KINETICS STUDIES OF SITE-SPECIFICALLY AND RANDOMLY IMMOBILIZED ALKALINE-PHOSPHATASE ON FUNCTIONALIZED MEMBRANES

A comparison of enzyme activities has been made between a site-specifi cally immobilized and a randomly immobilized bacterial alkaline phosph atase (BAP) on macroporous membranes. An octapeptide tag (FLAG(R))dagg er was attached at the N-terminus of alkaline phosphatase by recombina nt DNA techniques (gene fusion) to yield BAP that is modified in a sit e-directed fashion (SDBAP). The corresponding antibody (antiFLAG(R)) w as immobilized on an aldehyde-modified polyethersulfone (MPS) membrane via protein A. Immobilization of SDBAP on this membrane result in a m embrane-protein A-antiFLAG-SDBAP linkage. This site-specifically immob ilized enzyme demonstrated a relative activity (RA), defined as the ra tio of immobilized activity (V-max) to the corresponding homogeneous e nzymatic activity, of 85% as compared with the randomly immobilized BA P which had an RA of 0 . 8%. BAP, when chemically conjugated to the FL AG peptide and immobilized via antiFLAG and protein A on the MPS membr ane, showed an RA of only 1 . 9%, demonstrating the effectiveness of s ite-directed immobilization. SDBAP was also immobilized on the MPS mem brane in the absence of protein A. In this case, the RA dropped to 22% , further explaining the effectiveness of ordered immobilizations as c ompared with random immobilizations. The ratio of immobilized enzyme a ctivity to the activity in the absence of added phosphate inhibitor fo r the immobilized BAP was three-fold higher than the corresponding hom ogeneous ratio, showing a reduction in product inhibition for the immo bilized enzyme.