intrinsic velocity but had no detectable effect on the Michaelis constants. In all but one case- these methods for membrane characterization are nondestructive and can be used repeatedly on a given membrane. These techniques provide the means for quantitative comparisons of immobilization methods and make possible temporal studies of immobilized enzyme inactivation.