Study On The Mechanism Of Lignin Peroxidase Metabolism By HMW-PAHs Degrading Fusarium Sp.ZH-H2

Polycyclic aromatic hydrocarbons(PAHs),especially high molecule weight polycyclic aromatic hydrocarbons(HMW-PAHs)containing 4 or more rings,with the increase of the number of benzene rings,the ability of carcinogenesis,teratogenesis and mutagenicity becomes more prominent,thus it has caused greater environmental risks.The mechanism of degradation of PAHs is mainly the metabolic enzyme system.It is very important to optimize the regulation of environmental conditions,promote the metabolic degradation enzymes of the strain and mine the corresponding key genes to enhance the removal efficiency of HMW-PAHs.In the early stage of this research group,a fungi Fusarium sp.ZH-H2 that efficiently degraded HMW-PAHs was screened,and research confirmed that ZH-H2 secretes only lignin peroxidase(LiP)where HMW-PAHs is the only carbon source stress LiP is a key enzyme for removing HMW-PAHs.However,the environmental conditions under which the fungi can significantly improve LiP metabolism,the docking mode of HMW-PAHs when LiP catalyzes HMW-PAHs,and the key genes for LiP metabolism are still unclear.Therefore,in this paper,the fungi ZH-H2 is taken as the research object.Through the culture experiment,the environmental conditions of ZH-H2 metabolism of LiP and the optimal combination of positive induction are studied,and the gene regulation mechanism of LiP metabolization and HMW-PAHs degradation was studied from the perspective of molecular biology.The main results are as follows:(1)Through the cultivation experiment,the environmental conditions of Fusarium ZH-H2 metabolism LiP were optimized.It was recommended that the optimal combination of environmental conditions for ZH-H2 metabolism of LiP was temperature 15?,150 rpm/min,pH 7,and the culture period was 7 days;suitable carbon and nitrogen sources were starch and ammonium tartrate respectively,and the carbon to nitrogen ratio was 50:1,LiP enzyme activity was up to 34767.02 U/L,which was 90.35%higher than CK.(2)Through single-factor and two-factor experiments,an advantageous combination of inducers that promote the metabolism of Fusarium ZH-H2 LiP was proposed.The single-factor treatment enzyme activity of 0.50 mmol/L Cu(?),0.10 mmol/L Mn(?),5.00 mmol/L resveratrol,0.01 mmol/L reactive oxygen species was the highest,up to 28315.41 U/L,significantly higher treated 2.29 times in CK.The combination of the two factors with Cu(?)(0.50mmol/L)-resveratrol(10.00 mmol/L)showed the highest enzyme activity,reaching 40501.79 U/L,which was 1.62 and 2.76 times higher than the corresponding single factor,showing Significant synergies.(3)Through the degradation test of HWM-PAHs,the effect of the superior combination of inducers on the degradation of HWM-PAHs by ZH-H2 was clarified.The removal rate of the total amount of HMW-PAHs without the addition of the inducer CK was 68.07%,while the total removal of HMW-PAHs under the combination treatment of Cu(?)-resveratrol reached 78.98%,an increase of 16.02%over CK,This combination was particularly outstanding for the degradation of monomer BaP,up to 95.08%.(4)Through the combination of theoretical chemistry and computer simulation,the binding force and free energy of the combination of pyrene and LiP were determined.The pyrene of HMW-PAHs is more easily bound to the hydrophobic side of LiP in a non-polar solvent environment,and the residues that interact with pyrene were mainly the hydrophobic groups Trp171 and Phe267.The secondary effects of pyrene and Phe267 forming stacking force,vander Waals potential energy,electrostatic force,etc.can promote the reaction.The combined free energy with LiP was-8.52 kcal/mol,of which van der Waals potential energy contribution is-14.18 kcal/mol,and electrostatic contribution was-5.50 kcal/mol.(5)The results showed that there were significant changes in 63 genes of ZH-H2 induced by exogenous substances.Among them,40 genes were up regulated and 23 genes were down regulated.The sequence of up-regulated genes related to peroxidase metabolism was as follows TRINITY_DN7339_c0_g1.The functional enzyme genes in go functional annotation include peroxidase activity,cytochrome P450 enzyme,heme binding and others.KEGG pathway analysis includes carbohydrate metabolism pathway,peroxidase metabolism,hydrocarbon,oxygen-containing derivatives metabolism,and others.It further indicated that the enzyme played an important role in the degradation of HMW-PAHs.In summary,this study clarified the environmental conditions of ZH-H2 metabolism of LiP and the optimal combination of positive induction,revealed the combined force and free energy of the combination of pyrene and LiP,and clarified the gene expression and metabolic mechanism of strains degrading HMW-PAHs.