Enzyme Immobilization and Conjugation

 Immobilization and conjugation refers to the covalent attachment of an enzyme to a solid support or to a soluble molecule through use of bifunctional crosslinking reagents. The use of immobilized enzymes in the medical, pharmaceutical, chemical, and food industries continues to increase dramatically. Future exploitation of immobilized or conjugated enzymes will undoubtedly result in the development of new biosensors and bioreactors for use in such areas as diagnostics (in vivo monitoring of metabolites, drugs, and proteins); environmental monitoring (detection of pathogens, toxins, and pollutants); separation sciences; the synthesis of complex, chiral organic compounds; and the detection of macromolecular interactions (reviewed in Refs. 1 and 2. (

Among the most widely used bioconjugates are reporter enzymes covalently crosslinked to antibodies. These conjugates have been pivotal in the development of enzyme-linked immunosorbent assay (ELISA) systems, which allow detection of an immense variety of analytes through their specific recognition by appropriate antibodies. The two most commonly used reporter enzymes in ELISAs are alkaline phosphatase and horseradish peroxidase (HRP). The characteristics of these enzymes allow for different strategies in their conjugation to antibodies. For example, HRP contains saccharide groups that can be readily oxidized to aldehydes, which, in turn, are conjugation targets for free amino groups on the antibody. Alternatively, because HRP contains few lysine residues, it can be conjugated to antibodies by glutaraldehyde without significant formation of insoluble products. Several heterobifunctional crosslinking reagents, including N-hydroxysuccinimide ester/maleimide reagents, which selectively link amino and thiol groups, are also widely used in enzyme–antibody conjugation (3).

Enzymes can be immobilized by conjugation to a variety of different types of supports having different properties and uses. For instance, in aqueous solution polyethylene glycols have a large exclusion volume, which encompasses those molecules conjugated to them. Thus, proteinases and antibody molecules can be excluded from enzymes conjugated to these polymers, a property that can be beneficial for applications that require exposure of the conjugates to biological fluids (4. (Reversible solubilization of an enzyme can be achieved through its conjugation to liposomes, using a reagent such as a carbodiimide; reversible precipitation and solubilization can be brought about by manipulation of the dielectric strength of the solution. Biosensor technology utilizes proteins that are immobilized by bifunctional crosslinking reagents to diverse supports (5), including carboxymethylated dextran-coated gold film, metal-coated nylon mesh, and carboxymethyl cellulose. Immobilization of enzymes on novel solid-phase matrices, such as porous zirconium, silicates, and colloidal gold, has been used in the construction of bioreactors for the industrial generation of numerous compounds. Many supports have functional groups that must be chemically activated prior to conjugation with protein; this allows activation to be carried out under harsh conditions that would otherwise be detrimental to the protein.

References

1. E. Katchalski-Katzir (1993) Trends Biotechnol. 11, 471–478. 

2. F. Svec and J. M. Frecht (1996) Science 273, 205–211. 

3. G. T. Hermanson (1996) Bioconjugate Techniques, Academic Press, San Diego, pp. 461–469. 

4. H. C. Berger and S. V. Pizzo (1988) Blood 71, 1641–1647. 

5. G. T. Hermanson (1996) Bioconjugate Techniques, Academic Press, San Diego, pp. 593–629.