Study On Separation And Purification Of Zearalenone Degrading Enzyme And Optimization Of Degradation Conditions

Background: Zearalenone(ZEN)is an estrogen like mycotoxin,which can be produced by many Fusarium species.It can cause a variety of toxic effects,especially reproductive toxicity,causing great harm to plants,animals and even human beings.ZEN is one of the most seriously polluted mycotoxins.The detection rate of ZEN in various substances in nature and living environment,especially in cereals and its by-products,has always been at a high level.ZEN can cause huge economic losses to agricultural planting and animal husbandry.In the process of detoxification of ZEN,the traditional physical and chemical methods are not ideal,because the two methods are not efficient and often have some side effects.Biological detoxification has gradually become a new choice for detoxification due to its safety and efficiency.As a new type of biocontrol factor,the research of Bacillus velezensis has been involved in many fields.With the deepening of the research,it will have broad application prospects in disease prevention and control,pesticide development,animal food additive application and so on.Objective: To study the mechanism of a strain of Bacillus velezensis A2,which can degrade ZEN efficiently,so as to provide theoretical basis for the biological detoxification of ZEN,and lay a foundation for the clinical application of biological detoxification of Bacillus velezensis A2.Methods: Using ZEN as the sole carbon source,the residual amount of toxins in each component of Bacillus velezensis A2 co-cultured with ZEN for 72 h was detected by HPLC to locate the detoxification active substance.Different concentrations of ammonium sulfate were used to extract the best reaction components after crude extraction,incubated with ZEN for 24 h.The residual amount of toxin was detected by HPLC to determine the optimal ammonium sulfate concentration.The degradation conditions of the crude degrading enzyme extract were optimized through different reaction times,p H,and temperature.Determine the optimal reaction conditions for degrading enzymes.After denaturing the extracted protein,gel electrophoresis,Coomassie brilliant blue staining and slight decolorization,the bands of the gel entry are subjected to protein profile detection.Results: HPLC detection of the toxin residues in the fermentation broth,bacteria,intracellular fluid,cell components and inactivated bacteria components of Bacillus velezensis A2 after co-cultivation with ZEN for 72 h showed that the active substances that can degrade ZEN toxins exist in the fermentation broth of the strain,the degradation rate exceeds 80 %.After the fermentation broth protein with different concentrations of ammonium sulfate is extracted and incubated with ZEN,It was found that using 80% ammonium sulfate to extract the protein in the sample can extract more degrading active substances.Through crude extraction of degrading enzymes,the research on optimization of degradation conditions for the degrading enzymes found that the optimal degradation conditions for the degradation enzymes are p H 8 and 37 ? for 36 h.After detecting the protein profile of the gel entry band,according to the GO analysis method,it is inferred that NADPH dehydrogenase and phenolic acid decarboxylase enzymes are ZEN degrading enzymes produced by Bacillus velezensis A2 strain.Conclusion: The crude enzyme extracted from the fermentation supernatant of Bacillus velezensis A2 can effectively degrade ZEN,and the optimal reaction conditions are p H 8 and37 ? for 36 h.According to the results of mass spectrometry,ZEN degrading enzymes are NADPH dehydrogenase and phenolic acid decarboxylase.