Heterologous Expression And Characterization Of A Novel P450 Monooxygenase From Sphingomonas Wittichii

Cytochrome P450 monooxygenases are the most powerful biocatalysts with broad-spectrum,and widely distributed in nature.They participates in the metabolism of many endogenous substances and exogenous substances such as drugs and environmental compounds in vivo,and catalyze a variety of chemical reactions in vitro.In this research,we searched the P450 gene derived from Sphingomonas wittichii RW1(P450_SpMO for short)in order to exploit more cytochrome P450 with high efficiency and stability,it was heterologously expressed in Escherichia coli and carried out basic researches on enzymatic catalytic properties.The main conclusions are as follows:（1）The P450_SpMO plasmid was recombined through genetic engineering,and then the P450_SpMO enzyme was heterologously expressed in E.coli.It was found that when the6His-tag was designed at the N-terminus,the expression and purification of the target protein both better than on the C-terminus.（2）P450_SpMO is a novel P450 enzyme belonging to the CYP116B subfamily of Class VII,with a typical P450 absorption spectrum.Enzymatic properties of recombinant P450_SpMOpMO showed that the optimal pH and temperature were 6.5 and 35°C,and had good thermal stability at 30°C.The measured Mie constant K_m was 0.626 mmol/L,and the catalytic efficiency k_cat/K_m was 2.866 mmol/（L·min）.（3）Recombinant P450_SpMO can oxidize sulfides,and catalyze O-dealkylation of methoxy-substrates.In addition,factors such as electron conjugation and steric hindrance caused effect on the catalytic activity of recombinant P450_SpMO.The above conclusions indicated that recombinant P450_{Sp MO} is a multifunctional P450monooxygenase,which has great application potential in organic synthesis.