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

A comparison of Michaelis-Menten kinetic parameters, K-m and V-max has been made between a randomly immobilized and a site-specifically immo bilized beta-galactosidase on macroporous membranes. A biotinylated be ta-galactosidase conjugate (SDBG), was prepared by posttranslational m odification 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 beta-galactosidase. Avidin, which has a very str ong interaction with biotin, was immobilized on a pre-activated aldehy de modified polysulfone (MPS) membrane; both, commercial biotin-labele d beta-galactosidase and the enzyme conjugate mentioned above, (SDBG) were immobilized on this membrane separately. The immobilized beta-gal actosidase showed a dramatic drop in activity for the directly, random ly 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 be ta-galactosidase, immobilized through an avidin-biotin complex as a sp acer was 12.6% compared to a corresponding RA of SDBG of 25%. Thus, si te-directed immobilization of beta-galactosidase offers significant ad vantages over random immobilization. The diffusion-reaction process wh ich occurs inside the pores of a membrane was modeled to extract intri nsic data from the experiments performed. The values of the effectiven ess factor for directly attached SDBG were closely matched with the va lues of x(k)/x, the reaction-limited reactor length.