Separation,Purification,Gene Cloning And Expression Of Aflatoxin Degradation Enzyme From Bacillus Subtilis

Experiment 1:The aim of the study was to analyze and evaluate the contamination situation of aflatoxin(AF)in ingredients and complete feeds between years,seasons and regions of our country using high performance liquid chromatography(HPLC)method.Moreover,the change rule of AF content in maize growth and storage process was studied.Results showed that 1)in the Beijing area aflatoxin contamination were relatively common in 6 kinds of feed and 12 kinds of feed ingredients in 2013-2015.The content and contamination rate of AF in feed and feed ingredients were highest in 2013.Thought 3 years,DDGS and silage had higher over standard rates than other feed ingredients.2)different seasons and regions would affect the content of AF in feed raw materials.The corn in summer would easily contaminated by AF than in winter.The content of AF in the southwest and the south of China’s corn were higer than northeast and north of China’s corn.3)AF content in corn was influenced by growth environment and storage conditions,AF easily polluted grain in the process of grain growth’s period.An aflatoxin degradation enzyme from Bacillus subtilis ANSB060,designated as Bacillus subtilis aflatoxin degradation enzyme(BADE),have been prepared,separated,purified and identified in experiment 2.BADE was purified from the supernatant of B.subtilis using two times ion-exchange chromatography and hydrophobic chromatography.The apparent molecular mass of BADE was estimated to be 32 kDa by SDS-PAGE.An overall 193-fold purification of the enzyme with a recovery of 35.4%and a final specific activity of 75.25×103 U/mg was obtained using the present purification protocol.And we identified the 21 fragment of amino acid sequence of BADE using gel enzyme degradation and LC-MS.Experiment 3:The aim of this research was to clone gene of Bacillus subtilis aflatoxin degradation enzyme(BADE)and express BADE gene in E.coli Rosseta(DE3).In order to clone gene of BADE,we using amino acid sequence of peptide to design primers,the whole sequence of BADE gene was obtained via PCR.The full length of BADE DNA sequence was 870 bp,encoding 289 amino acids.After analysis,we found that the molecular weight of BADE was about 33.3 kDa,and isoelectric point was about 5.29.The amino acid sequence deduced from BADE gene had a signal peptide(splice site between 29th and 30th amino acid),4 putative phosphorylation sites and no putative N-giycosylation site.We express BADE gene in E.coli Rosseta using the expression vector pET30a.First,the BADE gene and expression vector pET30a were both cleaved by Ⅰ and Ⅰ,and then were used to construct the expression plasmid pET30a-BADE.We using PCR,Finally,the expression plasmid was transformed into E.coli Rosseta.By IPTG inducing,the recombinant protein rBADE was expressed successfully.The optimal expression condition were 0.3 mM IPTG and induction time was 2 h.1 L fermentation culture obtained 1 370 mg rBADE was which having a total activity of450 000 U and a specific activity of 328.5 U/mgto degrade AFB1.In experiment 4 characteristics of rBADE were preliminarily studied and the Ames test was analyzed.Results showed that rBADE have a broad range of pH values,between 6.0 and 8.5,and temperatures,between 25 and 35℃,this enzyme exhibits the largest amount of activity at 30℃ and pH 6.5,with Km=1.55×10-5 mol/L.We have tested the mutagenicity of degradation product of rBADE degrading AFB1 for 108 h using Ames test,and found the degrading products had low toxicity.The results shown that rBADE was a safe AF-detoxification agent.Experiment 5:The toxic effect of AF and zearalenone(ZEA)and their combination on laying performance,egg quality and toxins residues in eggs,as well as the efficacy of Bacillus subtilis biodegradation product(BDP)for ameliorating these effects in layers were evaluated.Layers were submitted to a two phase experiment.The first phase was an intoxication period(18-23 wk)with birds fed 7(3×2+1)diets(3 treatments with mycotoxins:AF(123.0 μg/kg),ZEA(260.2 μg/kg),or AF+ZEA(123.0+260.2 μg/kg);2 treatments with or without BDP(1 000 g/t);and a control group contained neither toxins nor BDP.The next phase was a recovery period(24-29 wk)in which birds were fed a toxin-free diet.In the intoxication period,AF and AF+ZEA groups exhibited lower egg production,feed intake and shell thickness,and higher AFB,,AFB2 and AFM1 residues as compared with the control group(P<0.05).In addition,AF and ZEA exerted synergistic effects on egg production and feed intake.Moreover,AF+ZEA had a continuous toxic effect on laying performance in the recovery phase.Addition of BDP offset these negative effects,showing that BDP has a protective effect on layers fed contaminated diets.