Expression Of Zearalenone Degrading Enzyme Gene ZLHY-6 In Bacillus Subtilis

Zearalenone(ZEN)is a toxic secondary metabolite produced by fungi of the genus Fusarium(mainly Fusarium graminearum and Fusarium culmorum),which widely pollutes corn,wheat,barley and other food crops and their by-products,ZEN seriously endangers the health of humans and animals,and causes huge economic losses and adverse social impacts.ZEN detoxification has become an urgent need.ZEN detoxification methods include physical,chemical,and biological methods.However,in the detoxification process,physical and chemical detoxification often changes the nutritional structure of food and feed and may cause secondary pollution to the environment.In contrast,biological enzyme detoxification has the advantages of specificity,environmental friendliness,and mild reaction conditions.In this study,the ZEN degrading enzyme gene ZLHY-6 was used as the target gene,and the p MA5plasmid and Bacillus subtilis 168 strain were modified and constructed to obtain an engineered strain that efficiently secretes zearalenone degrading enzyme,which is expressed in Bacillus subtilis.Mycotoxin degrading enzymes provide a reference,provide certain technical support for the industrial production of zearalenone degrading enzymes,and provide ideas for the effective removal of mycotoxins in agricultural products such as grains.The mian findings are as follows:(1)Synthesize the ZEN degrading enzyme gene ZLHY-6,clone the ZEN degrading enzyme gene ZLHY-6 and the promoter P43 into the Bacillus subtilis expression vector p MA5 through one-step cloning and overlap extension PCR,and construct single promoter(Hpa II)recombination respectively Expression vector and dual promoter(P43,Hpa II)recombinant expression vector.Then the constructed recombinant plasmid was introduced into E.coli JM109 by the heat shock method,and positive clones were screened by ampicillin,the positive clones were digested and verified by electrophoresis,and the positive clones that were initially verified to be correct were sent to BGI for sequencing verification.The sequencing results were consistent with the expected results,proving that the recombinant plasmid was constructed successfully.After identification,we successfully constructed an expression vector p MA5-zlhy-6 containing a single promoter(Hpa II)and an expression vector p MA5-P43-zlhy containing a dual promoter(P43,Hpa II).A two-step method(GM?,GM?)was used to prepare Bacillus subtilis competent cells,and then the obtained recombinant vector was transformed into Bacillus subtilis 168,and positive transformants were screened by kanamycin plates.(2)The promoter P43 from Bacillus subtilis and ZLHY-6 genes were fused with lox71-spc-lox66resistance gene expression cassette by overlap PCR.The fusion fragment was inserted into the amylase gene site of Bacillus subtilis 168 by double exchange integration.Finally,the antibiotic resistance gene,Spc gene was knocked out by the Cre/lox system,generating the recombinant Bacillus subtilis BZ-zlhy expressing zearalenone degrading enzyme based on chromosomal integration free of antibiotic resistance genes.(3)The protein expression of different transformants was compared by SDS-PAGE,and the enzyme activity of the supernatant was tested.It was found that the highest enzyme activity of the recombinant strain with double promoters was 2.2?g·m L^-1,which was 1.2 times higher than that of the single promoter strain.The degradation rate of zearalenone(4?g·m L^-1,30 min)by the zearalenone degrading enzyme expressed by the double-promoter recombinant strain was 65.1%.As for the recombinant Bacillus subtilis BZ-zlhy,its expression level was the lowest(0.4 U·m L^-1).(4)The effect of different conditions on the degradation of ZEN by zearalenone degrading enzyme was explored.The effects of initial p H,temperature,metal ions,etc.on the degradation of ZEN by the zearalenone degrading enzyme were analyzed.It is clear that optimal reaction temperature of the enzyme is 37°C,and the optimal p H is 7.5.Ions Li⁺and Mg²⁺can promote the enzyme activity,however,ions Cu²⁺,Ca²⁺,Mn²⁺,Fe²⁺have an inhibitory effect on the enzyme activity.