Cross-Linked Enzyme Crystals (CLECs) exhibit remarkable characteristics, that makes them extremely useful in organic synthesis in general and in the synthesis of optically pure compounds in particular. CLEC formulation has been limited to hydrolases, although commercial interest in more complex enzymes such as cofactor dependent dehydrogenases is high. In Phase I, using alcohol dehydrogenase as a prototype, we demonstrated that a cross-linked crystalline enzyme/cofactor complex is catalytically active, that the enzyme is stabilized to organic solvent, thermal, and proteolytic inactivation, and that the cofactor can be repeatedly regenerated while remaining tightly bound to the apoenzyme. In Phase II, we will continue development of CLECs of horse liver alcohol dehydrogenase and a thermostable microbial alcohol dehydrogenase, and begin development of L-lactate dehydrogenase and L-leucine dehydrogenase CLECs. CLEC formulation; including enzyme purification, crystallization, cross-linking and drying, will be scaled to the 100 gram level. The stability, substrate specificity, and performance of the dehydrogenase CLECS in reactions of commercial interest will be studied.
Alcohol dehydrogenases from various sources have a great potential in preparative organic synthesis as they have a broad substrate specificity and can produce valuable chiral building blocks in high optical purity. ADH-CLECs may become major catalysts in the synthesis of optically pure drugs. The market represents one of the fastest growing sectors of the pharmaceutical industry and is expected to reach $40 billion by the end of the century.
