The Acting Mechanisms And High-level Expression Of Broad-spectrum Enzymes Detoxifying Major Mycotoxins In Feed

Mycotoxins are fungal secondary metabolites which are threatening the health of the humans and animals.Among the numerous of detoxification methods,enzymatic treatment is an attractive method for mycotoxin detoxification because of its advantages of sustainability and environmental friendly.Most discovered enzymes are reported to degrade only a certain type of mycotoxins.This is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed.Firstly,we found manganese peroxidases（MnPs）from different lignocellulose-degrading fungi could all degrade four major mycotoxins（Aflatoxin B₁,AFB₁;Zearalenone,ZEN;Deoxynivalenol,DON;Fumonisin B₁,FB₁）.Laccase has a lower redox potential than MnP.The ability and mechanism of laccase to degrade mycotoxins was investigated.Finally,we studied the high-level expression of manganese peroxidase from Moniliophthora roreri in Pichia pastoris and laccase from Melanocarpus albomyces in Trichoderma reesei through fed-batch cultivation.By the study above,we wanted to have some understanding of the broad-spectrum mycotoxin-degrading ability of manganese peroxidase and laccase-mediator system.Details are as follows:Seven MnP genes from Irpex lacteus CD2,Phanerochaete chrysosporium,Ceriporiopsis subvermispora were recombinantly produced in Escherichia coli and one from Nematoloma frowardii was purchased from Sigma-Aldrich were tested for their ability to degrade mycotoxins.The results indicated that all eight MnPs were found to be able to degrade four major mycotoxins,AFB₁,ZEN,DON,and FB₁.For mycotoxins degradation,MnPs with transformation rates varying from 78.4-100.0%for AFB₁,42.2%-94.3%for ZEN,42.9-96.3%for DON,22.2-43.6%for FB₁,respectively.Laccase BsCotA from Bacillus subtilis was tested for their ability to degrade AFB₁ and ZEN directly.AFB₁ and ZEN（5μg/mL each）were individually incubated with 0.03 U/mL BsCotA and the transformation rates were 18.2%for AFB₁ and 17.6%for ZEN.Nine chemical compounds with a defined chemical structure were first screened for a possible mediator role,which may assist in degrading AFB₁ and ZEN.In this screening,the highest degradation rate was obtained with methyl syringate.The degradation efficiencies were 98.0 and 100.0%for AFB₁ and ZEN,respectively.We chose several plant extracts to test their efficacy in aiding BsCotA to degrade AFB₁ and ZEN.Among the tested plant extracts,the Epimedium brevicornu（epimedium）and Schizonepeta tenuifolia（schizonepeta）extracts were effective mediators for BsCotA-mediated AFB₁ and ZEN degradation.The AFB₁ and ZEN also could be degraded by the Ganoderma sp.laccase in the presence of mediators above.After the ability of broad-spectrum mycotoxin-degrading by manganese peroxidase and laccase-mediator systems had been elucidated,we attempted to study the mechanisms of degradation of AFB₁ and ZEN by IlMnP5 and IlMnP6 using different organic acid systems and radical scavengers.The results showed that radicals played an important role in mycotoxin degradation and MnPs could also reacted with ZEN directly.On this basis,we found that the rate of dye RB5 decolorization by MnPs was positively related to that of mycotoxin degradation,dye decolorization could then be used as a safe indicator,speeding up the discovery of novel MnPs with mycotoxin-degrading ability or evolving MnPs with higher degrading ability.Then,AFB₁ and ZEN transformation products of IlMnP5 and IlMnP6 were analyzed by using UHPLC-MS/MS.The oxidation mechanisms and products of laccase were depended on the structures of substrates.Using BsCotA-methyl syringate as a model system,AFB₁ and ZEN transformation products of BsCotA-methyl syringate were analyzed by using UHPLC-MS/MS.Experimental results showed that the laccase-methyl syringate system resulted in oxidation of mycotoxins and a coupling of phenoxy radical to these products.Finally,hydra,engineered Saccharomyces cerevisiae strain BLYES,and Caco-2 were used to determine the residual toxicity of AFB₁ and ZEN treated with MnP and laccase.From these experiments,a preliminary impression was deduced that the action of MnP and laccase may lead to detoxification of the mycotoxins.We studied the high-level expression of manganese peroxidase and laccase.Two expression systems commonly used in feed enzyme industrial production,P.pastoris and T.reesei,were used as hosts for the expression of MnP and laccase which provided references to further production.Expressing the manganese peroxidase gene from M.roreri in P.pastoris strain X33 through fed-batch cultivation.The manganese peroxidase activity up to 2,074.1 U/L when cultured for 108 h.The laccase gene from M.albomyces was expressed in T.reesei SUS1.In the fed-batch cultivation,the maximal laccase activity reached 11,703.7 U/L when cultured for 91 h.