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
Rok vydání: 2004
Předmět:
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