Heterologous Expression Of Trametes Versicolor Aflatoxin B₁ Degrading Gene And Preliminary Study On The Mechanism Of AFB₁ Degradation

Aflatoxin B₁(AFB₁)widely existing in nature seriously threatens to human and animal health due to its potential carcinogenic properties.Enzymatic degradation was an effective and environmentally friendly alternative.However,most of the currently discovered enzymes were wild enzymes,which were difficult to be widely used and developed due to the instability of wild enzymes.Therefore,in this study,AFB₁ degrading gene from Trametes versicolor(TV-AFB₁D)was isolated and cloned,the p ET-28a(+)-TV-AFB₁D prokaryotic expression vector and p PIC9K-His-TV-AFB1D eukaryotic expression vector were successfully constructed.The enzymatic properties of TV-AFB₁D and the effect of degrading AFB₁ were investigated,and the application of TV-AFB₁D in AFB₁-contaminated peanuts.This study provides a basis for further exploring the application of TV-AFB₁D in AFB₁ degradation,and the main conclusions are as follows:(1)Cloning and informatics analysis of TV-AFB₁D gene.The TV-AFB₁D gene was obtained from Trametes versicolor by reverse transcription PCR amplification,and the recombinant cloning vector p EASY-TV-AFB₁D was constructed,transferred into E.coli DH5?,sequenced to verify the correct sequence and analyzed.TV-AFB₁D was identical to Trametes versicolor FP-101664 SS1(sequence no:txid717944)with 99.76%homology.TV-AFB₁D was found to have the highest homology with with the aflatoxin degrading enzyme of Trametes pubescens by protein evolutionary tree.The target protein consists of 696 amino acids,with the molecular formula C₃₄₇₈H₅₄₂₂N₉₁₆O₁₀₅₂S₇ and the relative molecular mass of 33572.34.It is a hydrophilic protein,which does not contain a signal peptide.TV-AFB₁D contains 45.26%alpha helix,12.93%beta-fold,41.81%random coil,and no unconventional secondary structure in TV-AFB₁D.(2)TV-AFB₁D expression optimization and enzymatic characterization.The expression vector p ET-28a(+)-TV-AFB₁D was constructed and successfully introduced into E.coli BL21(DE3)for expression.4 h and 0.8 mmol·L^-1 IPTG were optimal conditions for induction in recombinant E.coli.After Ni²⁺purification and SDS-PAGE result showed that the molecular weight of protein was 77 k Da.Enzymatic characterization showed that the optimum p H was 7 and optimum temperature was 32? of TV-AFB₁D,respectively,and its residual enzyme activity was best and stability was highest within p H 6-8 and 30-34?.Enzyme kinetic parameters:y=0.01671x+1.80756(R²=0.994,K_m=9.24 mmol·L^-1,V_max=553.23 m M·min^-1).The relationship between the initial catalytic rate of the reaction and the reaction temperature was:lnv=16.51-3338.73/T(R²=0.999).The circular dichroism study indicated that t the percentages of random coil,helix,and antiparallel structures could be the main factors affecting the activity and stability of TV-AFB₁D.(3)Study of TV-AFB₁D catalyzed AFB₁.HPLC showed the generation of new product after catalysis,and 67.4%of AFB₁ was degraded by the catalytic time of 5 h.Molecular docking model indicated the existence of hydrogen bonding force between TV-AFB₁D and AFB₁.LC-TOF-MS showed the generation of new product,and the molecular formula of the products was possible by speculation C₁₂H₁₂O₂ or C₁₃H₁₂O₄.(4)Construction of recombinant P.pastoris GS115 and preliminary application in AFB₁-contaminated peanut.The p PIC9K-His-TV-AFB₁D expression vector was constructed by double enzyme ligation and was transformed into P.pastoris GS115 by electroporation to construct the phenotype His⁺Mut⁺.After 84 h of induction,Ni²⁺purification and SDS-PAGE indicated the appearance of a specific protein band of 77 k Da and measured enzyme activity of 17.5 U/m L.The induction conditions of TV-AFB₁D expressed by recombinant P.pastoris GS115 were optimal at a methanol concentration of0.8%,induction time of 84 h and temperature of 28?.TV-AFB₁D catalyzed 12 h and achieved a degradation rate of 75.9%.For AFB₁contamination in peanut,the degradation rate reached 48.5%after 18 h of catalysis.By temperature exploration,it was found that the best degradation of AFB₁in contaminated peanuts was achieved at 34?.