Broad-spectrum And Efficient Degradation Of Six Major Mycotoxins In Feed By Laccase Lac-W

Multiple mycotoxin co-contamination is widespread all over the world and poses a serious threat to human and animal health.It is a great challenge to solve the mycotoxin co-contamination in a green and efficient way worldwide.Compared with traditional physical or chemical methods,enzymatic method has gradually become an important and effective method to control mycotoxin co-contamination due to its strong specificity and environmental friendliness.In this study,laccase Lac-W,which can degrade six major mycotoxins in feed,was found for the first time.Therefore,this study took laccase Lac-W as the research object to investigate its degradation effect on major mycotoxins in feed and its preliminary application.First of all,laccase Lac-W from Weizmannia coagulans 36D1 is expressed in E.coli BL21(DE3).The purified recombinant Lac-W was obtained by Ni column.Using ABTS as substrate,the specific enzyme activity of Lac-W was 0.142 U/mg.UV-vis spectrum analysis showed that Lac-W had a characteristic absorption peak of Cu ion at 610 nm.The Km value of Lac-W to ABTS is 81.02 μM,kcat is 3.41 s-1,and kcat/Km is 4.208×104 M-1·s-1.The enzymic properties of Lac-W were studied and the results showed that Lac-W had high activity between 0℃ and 100℃,and its relative activity was 110% at 0℃ and 70% at 100℃.Lac-W has excellent properties such as high activity,high thermal stability,wide range of temperature,p H and metal ion tolerance.Subsequently,Lac-W has the ability to degrade major mycotoxins in feed,and can directly degrade six mycotoxins in the absence of mediators,including AFB1,ZEN,DON,T-2 toxins,FB1 and OTA.Taking AFB1 as an example,the optimal condition for degradation of mycotoxin by Lac-W was determined as adding 1 U/L Lac-W into Tris-HCl buffer(p H9.0),and standing at room temperature for 24 h.Under these conditions,the degradation rates were AFB1(93%),ZEN(60%),DON(34%),T-2 toxin(19%),FB1(18%)and OTA(12%),respectively.Molecular docking showed that Lac-W had a large enough substrate binding pocket to allow the entry of six different mycotoxins,which was consistent with the property that Lac-W could degrade six mycotoxins.However,the affinity between Lac-W and the six mycotoxins was different(AFB1>ZEN>DON>T-2 toxin >FB1>OTA),which may account for the different degrading efficiency of Lac-W to degrade the six mycotoxins.Furthermore,the structure of Lac-W-mediated AFB1 degradation product was identified by UPLC-MS/MS,and the results showed that the degradation product of AFB1 was AFQ1.Cytotoxicity of AFB1 degradation products was determined by cell survival rate and cell apoptosis test.The results showed that compared with AFB1,Lac-W-mediated AFB1 degradation products had no effect on the vitality of IPEC-J2 cells,and did not cause the apoptosis of IPEC-J2 cells,indicating that AFB1 could be detoxicated by Lac-W.Finally,the Lac-W-AS system was successfully constructed by screening small molecular mediators,and the degradation conditions were optimized to achieve the complete degradation of AFB1 and ZEN.Lac-W-AS system was applied to pregnant full feed,DDGS and cotton meal,and the degradation of excessive mycotoxins(ZEN and DON)was also achieved.In conclusion,laccase Lac-W,which can degrade six mycotoxins,was found in this study for the first time,and the Lac-W has advantages such as high activity,high thermal stability,wide range of temperature,p H and metal ion tolerance,so that it has great application potential in the detoxification of food and feed co-contaminated by multiple mycotoxins.This study can provide theoretical basis for the exploration of laccase and its related mycotoxin degrading enzymes with broad-spectrum and high catalytic activity,and provide solutions for the common mycotoxin co-contamination problem in animal husbandry industry.