Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries
Autor: | Sabine L. Flitsch, Nicholas John Turner, Sarkar Manish, Robert Speight, Hanamanthsa S. Bevinakatti, Chris Winkel, Fred E. Hancock |
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Rok vydání: | 2004 |
Předmět: |
chemistry.chemical_classification
biology Cytochrome Chemistry Organic Chemistry Active site Cytochrome P450 Directed evolution Catalysis Amino acid Inorganic Chemistry Hydroxylation chemistry.chemical_compound Enzyme Biochemistry biology.protein Physical and Theoretical Chemistry Function (biology) |
Zdroj: | Tetrahedron: Asymmetry. 15:2829-2831 |
ISSN: | 0957-4166 |
DOI: | 10.1016/j.tetasy.2004.06.053 |
Popis: | The selection of cytochrome P450 enzymes from large variant libraries, and the subsequent use of these enzymes in preparative scale biotransformations, remains a formidable challenge due to the complexities of the associated electron transport systems. Here, a powerful approach for the generation and screening of P450cam libraries for new function is presented that is both flexible and robust. A targeted library was generated wherein only the P450cam active-site amino acids Y96 and F98 were fully randomized and biotransformations, using a novel P450cam whole-cell system, were screened by GC–MS for the hydroxylation of diphenylmethane. One in 50 of the reactions screened, including 16 different variants, produced 4-hydroxydiphenylmethane with up to 92% conversion observed in the case of the Y96A variant. These results demonstrate a primary example of the screening of P450cam libraries in a format that is compatible with extension to preparative scale reactions. |
Databáze: | OpenAIRE |
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