The matrix is activated and precoupled with an affinity ligand. The target enzyme is added.

The enzyme is conjugated to another molecule, which in turn has affinity toward a matrix. The bioconjugate can be prepared by chemical cross-linking or obtained as a fusion protein.

The enzyme is not subjected to harsh conditions. If the affinity ligand is a substrate/product ana logue, the chances of enzyme dissociating from the affinity ligand are high. A more generic approach is possible for preparing a series of immobilized enzymes without varying the affinity pair.

Bioaffinity Immobilization

Fig. 1. Two commonly used strategies for bioaffinity immobilization of proteins. (A) The enzyme (E) has affinity for an affinity ligand (-P) linked to the matrix. (B) The enzyme (E) is linked to a fusion tag (T), which has affinity for an affinity ligand (-P) linked to the matrix.

Fig. 1. Two commonly used strategies for bioaffinity immobilization of proteins. (A) The enzyme (E) has affinity for an affinity ligand (-P) linked to the matrix. (B) The enzyme (E) is linked to a fusion tag (T), which has affinity for an affinity ligand (-P) linked to the matrix.

This adherence can be achieved by choosing affinity pairs that have relatively higher association constants, and/or operational conditions favoring zero leaching. Mattiasson (2) wrote an excellent introduction to the method and listed association constants of some frequently used affinity pairs. More recently, Saleemuddin (3) has reviewed this approach with a focus on affinity immobilization on monoclonal/polyclonal antibodies and lectins. A review with a sharper focus on the use of concanavalin A (Con A)-based supports is also available (4). It should be mentioned that the classical view of the affinity of biological molecules has now been replaced with a broader concept, according to which a ligand may not have any biological relationship with the target protein, either in vivo or in vitro (5). Thus, a dye, a metal ion complex, or a peptide may all be useful affinity ligands (6-8). In many cases, this provides a successful alternative to more costly affinity ligands such as lectins and monoclonals, although the availability of efficient bioseparation protocols should result in lowering the cost of affinity ligands like lectins and antibodies (1).

The precoupling of affinity ligand to a matrix can be carried out with any cova-lent coupling method that is generally used for obtaining affinity media (9,10). If the affinity ligand itself is a biologically active protein (e.g., an antibody or a lectin), the design of this precoupling step has to be such that optimum accessibility of binding sites on this affinity ligand (in the precoupled form) to the enzyme (to be immobilized) is possible. Thus, oriented immobilization of proteins as affinity ligands is aimed at linking such affinity ligands (to the matrix) via a site that leaves the binding site free. For example, linking antibodies via Fc portion leaves their binding sites free for interacting with enzymes (11). A recent work uses site-specific attachment by use of site-directed mutagenesis as another approach to attach the protein affinity ligand in such a way that its binding site is free for "affinity recognition" of the enzyme (12). Biotin-avidin or biotin-streptavidin technology (13) is now well developed. If the matrix is labeled with avidin/streptavidin or biotin, the enzyme tagged with biotin or avidin/streptavidin, respectively, would allow bioaffinity immobilization with high binding constants (12). The linking of the enzyme with one of the members of the affinity pair can also be done by chemical cross-linking (14). Alternatively, fusion proteins can be obtained by recombinant methods (15,16).

"Affinity layering" is a relatively recent approach in bioaffinity immobilization (17,18). This interesting approach allows large amounts of glycoenzymes to be immobilized by creating alternate layers of Con A and the glycoenzyme. Gemeiner et al. (19) described a "superactive" immobilized invertase by a somewhat similar approach.

Immobilization of various enzymes on Con A-derivatized supports has been reported to significantly enhance their stability under various denaturing conditions, during storage, and even for long-term continuous operation (20).

The use of fusion proteins opens several exciting possibilities in the context of bioaffinity immobilization. Use of histidine-rich affinity tails to immobilize enzymes on immobilized metal affinity supports (21) or chimeric enzymes with cellulose-binding domains (22,23) are two well-known examples of this generic approach. A recent work uses a histidine-tagged fragment of protein A, which showed high affinity for IgG (24).

The proteins/enzymes immobilized by bioaffnity immobilization have, more often, been used for bioanalysis. The protocol (25) given in this chapter illustrates the application of the approach in bioconversion. Table 2 catalogs other applications. Anspach and Altmann-Hasse (26) have described the immobilization of Escherichia coli penicillin G aminohydrolase on immobilized metal chelate supports. The fusion protein consisting of Protein A and P-lactamase immobilized on IgG-Sepharose was found to hydrolyze penicillin G more efficiently than the covalently immobilized enzyme (27). Similarly, the immobilization of cellulose-

Table 2

Applications of Enzymes/Proteins Immobilized Through Affinity Interactions

Table 2

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