Study On The Aflatoxin B₁ Degradation Enzymes And Degradation Mechanism

Aflatoxins are a class of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus.Aflatoxin B₁?AFB₁?is one of the most toxic mycotoxins found so far and widely distributed to humans and animals.Carcinogenic,teratogenic,and mutagenic.In this study,Cladosporium uredinicola was used to degrade AFB₁,and the catalytic sites and degradation products of AFB1 were analyzed.The AFB1 degrading enzyme was isolated and purified and its enzymatic properties were initially explored.The specific research content is as follows:?1?The corn cobs culture medium can enhance the enzyme activity of the AFB₁ degrading enzyme in the supernatant of the spores of Fusarium solani,and analyzed the effects of carbon sources on the fermentation of Cladosporium uredinicola and the AFB₁enzymatic reaction.Corncob powder is hydrolyzed to produce furfural,vanillin and other derivatives after high temperature treatment.100?g/mL furfural can increase the enzyme activity of AFB₁ by about 100%.80?g/mL vanillin can increase the enzyme activity of AFB1 by about 85%;furfural can reduce the activity of AFB₁ degrading enzyme by about30%.Vanillin The AFB₁ degrading enzyme activity can be reduced by about 20%.These results indicated that these derivatives could induce multiple AFB₁ degrading enzymes in the spores of A.sphaeroides.AFB₁ multiple sites were simultaneously degraded,and the enzyme activity of the AFB₁ degrading enzyme in the fermentation supernatant was significantly increased.It is inferred that these AFB₁ degrading enzymes mainly catalyze the furan ring and benzene ring on the AFB1?2?Two degradation products,P1 and P2,were obtained by degradation of AFB₁ by fermentation supernatants from the fermentation of Cladosporium uredinicola.in Catch's medium.Structures of the two degradation products were characterized.High-resolution liquid chromatography?LC-Q-TOF MS?and tandem mass spectrometry?MS/MS?analysis showed that the molecular weights of P1 and P2 were 406 and 342,respectively,and the molecular formulas were C₁₉H₁₈O₁₀ and C₁₈H₁₄O₇,and the molecules of P1 and P2 in AFB₁.Furan double bond sites generate new branches through addition or oxidation reactions.?3?Through software simulation analysis and cytotoxicity tests,the toxicity of the two degradation products was lower than that of AFB₁.The theoretical toxicity prediction using TEST software found that the rat's minimum toxic dose was 29.01mg/kg.In contrast,the P1 rat has a minimum toxic dose of 102.5 mg/kg,while the P2rat has a minimum toxic dose of 26.48 mg/kg;using the cytotoxicity test,the AFB₁,P1,and P2 are added at 10?g/mL.In the cell culture medium,intense diffuse necrosis and nucleus pyknosis of the cells treated with AFB₁ were found,whereas cells treated with P1 and P2 did not show significant necrosis.P1 and P2 are less toxic than AFB₁,suggesting that P2 is an intermediate metabolite in the formation of P1.?4?The supernatant of the fermentation of the spores of Cladosporium uredinicola was collected by lyophilization,concentrated and purified to obtain two AFB₁degrading enzymes?E1 and E2?.After SDS-PAGE,the molecular weights of E1 and E2were estimated to be about 65kD and 30kD,respectively.The optimal reaction temperatures for E1 and E2 were 45°C,the optimal reaction p H was 7.5 and 7.0,the Km values were 44.89?g/mL and 42.908?g/mL,and the maximum responses were196.0784 ng/min and 125 ng/min,respectively.E1 can catalyze the furan ring and the benzene ring,E2 mainly catalyzes the furan ring,and the detection of enzyme 1 by 2?-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid?ABTS?is most likely a Oxygenase.