? NADP-dependent oxidoreductases are an important class of enzymes for the synthesis of ? enantiomerically pure, complex, chiral compounds. For these enzymes to be commercially viable, the nicotinamide cofactor must be regenerated in situ. To date, the most successful NADPH regeneration method involves the oxidation of glucose with glucose dehydrogenase. This scheme has some major drawbacks including the production of equimolar amounts of by-product and the necessity for pH control. In Phase 1, we propose to use directed evolution methods to create a formate dehydrogenase (FDH) that is able to accept, with high rates, NADP+ as a cofactor. The key advantages of an FDH-based recycle method are the low cost of reductant (formate), the absence of by-products (only carbon dioxide), and lack of need for pH control. A patent-pending assay for use in high throughput screening has already been developed and validated for the detection of NADP-utilizing formate dehydrogenases. These new enzymes will be produced by directed evolution and characterized in terms of the location of the mutation(s) and kinetic parameters. Success in Phase 1 will lay the foundation for developing formate dehydrogenases with additional improved properties, such as higher specific activity, broader pH optima, higher stability and reduced product inhibition. ? ?
