| Cytochrome P450s(CYP)is probably the most diverse biological catalyst.White rot fungi can degrade many refractory organic pollutants.Besides its lignin degrading enzyme system,the degradation function of cytochrome P450s(CYP)enzyme system has attracted more and more attention.White rot fungi can detoxify many kinds of exogenous compounds depending on CYP enzyme system,such as polycyclic aromatic hydrocarbons,phenols,steroids and alkanes.Trametes hirsuta AH28-2 and Trametes versicolor are both white-rot fungi.Previous studies in the laboratory showed that the transcription level of several cyp genes in Trametes hirsuta AH28-2 cells were up-regulated after the induction of ortho-toluidine.It is suggested that the CYP system is involved in the response to the exogenous aromatic compounds.NADPH-cytochrome P450 reductase is a kind of redox reductase supplemented by FMN and FAD.CPR is the main electron donor of CYP,and its electron transfer to CYP is the rate-limiting step.CYP is a kind of enzymes that can binds to CO and contains heme and thio-hydroxyl groups with UV absorption peaks near 450 nm.CYP has a very broad spectrum of catalytic reaction ability.It has been reported that CYP can carry out reactions include hydroxylation reaction,oxidation of hetero atoms,alum reaction,dehydrocarbon reaction,epoxidation reaction,aromatic hydroxylation reaction,reduction reaction,dehalogenation reaction and so on.Single expression,purification and enzyme characterization of CPR and CYP will facilitate constructing the in vitro enzyme reaction system of CYP,studying on the interactions between CYP and CYPs from different families,and comparing the differences between CPR and other CYP electron transporters that are not dependent on CPR(such as cytochrome b5 and NADH dependent cytochrome b5 reductase).There have been some reports about the heterologous expression and functional identification of CPR in E.coli.For efficient and soluble expression of CPR in E.coli cells,the usual strategy is to modify or remove the N-terminal hydrophobic-membrane binding region.The CYP of eukaryotes is also a membrane binding protein.The expression of soluble and active form of C YP in Escherichia coli is also usually achieved by the substitution or removal of its N-terminal hydrophobic region.In this study,the full-length CPR gene and a truncated CPR protein lacking the predicted membrane anchor region(residues 1-24),named CPRA24 of Trametes versicolor was heterologous expression in E.coli respectively.Finally,we obtained the soluble expressed CPRA24 protein.The protein was purified and its enzymatic characteristics analysis results showed that the molecular weight consistent with the theoretical size of 78.35 kDa.The specific activity after nickel ion affinity chromatography and molecular sieve chromatography was 5.82 U/mg.The analysis of enzymatic properties showed that the optimum temperature and pH of CPRA24 were 35 ? and 8.0,respectively.The analysis showed that CPRA24 protein had some degree of tolerance to some metal ions and organic solvents.The kinetic parameters Km and kcat of recombinant CPRA24 for NADPH at 35 ? and pH 8.0 were 19.7?M and 3.31/s,respectively.The kinetic parameters Km and kcat for the substrate cytochrome c were 25.9?M,10.2/s,respectively.In this study,a CYPThCYP53-1 in Trametes hirsuta AH28-2 was expressed in E.coli.The recombinant ThCYP53-1 was finally expressed by removing the N-terminal membrane anchor region and using the sumo fusion tag.The ThCYP53-1 was co-expressed with the chaperone pGro7 to be expressed as a soluble and active form.ThCYP53-1 belongs to the CYP53 family.It has been reported in the literature that this family of enzymes can add hydroxyl groups at the 4 position of the aromatic benzene ring.This study will provide guidance for further constructing in vitro enzymatic reaction of CYP system,exploring the interaction between CPR and different CYPs,and studying the function of CPR and CYPs in the degradation of environmental organic pollutants. |
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