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Abstract
Grant Number: 1R43GM067536-01 PI Name: HUA, LING PI Email: lhua@biocatalytics.com PI Title: Project Title: Evolving Improved Formate Dehydrogenases Abstract: DESCRIPTION (provided by applicant): 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.
Thesaurus Terms:
NAD(H) phosphate, aldehyde /ketone oxidoreductase, cofactor, directed evolution, formate, technology /technique development
chemical synthesis, method development
genetic library, high performance liquid chromatography, polymerase chain reaction
Institution: BIOCATALYTICS, INC. 129 N HILL AVE, STE 103 PASADENA, CA 911051955 Fiscal Year: 2003 Department: Project Start: 01-JAN-2003 Project End: 30-JUN-2003 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1
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