Screening,Degradation Characteristics And Application Of Deoxynivalenol-Degrading Bacteria

Deoxynivalenol,also known as vomitoxin,is a Trichothecene group B toxins.DON has acute toxicity,chronic toxicity,immunotoxicity and reproductive toxicity,and can cause dizziness,unresponsiveness,loss of appetite,diarrhea and central nervous disorders in humans and animals,seriously endangering their health.DON toxin has been detected worldwide and has caused significant economic losses.The aim of this study is to screen DON degrading bacteria,study their degradation characteristics and degradation mechanisms,clone DON-degrading genes,develop DON degrading bacteria preparations and enzyme preparations,with a view to apply them in feed detoxification and provide degrading bacteria and degrading enzyme resources for DON biodegradation.The main studies are as follows:1)A DON-degrading strain was screened and systematically identified:A strain of DON-degrading bacteria was screened from wheat plots contaminated with wheat Fusarium Head Blight and named as A4.A4 could degrade 9.7μg·m L^-1 of DON in 10 hours,and the degradation rate reached 97%.On LB solid medium,the single colony of A4 appeared waxy yellow and slightly transparent;the bacterium was full,with smooth surface and neat edges;the cells of the bacterium were short rod-shaped,about 1.2μm in length and 0.4μm in width and without flagella.Through morphological characteristics,physiological and biochemical characteristics and molecular biology results,A4 was finally identified as Devosia sp.The growth characteristics experiments showed that the optimum growth temperature was 35℃,p H 7.0 and Na Cl concentration 2.0%,while the optimum temperature and p H for the degrading DON was 35°C and 8.0.The degradation products of DON by A4 were obtained by extraction and ethen purified,and identified by mass spectrometry and nuclear magnetic resonance techniques.The DON degrading product was identified as 3-keto-DON.2)Cloning,expression and enzymatic characterization of DON-degradation gene:The whole genome of A4 was sequenced,analysed for possible degrading genes.The DON-degradation gene a4dd was amplified by PCR,and then heterologously expressed in Escherichia coli.The results of SDS-PAGE gel electrophoresis showed that the size of A4dd was about 70KDa.The results of enzymatic properties showed that the optimum temperature for DON degradation by A4dd was 40°C and the optimum p H was 8.0-10.0.The metal ions Mn²⁺,Ca²⁺and Mg²⁺promoted the DON degradation;the enzyme kinetic constant for the degradation of DON by A4dd was Vmax=39.95 n M·min^-1,Km=144.2 m M.It was determined that amino acid D311 was the key catalytic site for degrading enzyme A4dd by bioinformatics predicting the key catalytic amino acid sites and verifying the mutational degradation effect of the binding points.3)Preliminary study and application of DON degrading bacterial and enzyme preparations:Bacillus subtilis WB800 competent cells were prepared,the recombinant strain WB800-p AX01-a4dd was constructed and was induced to express the DON-degrading enzyme protein A4dd.SDS-PAGE gel electrophoresis showed that no protein bands appeared at 70 KDa,and the results of HPLC showed that the recombinant strain was not effective in degrading DON,which indicates that A4dd was not successfully expressed in Bacillus subtilis.In order to be able to develop practical DON degradation products,enzyme preparations were developed by degrading enzymes expressed in E.coli.It was clarified that when the concentration of sodium alginate solution was 1%,the concentration of calcium chloride solution was 2%,and the immobilization time was 9 min,the degradation effect of enzyme preparations was the best.