Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity

Luo Liu (1) , Rolf D. Schmid (1) , Vlada B. Urlacher (1)

1 Institute of Biochemistry and Technical Biochemistry = Institut für Biochemie und Technische Biochemie [Stuttgart]

Cytochrome P450 monooxygenase CYP116B3 from   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  . 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).

Mots clés: Cytochrome P450; Dealkylation; Directed evolution; High-throughput screening

Luo Liu, Rolf D. Schmid, Vlada B. Urlacher. Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity. Biotechnology Letters, 2010, 32 (6), pp.841-845. ⟨10.1007/s10529-010-0233-9⟩. ⟨hal-00574012⟩