Heterologous Expression And Characterization Of CYP116B256v2:A Cytochrome P450 From Variovorax Paradoxus S110

The cytochrome P450 monooxygenases?P450s or CYPs?are a superfamily of heme-containing enzymes which are widespread in nature.P450s are highly attractive biocatalysts due to their ability to catalyse a wide variety of reactions including hydroxylation,epoxidation and O-dealkylation.In particular,it can catalyse the regio-and stereoselective oxidation of inactive C-H bonds under mild conditions,making P450s very attractive in the organic synthesis and pharmaceutical industries when in comparison with chemical catalysts.In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology,two P450s from Sphingomonas wittichii RW1 and Variovorax paradoxus S110 were mined,cloned and expressed.It was found that only a novel self-sufficient cytochrome P450 monooxygenase(P450_VpMO)from Variovorax paradoxus S110,belonging to a self-sufficient CYP116B class,was able to be recombinantly overexpressed in Escherichia coli.The purified P450_VpMOpMO with Soret peak was shifted to 450 nm from 420 nm when it was reduced by sodium dithionite and bond with carbon monoxide?CO?upon formation of the Fe?II?-CO complex.Furthermore,the demethylation of 4'-methoxyacetophenone by P450_VpMO as the pattern reaction showed that the optimal pH and temperature for P450_VpMO demethylation reaction were 8.0 and 45°C,respectively.P450_VpMO is relatively stable at temperatures below 35°C.The K_m and k_cat of P450_VpMO toward 4'-methoxyacetophenone is 0.458 mmol·L^-1 and 2.438 min^-1,respectively.Next,we analyzed its substrates profile,showing that P450_VpMOpMO was a versatile biocatalyst as it can promote styrene oxidation,sulfide oxidation and O-dealkylation.Interestingly,P450_VpMO was able to facilitate the O-dealkylation reaction with a range of substrates.For 4'-methoxyacetophenone,it was with a great conversion efficiency at 91%.Last,it also could degrade Trimethoprim,an antibiotic contamination.All of above results showed that P450_VpMO is a great biocatalyst in biotransformation reaction.