Enzyme microcrystals (less than 100 um) grown from aqueous solution and cross- linked with a bifunctional agent such as glutaraldehyde exhibit remarkable characteristics that are superior to both soluble and conventionally immobilized enzymes. Such cross-linked enzyme crystals (CLECs) remain active in environments that are otherwise incompatible with enzyme function, including prolonged exposure to high temperatures, extremes of pH, near-anhydrous organic solvents and aqueous-organic solvent mixtures. CLECs are also remarkably stable against autolysis and exogenous protease degradation. This unique set of properties makes CLECs extremely useful in organic synthesis in general and in the synthesis of optically pure compounds in particular. The authors have demonstrated the synthetic utility of CLECs in the synthesis of aspartame, catalyzed by thermolysin and in the synthesis of optically pure compounds, catalyzed by lipase from Candida rugosa. In this Phase I study, CLECs of a cofactor-dependent enzyme alcohol dehydrogenase (ADH) from two different sources will be investigated to demonstrate the feasibility of using CLECs of cofactor-dependent enzymes for practical stereoselective synthesis. ADH was chosen because it is one of the most studied, as well as useful and versatile cofactor-dependent enzymes for stereoselective organic synthesis.
