SITE-DIRECTED AND RANDOM ENZYME IMMOBILIZATION ON FUNCTIONALIZED MEMBRANES: KINETIC STUDIES AND MODELS

(J. Membrane Sci., IN PRESS (1995))

S. Vishwanath and D. Bhattacharyya1, Department of Chemical and Materials Engineering, W. Huang and L. G. Bachas, Department of Chemistry; University of Kentucky, Lexington, KY 40506-0046

Abstract

A comparison of Michaelis-Menten kinetic parameters, Km and Vmax has been made between a randomly immobilized and a site-specifically immobilized -galactosidase on macroporous membranes. A biotinylated -galactosidase conjugate (SDBG), was prepared by posttranslational modification of a recombinant fusion protein in E. coli. This conjugate had biotin attached at a specific location on a polypeptide tag fused to the N-terminus of -galactosidase. Avidin, which has a very strong interaction with biotin, was immobilized on a pre-activated aldehyde modified polysulfone (MPS) membrane; both, commercial biotin-labeled -galactosidase and the enzyme conjugate mentioned above, (SDBG) were immobilized on this membrane separately. The immobilized -galactosidase showed a dramatic drop in activity for the directly, randomly immobilized case; a relative activity (RA) of 1.8% compared to the RA of SDBG which was 87.7%. The RA of the commercial biotin-labeled -galactosidase, immobilized through an avidin-biotin complex as a spacer was 12.6% compared to a corresponding RA of SDBG of 25%. Thus, site-directed immobilization of -galactosidase offers significant advantages over random immobilization. The diffusion-reaction process which occurs inside the pores of a membrane was modeled to extract intrinsic data from the experiments performed. The values of the effectiveness factor for directly attached SDBG were closely matched with the values of xk/x, the reaction-limited reactor length. This research is supported by the National Science Foundation.

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