Prokaryotic Expression Of Spodoptera Exigua Flavin Monooxygenase And Its Insecticide Metabolism

Beet armyworm(Spodoptera exigua)is an important vegetable pest.It has developed high resistance to many insecticides in China.Increasing detoxification and metabolism is an important mechanism conferring to insecticide resistance,especially P450s and esterases.Recent studies have shown that besides P450s and esterases,flavin monooxygenase(FMO)is also associated with insecticide resistance of beet armyworm,so the prokaryotic expression and insecticide metabolism of FMO of beet armyworm should be studied.In this paper,the prokaryotic expressions of SeFMOs were reported for the first time,and the soluble FMO enzymes were obtained.After purification,the activities of SeFMOs in prokaryotic expression were determined,and the metabolic activities of SeFMO enzymes to insecticides were analyzed.Finally,the metabolic activities of SeFMOs were verified by high performance liquid chromatography.Results are summarized as follows:1.Prokaryotic expression and purification of Spodoptera exigua flavin monooxygenasesUsing prokaryotic expression system,SeFMO2 and SeFMO3 genes were successfully reconstituted onto pET32a vectors.The optimum conditions for soluble expression of SeFMO2 and SeFMO3 were determined by optimizing the expression conditions.More soluble proteins were expressed in the supernatant after 8 hour induction with 0.5mM IPTG at 25℃.Then 100 mM imidazole solution was used for elution to obtain purified SeFMO2 and SeFMO3.Although the construction of SeFMO 1 expression vector was successful,the expression protein was in precipitation under various induction conditions,and no soluble expression was found.The expression conditions need further exploration.2.Enzyme activities of SeFMO2 and SeFMO3The N-oxidation and S-oxidation activity of SeFMO2 and SeFMO3 were determined by NADPH oxidation.The results showed that the two SeFMO enzymes expressed in the prokaryotic medium exhibited oxidative activity against both S-oxidizing substrates methimazole and N-oxidizing substrate benzydamine.The Km values of the metabolic kinetic parameters of SeFMO2 and SeFMO3 were 37.60± 8.28μmol/L and 3.00±0.88μmol/L for methimazole oxidation,respectively,and those of SeFMO2 and SeFMO3 were 165.98±10.45μmol/L and 17.71±2.48μmol/L for benzydamine oxidation,respectively.Based on the Km value the S-oxidation and N-oxidation activities of SeFMO3 were higher than that of SeFMO2.3.Metabolic activities of SeFMO2 and SeFMO3 on insecticidesThe metabolic activities of SeFMO2 and SeFMO3 to insecticides were also measured by NADPH consumption.The results showed that SeFMO2 and SeFMO3 had higher metabolic activities on chlorfenapyr,thiodicarb and chlorpyrifos,but less or no metabolic activities on other five insecticides were observed.The metabolic activities of SeFMO2 on thiodicarb,chlorfenapyr and chlorpyrifos were 4.6 nmol/min/mg protein,7.6 nmol/min/mg protein and 12.4 nmol/min/mg protein,respectively,while those of FMO3 to chlorfenapyr,thiodicarb and chlorpyrifos were 7.1 nmol/min/mg protein,12.8 nmol/min/mg protein,and 26.9 nmol/min/mg protein,respectively.The metabolic activities of SeFMOs on insecticides indicated that SeFMOs had detoxification and metabolism function to insecticides.4.Studies on insecticide metabolism of SeFMO2 and SeFMO3Further,we used high performance liquid chromatography to analyze the clearance of insecticides by SeFMO enzymes.The concentration of insecticides in reaction system were reduced when incubating with SeFMO enzymes.The results showed that the content of chlorpyrifos,chlorfenapyr and thiodicarb were decreased by 11.8%,11.5%and 27.8%.when incubating with SeFMO2,respectively,and decreased by 28.8%,23.1%and 30.2%when incubating with SeFMO3 enzyme,suggesting SeFMOs have the detoxification abilities to these insecticides.In conclusion,the prokaryotic expression of SeFMO2 and SeFMO3 was successfully achieved for the first time.The purified SeFMO2 and SeFMO3 enzymes had S-oxidation and N-oxidation activities to FMO model substrates.The liquid chromatography analysis also showed that SeFMO2 and SeFMO3 had detoxification and metabolism abilities to chlorfenapyr,thiodicarb and chlorpyrifos.The metabolic products of thiodicarb by SeFMO were identified,the oxidative reaction take placed on the thioether to form the sulfone compounds of thiodicarb.SeFMOs have the S-oxidation and N-oxidation activities against exogenous compounds,and FMO enzyme is one of the detoxification enzymes in insects.