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 immobilized β-galactosidase on macroporous membranes. A biotinylated β-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 β-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 β-galactosidase and the enzyme conjugate mentioned above, (SDBG) were immobilized on this membrane separately. The immobilized β-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 β-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 β-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 x_k x, the reaction-limited reactor length.