Future Opportunities

A major factor driving the commercialization of an immobilized enzyme process is the ability to produce a product that cannot be produced by other means.

This may be the case for production of modified proteins designed for specific functionality in a food—for example, modification of the emulsifying, gelling, or foaming properties of a protein ingredient by limited proteolysis (14, 15,44, 45). If the proteolysis is carefully controlled, large oligopeptides with native-like structure can be released that have more desirable functionality than the parent protein (14, 15). Use of an immobilized proteinase allows precise control of the extent of proteolysis without a downstream enzyme inactivation step such as a heat treatment. Because excellent functionality is destroyed by heat treatments that cause unfolding and aggregation, such a downstream process is often counterproductive. Another potential example is the alteration of the structural stability or gelling characteristics of a protein by limited crosslinking using transglutaminase as a catalyst (46, 47). As in the case with proteinases, use of immobilized enzyme avoids the need for a downstream enzyme inactivation step to prevent eventual gel formation and to stop reaction at the desired level of crosslinking.

Another major economic factor is the cost of enzyme purification and immobilization. Designing the enzyme for bioselective adsorption can minimize this cost. In the future, many enzymes will most likely be recombinant proteins designed with consideration of specific catalytic requirements such as specificity, thermal stability, pH optima, and cofactor requirements. In addition, it would not be difficult to design structures allowing ease of purification and immobilization. A number of fusion proteins have been genetically designed for this purpose. The cellulose-binding domain of Cellulomonas fimi exoglucanase has been fused to the gene for p-glucosidase allowing this fusion protein to bind to cellulose (48). Similarly, the starch-binding domain of Aspergillus glucoamylase was fused to p-galactosidase, resulting in a fusion protein that bound to starch (49). The streptavidin-biotin interaction represents an even stronger bio-specific interaction with a dissociation constant of the order of 10-15 M-1. Genetic constructs have been prepared that allow expression of streptavidin-p-galac-tosidase (50), streptavidin-lipase (51), streptavidin-transglutaminase (52), and trypsin-streptavidin (53). Biotin can be covalently attached to a variety of supports using commercially available biotinylation reagents. Streptavidin-enzyme fusion proteins were purified and immobilized in one-step from crude recombinant cell lysates (50-53). Furthermore, the bio-tinylated supports can be regenerated and repeatedly used by desorption of spent enzyme and readsorption of fresh recombinant fusion enzyme (54).

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