Kinetic Studies of Site-Specifically and Randomly Immobilized Alkaline Phosphatase on Functionalized Membranes

Abstract

A comparison of enzyme activities has been made between a site‐specifically immobilized and a randomly immobilized bacterial alkaline phosphatase (BAP) on macroporous membranes. An octapeptide tag (FLAG™) was attached at the N‐terminus of alkaline phosphatase by recombinant DNA techniques (gene fusion) to yield BAP that is modified in a site‐directed fashion (SDBAP). The corresponding antibody (antiFLAG™) was immobilized on an aldehyde‐modified polyethersulfone (MPS) membrane via protein A. Immobilization of SDBAP on this membrane result in a membrane–protein A–antiFLAG–SDBAP linkage. This site‐specifically immobilized enzyme demonstrated a relative activity (RA), defined as the ratio of immobilized activity (Vmax) to the corresponding homogeneous enzymatic activity, of 85% as compared with the randomly immobilized BAP which had an RA of 0·8%. BAP, when chemically conjugated to the FLAG peptide and immobilized via antiFLAG and protein A on the MPS membrane, showed an RA of only 1·9%, demonstrating the effectiveness of site‐directed immobilization. SDBAP was also immobilized on the MPS membrane in the absence of protein A. In this case, the RA dropped to 22%, further explaining the effectiveness of ordered immobilizations as compared with random immobilizations. The ratio of immobilized enzyme activity to the activity in the absence of added phosphate inhibitor for the immobilized BAP was three‐fold higher than the corresponding homogeneous ratio, showing a reduction in product inhibition for the immobilized enzyme. © 1997 SCI.

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

“…Therefore the association of alkaline phosphatase with the soil phenolic support did not change the spatial conformation of the active site, nor did it block access of the substrate. 54 This contrasts with results reported by Ruggiero and Radogna 19 showing that humic acids increased the K m value for tyrosinase. The effect of the immobilisation process on the maximum rate of phosphatase activity (V max ) was not measured, because it was not possible to determine the protein bound to the humic matrix and to express the V max values as specific activities.…”

Affinity Membranes as a Tool for Life Science Applications

“…As a result, we determined a sixfold increase of the specific activity in favor of the directed enzyme coupling. Vishwanath and coworkers15 already noted, for an alkaline phosphatase with an octapeptide tag (FLAG tag), that the ordered immobilization to its receptor on a macroporous membrane led to an increase in enzyme activity (minimum 10%) as compared with random immobilization.…”

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…In some instances, the antibody is not designed for the target protein, but for a specific domain, which is introduced in the target protein sequence using sitedirected mutagenesis (Solomon et al, 1991, Vishwanath et al, 1997, Wang et al, 2001b, Witkowski et al, 1993. This permits to always have the desired orientation, but it will hardly have a very positive effect on enzyme properties.…”