Recombinant Expression Of Zearalenone Degradation Enzyme And Its Application

Zearalenone(ZEN)is a non-steroidal mycotoxin,which widely existed in food crops and its processed products.ZEN degradation enzyme can effectively convert ZEN into low-toxic or non-toxic products,which can effectively solve the problem of ZEN pollution.However,there lack of genetic resources for ZEN degradation enzyme,and the expression level of ZEN degradation enzyme remains generally low.In this paper,Aspergillus niger,Bacillus subtilis and Escherichia coli were used as expression hosts to express ZEN degradation enzymes(ZHD101,ZHD-P,SPSK and Prx).Finally,the high-level intracellular expression and surface-display of the ZEN-degrading enzyme gene in E.coli were realized.A new enzyme ZHD-P with good ZEN-degrading function was discovered and its enzymatic properties were explored.In addition,we established a method by using Photobacterium Phosphoreum to quickly detect the concentration of ZEN in the sample,reflecting the biological toxicity of the product obtained after ZEN was treated with enzyme.The main research contents are as follows:(1)Recombinant expression of ZEN degradation enzyme.Expression vectors were constructed and transformed into the host,including A.niger SH-2,B.subtilis 6051?10,and E.coli BL21.(1)Recombinant expression of ZEN degradation enzyme in A.niger SH-2:The recombinant ZHD101,ZHD-P,SPSK and Prx had low ZEN degradation activities,and the degradation rates were 18.76%,20.78%,6.38%,41.06%,respectively.There was no target protein detected by SDS-PAGE,suggesting that ZEN-degrading enzyme were poorly expressed in A.niger SH-2.(2)Recombinant expression of ZEN degradation enzyme in Bacillus subtilis:The recombinant ZHD101,ZHD-P,SPSK and Prx had low ZEN degradation activities,and the degradation rates were 9.37%,9.61%,6.17%,22.40%,respectively.There was no target protein detected by SDS-PAGE,suggesting that ZEN-degrading enzyme were poorly expressed in Bacillus subtilis 6051?10.(3)Recombinant expression of ZEN degradation enzyme in Escherichia coli:The recombinant ZHD101,ZHD-P and Prx had good activities to degrade ZEN,with degradation rates of100%,while recombinant SPSK had a lower activity to degrade ZEN with a degradation rate of 23.91%.SDS-PAGE results showed that the four ZEN-degrading enzyme transformants produced obvious target proteins,suggesting that ZEN-degrading enzymes(ZHD101,ZHD-P and Prx)were effectively expressed in E.coli.(4)Surface display of ZEN degradation enzyme in Escherichia coli:The surface-displayed ZHD101 and ZHD-P had good ZEN degradation activities,with degradation rates of 100%,while the surface-displayed SPSK and Prx had weak activities,with degradation rates of 11.86%and 42.05%,respectively.(2)Purification by nickel column affinity chromatography and characteristics of the enzymatic properties of ZHD-P.After purification,the specific enzymatic activities of the recombinant enzymes ZHD101,ZHD-P and Prx reached the activity of 124.69 U/mg,191.94U/mg and 0.08 U/mg,respectively.The recombinant SPSK had no obvious ZEN degradation activity.The optimal reaction temperature of the intracellular ZHD-P was 45°C,and the optimal reaction p H was 7.5-9.0.Ba²⁺,K⁺,Mg²⁺,and Na⁺had little influence on its catalytic activity,and Ca²⁺,Co²⁺,Cu²⁺,Mn²⁺,Ni²⁺,Zn²⁺had strong inhibitory effects.The optimum temperature of the surface-displayed ZHD-P was 40?,and the optimum p H was 9.0;Ca²⁺,K⁺,Mg²⁺,Mn²⁺and Na⁺had promoting effects,while Ba²⁺,Co²⁺,Cu²⁺,Ni²⁺,and Zn²⁺had strong inhibitory effects.(3)Detection of the ZEN degradation effect based on the luminescence intensity of P.phosphoreum T3.There was a linear quantitative relationship between the concentration of ZEN and the luminescence inhibition rate of P.phosphoreum T3,and the regression equation is obtained:=0.0069²-0.0190+7.9907(R²=0.9943,y is the luminescence inhibition rate,x is the concentration of ZEN).The biotoxicity of ZEN samples after enzymatic treatment was determined using P.phosphoreum T3.Results showed that all three ZEN degrading enzymes(ZHD101,ZHD-P and Prx)could effectively degrade ZEN into low-toxic products.Therefore,the degradation of ZEN by these three enzymes were effective detoxification.Therefore,the ZEN degradation enzymes(ZHD101,ZHD-P and Prx)expressed in E.coli cells and the surface-displayed(ZHD101 and ZHD-P)showed good ZEN degradation activity.The optimal reaction temperature of the recombinant enzyme ZHD-P was 45°C,and the optimal reaction p H was 7.5-9.0,at the same time it showed good stability in the range of25-40°C and p H 7.0-9.0;the optimum reaction temperature of the surface-displayed ZHD-P was 40?,the optimum reaction p H was 9.0,at the same time it showed good stability in the range of 30-45?and p H 5.0-11.0.In addition,there was a quantitative relationship between the concentration of ZEN and its luminescence inhibition rate against P.phosphoreum T3.The regression equation was:=0.0069²-0.0190+7.9907(R²=0.9943,y was the luminescence inhibition rate,and x was the concentration of ZEN).Three ZEN degrading enzymes(ZHD101,ZHD-P and Prx)could effectively degrade ZEN into low-toxic products,and these three enzymes were effective in detoxifying ZEN.