Abstract
Cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed high-throughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).
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We thank Marco Girhard and Clemens von Buhler for constructive discussions and critical review of the manuscript.
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Liu, L., Schmid, R.D. & Urlacher, V.B. Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity. Biotechnol Lett 32, 841–845 (2010). https://doi.org/10.1007/s10529-010-0233-9
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DOI: https://doi.org/10.1007/s10529-010-0233-9