Bioaffinity Immobilization

Ipsita Roy and Munishwar N. Gupta

Summary

In bioaffinity immobilization, the enzyme/protein is immobilized via bioaffinity interactions. A large number of affinity pairs such as lectin-sugar, antigen-antibody, and bi-otin-avidin are known. The use of affinity tags to create fusion proteins that can bind to the desired matrix expands the list further and leads to a more or less a general method. Two versions of bioaffinity immobilization are possible. In the first, the matrix is precoupled to an affinity ligand and the target enzyme is added. In the second, the enzyme is conjugated to another molecule that in turn has affinity toward a matrix. The bioconjugate of the enzyme can be obtained either by chemical cross-linking preparation or as a fusion protein. The concept of bioaffinity immobilization is illustrated with a protocol that utilizes the second approach. A conjugate of P-galactosidase with lectin, concanavalin A, was prepared by cross-linking with glutaraldehyde. The conjugate was bound to Sephadex G-50. The protocol was originally developed for creating an immobilized biocatalyst for lactose hydrolysis. Lactose hydrolysis is biotechnologically relevant for whey hydrolysis and for producing low-lactose milk (for consumption by lactose intolerant persons)

Key Words: Reversible immobilization; lectin bioconjugates; P-galactosidase; low-lactose milk; whey hydrolysis; lactose hydrolysis; fusion proteins; Con A.

1. Introduction

In bioaffinity immobilization, the protein/enzyme is linked to a matrix via biospecific interactions. There are two strategies that are followed for bioaffinity immobilization, which are outlined in Table 1 and illustrated in Fig. 1. In either case, an affinity pair is involved. In principle, all affinity pairs (affinity ligand-target protein) used in affinity chromatography (1) can also be used for affinity immobilization. However, in affinity chromatography the design of the protocol is geared toward ensuring adequate dissociation (of the target protein), whereas in affinity immobilization the immobilized molecule does not come off the matrix.

Table 1

Strategies Involved in the Immobilization of Enzymes/Proteins on a Matrix Via Bioaffinity Interactions

Strategy Procedure

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