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Biotechnol Lett. 2013 Oct;35(10):1663-8. doi: 10.1007/s10529-013-1254-y. Epub 2013 Jun 21.

Directed evolution of cytochrome P450 for sterol epoxidation.

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School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.


16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.

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