Cloning And Heterologous Expression Of Amine Oxidase Gene And Its Ability To Degrade Fumonisins

Fumonisins are mainly metabolites produced by fungi such as Fusarium,and are a kind of mycotoxins with relatively common pollution distribution.Toxigenic bacteria and fumonisins mainly exist in some corn,wheat,and other food crops and their products,which cause them to endanger the health of humans and animals along the food chain.In recent years,the food safety pollution of grains has been seriously harmed by mycotoxins.Among them,the proportion of fumonisins to food hazards is gradually increasing,and fumonisins B1(FB1)is the most toxic and serious pollution of the fumonisins.Therefore,how to degrade the harm of FB1 has become a research hotspot.Amine oxidase(AO)is a special protein that can catalyze the oxidative deamination of amines.At present,there is a lack of research on the degradation of FB1 by amine oxidase at home and abroad.Therefore,it is very necessary to study the degradation of FB1 by amine oxidase.The results of this paper are as follows:(1)After heterologous expression of the screened six amine oxidase genes in the E.coli expression system,the recombinant proteins were subjected to fumonisin activity analysis by high-performance liquid chromatography,and three new fumonisins were successfully obtained.Amine oxidases ACAO,ASAO,and NDAO of toxin-degrading capacity.(2)The degradation products of fumonisins by carboxylesterase and amine oxidase were preliminarily analyzed by LC-MS,and the pathway of fumonisin degradation by carboxylesterase and amine oxidase was speculated.(3)The enzymatic properties of amine oxidase ACAO,ASAO,and NDAO were analyzed respectively.The optimum temperature of amine oxidase ACAO and ASAO is 45℃,and that of NDAO is 40℃;amine oxidase ACAO can still maintain 71.86% relative activity in the high-temperature environment of 45℃ for 60 min;the optimum p H of amine oxidase ACAO,ASAO,and NDAO are all 9;when p H=10,amine oxidase ACAO and ASAO all showed relative activities of 99.36% and 93.60%,when p H=11,NDAO still had a relative activity of 91.51%,indicating that all these three enzymes have strong alkali resistance.(4)The optimal induction conditions of amine oxidase ACAO,ASAO and NDAO were analyzed respectively.For amine oxidase ACAO,the effects of different His tag positions and co-expression chaperone proteins on the expression of amine oxidase were analyzed,and it was found that the protein expression level of amine oxidase ACAO-C-p G-KJE8 increased by 321.77% compared with that before optimization;(5)Different flexible and rigid linker-linked fusion vectors were constructed for the genes of carboxyl lipase Fum DPS and amine oxidase ACAO,and the fusion proteins were successfully expressed.The results of HPLC demonstrated that all five fusion proteins were degraded.FB1 capabilities.Combined with the analysis of fusion protein expression and activity results,the expression of fusion protein connected by a rigid linker was higher than that of a flexible linker;comparing the length of the linker,it is found that a longer linker was more favorable than the expression of fusion protein;the activity of different linkerlinked fusion proteins of five groups varied,with the fusion protein consisting of the(G4S)3linker having the highest activity.