Function Of Cytochrome P450 In Degradation Of Refractory Organic Compounds By A White Rot Fungus

Cytochrome P450s are widely distributed hemoproteins involved in detoxification of many refractory organic compounds. The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited due to low expression level of P450 and problems in isolating and detecting fungal P450s. In this study, induction and function role of P450s in degradation of various refractory organic compounds by P. chrysosporium was studied, based on an optimized protocol to isolate and determine microsomal P450.The optimized protocol to isolate and determine P450 included cell disruption by high-speed disperser and glass homogenizer, differential centrifugation to isolate microsomal fractions, and measurement of CO difference spectra which were recorded after the samples were gassed and reduced according to the optimum parameters strictly.Induction of P450s in P. chrysosporium was observed upon addition of various xenobiotics, including n-hexane, phenobarbitone, benzoic acid, chlorobenzoic acid, toluene, monochlorobenzene, 2,4-dichlorophenol, pentachlorophenol, phenanthrene and naphthalene, respectively. The P450 contents in the microsomal fractions were up to 37~137pmol/mg. The induction of P450 was obviously affected by nutrient conditions, inducer concentrations, incubation time with inducers and rotator condition.The P450 of P. chrysosporium was able to degrade benzoic acid with specific activity of 29 mol/(min·mol P450) on average. However, P450 inhibitors have no effect on benzoic acid degradation, suggesting involvement of other enzyme. The extracellular manganese peroxidase (MnP) can not degrade benzoic acid.Pentachlorophenol could be oxidized by the P450 of P. chrysosporium at specific activity of 179 mmol/(min·mol P450) on average. Pentachlorophenol degradation in nutrient-limited cultures was mostly catalyzed by MnP and methyltransferase, and was not affected by the P450 inhibitor, piperonyl butoxide (PB). In nutrient-rich cultures, methylation was dominant fate of pentachlorophenol, and it increased upon addition of PB.The P450 of P. chrysosporium could oxidize phenanthrene to form the trans-dihydrodiol metabolite, and the specific activity was 638 mmol/(min·mol P450) on average. In nutrient-rich cultures, phenanthrene trans-dihydrodiols and phenanthrenols were detected as metabolites of phenanthrene, and PB could inhibit the degradation of phenanthrene. In nutrient-limited cultures, phenanthrene trans-9,10-dihyrodiol was the major detectable product. Phenanthrene degradation and the trans-9,10-dihyrodiol formation were significantly inhibited by PB, suggesting important role of P450 in phenanthrene metabolism. Phenanthrene was also degraded by MnP, and PB had no effect on MnP activities. Furthermore, PB inhibited degradation of naphthalene, pyrene, 2,4-dichlorophenol and 2,4,6-trichlorophenol, suggesting the important role of P450 in degradations of these compounds by P. chrysosporium.