Identification And Analysis Of Enzymes Involved In Zearalenone And Deoxynivalenol Detoxification

Zearalenone(ZEA)and deoxynivalenol(DON)are toxic metabolites produced by Fusarium spp.Fusarium graminearum widely exists in wheat,corn and other crops.Under suitable conditions,these fungi are easy to produce mycotoxins,which will cause great harm to the life and health of animals after eating,and also seriously threaten the safety of animal food.Therefore,the prevention and control of mycotoxins,especially biological control,has become a research hotspot.In this study,the functional genes of zearalenone degrading enzymes were further identified.At the same time,the screening of deoxynivalenol degrading microorganisms and the identification and analysis of their degrading enzymes and functional genes were carried out.Experiment 1:Identification and validation of genes encoding of zearalenone degrading enzymeBacillus amyloliquefaciens H6,which can efficiently degrade zearalenone,was screened and its transcriptome was analyzed.Bioinformatics analysis showed that BAMF_RS30125 gene a might be a candidate gene encoding degradation enzyme,so this gene was identified and verified.Preparation of recombinant protein:the recombinant expression vector was constructed,and the expression of recombinant protein was induced by IPTG.The results showed that the recombinant protein was mainly detected in the supernatant.SDS-PAGE and Western blot analysis showed that the recombinant protein was named ZTE138(zearalenone thioesterase 138).Enzyme linked immunosorbent assay(ELISA)and high performance liquid chromatography(HPLC)were used to detect the activity of recombinant protein.The results showed that the degradation rates of recombinant protein to ZEA were 46.15%and 69.28%at 36 h and 72 h,respectively;The degradation rate of ZEA was 34.20%and 59.79%at 36 h and 72 h,respectively.ZEA was treated with fermentationsupernatant containing recombinant protein and evaluated on MCF-7 cells.The results showed that the proliferation rate of MCF-7 cells in the control group was 45.23%,while that in the treatment group was only 20.65%.After 72 h of culture,the proliferation rate of MCF-7 cells in the control group and the treatment group was 93.89%and 34.43%,respectively.At 36 h and 72 h,the proliferation rate of MCF-7 cells in the treatment group was significantly lower than that in the control group(P<0.01),indicating that the degradation of ZEA produced substances with low or no estrogen activity.Experiment 2:Screening and identification of DON degrading strainsUsing deoxynivalenol as the sole carbon source,the faeces of herbivorous animals were used for primary screening and re-screening.The degradation rate of the selected microorganism was evaluated by the DON-ELISA kit.Screening and identification of microorganisms:after initial screening and rescreening,it was found that strain S12 had the highest degradation efficiency of DON,reaching 78.23%.The 16S rRNA gene sequencing of strain S12 showed that it had the highest homology with Acinetobacter pitti ST220(Access No.CP029610.1),and strain S12 was identified as Acinetobacter pitti by Gram staining and bacterial biochemical test.Detection of active ingredient distribution:the fermentation supernatant,intracellular liquid and bacterial suspension of Acinetobacter pitti S12 were prepared respectively,and their degradation efficiency was detected respectively.The results showed that the active substance of DON degradation by Acinetobacter pitti S12 was mainly distributed in the fermentation supernatant,and its activity was lost after heating and SDS treatment,indicating that it was an extracellular protease.Experiment 3:Target analysis of degradation of DON by Acinetobacter pitti S12In Experiment 2,the supernatant contained the degradation product of DON,which was detected and analyzed by Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.The results are as follows,The HPLC-MS mass spectra of the treatment group and the control group showed that the mass spectra of deoxynivalenol in the treatment group sample 002 and the control group sample 001 were different.The m/z of the control group was 265.8124,295.3528,326.7145 and 351.8541,which were[M-CH2O-H]-,[M-H]-,[M+CH2O]-and[M+COONa]-respectively.The ion peak m/z of treatment group was 314.9587,338.9412,355.5521 and 375.2857,which were[M+H2O]-,[M+CH3CO]-,[M+CH3COO]-and[M+PO3]-respectively.In the treatment group,the standard peak of deoxynivalenol disappeared.The molecular weight and formula of the ion peak m/z 338.9412[M+CH3CO]in the treatment group were analyzed,which showed that don transformed into 3-ADON or 15-ADON.Experiment 4:Transcriptome analysis of DON degrading bacteriaBecause Acinetobacter pitti is a conditional pathogen,it can not be directly used in food or feed additives.It is necessary to further study its degradation enzymes and coding genes,so the transcriptome sequencing analysis of Acinetobacter pitti was carried out.Microbial transcriptome sequencing:construct the transcriptome sequencing Library of Acinetobacter pitti S12 for transcriptome sequencing.After sequencing,the differentially expressed genes were screened out,and the functions of up-regulated genes in the differentially expressed genes were analyzed.The DLK06_RS 13370 gene was screened from the limited up-regulated genes with clear function(acyltransferase)may be related to the degradation of deoxynivalenol,which will be the focus of future research.In conclusion,we successfully identified a protease ZTE138(zearalenone thioesterase 138)from Bacillus amyloliquefaciens H6,and found that the protease has the ability of detoxification of zearalenone.A strain of Acinetobacter pitti S12 which can effectively degrade DON toxin was screened and identified.Combined with transcriptome analysis and HPLC-MS analysis,it was found that DLK06_RS13370(acyltransferase)in Acinetobacter pitti S12 may be the key gene involved in DON degradation,and it plays a biotransformation role by transforming DON into 3-ADON or 15-ADON.