Figure 5. A schematic diagram showing a typical affinity separation. The affinity ligand is represented by the black squares attached to the round support particles. The complementary structure of the target protein is represented by the square cavities carried by some feedstock components. Only these compounds can bind to the immobilized ligand while unwanted materials are washed through. Bound product can then be recovered in an elution step.

Because of the high degree of selectivity which is possible, affinity systems are among the most powerful separation methods available. Some are currently used on a preparative scale for the commercial purification of pharmaceutical and research products. However, there are economic and technical restrictions on the application of these methods to the purification of large-volume, low-cost products such as many of those required for food use. Many ligands are themselves biological compounds and are therefore expensive to synthesise or to purify from biomass. The specificity of the protein-ligand interaction can also be a disadvantage as method development may involve a great deal of research and development which is likely to be relevant only to a single product. Affinity chromatography will also suffer from those limitations of chromatographic methods which were discussed in the previous section, but using more expensive media with a shorter active life.

The use of group-specific ligands can help to overcome some of these problems. These ligands do not have specificity for a single enzyme, but for a group of enzymes which share areas of structural similarity. For example, cofactors such as NAD and AMP may be used to purify the many enzymes which have binding sites for these molecules (Mosbach et al, 1972; Lee, 1983). The introduction of anthraquinone dyes such as Cibacron Blue as group-specific ligands was an important development in affinity chromatography. These originated as textile dyes and are therefore available in bulk at low cost. Haeckel et al (1968) observed by chance that Cibacron blue bound to pyruvate kinase during gel filtration. It has since been used as an affinity ligand for the purification of a wide range of enzymes (Clonis, 1987). The specificity is therefore not absolute but this is off-set by the wide range of potential applications, and the commercial availability of bulk volumes of pre-prepared separation media. Dye-ligand chromatography is the most widely used form of affinity chromatography on a large scale (Jones, 1991). Sea wen & Atkinson (1987) listed 228 examples of dye-ligand chromatography published between 1980 and 1987. Of these over 40% described the purification of oxidoreductase enzymes such as alcohol dehydrogenase and malate dehydrogenase, and over 50% used Cibacron Blue as affinity ligand.

The techniques of molecular modelling have been used to establish that Cibacron Blue binds to the NAD binding site of horse liver alcohol dehydrogenase (Biellmann et al, 1979). This has enabled the synthesis of dye analogues with improved binding (Lowe et al, 1992). The ligand can thus be "tailor made" to fit the target enzyme. Group-specific ligands can thus be used as a starting point for the development of a series of ligands of higher selectivity. The rational design of affinity agents, either de novo or as analogues of established ligands, will certainly be aided by knowledge of the structure of binding domains in enzymes. Various techniques are now available for the determination of 3-dimensional molecular structures and these are being applied to the modelling of molecular interactions in food research (Kumosinski et al, 1991). As many of the enzymes involved in food technology share common functions (Table 1), they may make good subjects for the rational design of low cost ligands which can be synthesised on a large scale.

An alternative means of achieving selectivity in bioseparations is molecular imprinting. This involves the synthesis of polymer matrices which incorporate an intrinsic affinity for the species to be adsorbed. This is achieved by carrying out the polymerisation reaction in the presence of the target molecule which acts as a template around which the polymer is formed.

Interactions between monomer functional groups and the template molecules cause the resulting polymer to form in a conformation which is complementary to the template structure. Subsequent removal of the template molecule leaves cavities in the polymer matrix which are capable of specifically adsorbing the target species from solution (Wulff, 1993). This procedure is illustrated in Figure 6. The attraction of molecular imprinting is that separation matrices can be synthesised which incorporate the desired affinity in a stable form, and that specific adsorbents can be obtained for molecules for which no complementary ligands exist. This method has been investigated for the resolution of optical isomers of sugars (Wulff & Schauhoff, 1991) and amino acids (Ekberg & Mosbach, 1989).

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