Heterologous Expression And Charaterization Of Fumonisin B₁ Degradation Enzyme YD

Fumonisin(FB)is a kind of mycotoxin produced from genus fusarium under a certain temperature and humidity conditions.Fumonisin can interfere with the normal physiology metabolic function of organisms withina narrow concentration range.It can not only greatly contaminate grain crops which will cause reduction of output but also can cause huge damage to humans and animals.Research on the degradation and control of fumonisin has become a research hotspot all over the world.Toxin degradation of transforming mycotoxin to non-toxic or low toxic matter using microorganisms intracellular and extracellular enzyme has a lot of advantages of high efficiency,no loss of nutrition.The engineering strains YD preserved in the laboratory were selected as the research objects.The FBi degrading enzyme YD was purified by Ni+ affinity chromatography,and the specific activity of emzyme YD is 1.7 U/mg which was 9.4 fold than crude enzyme.The result-indicated that the optimum reaction temperature of degrading enzyme was 30 ℃and a better thermal stability below 10℃.The optimum pH for enzymatic hydrolysis reaction were 7-8,and a better thermal stability in the range from pH 6 to pH7.Mg2+can active the enzyme in high or low concentration,Cu2+、Ni+、Zn2+、Co2+、Mn2+and EDTA has an inhibitory effect on the enzyme.The Kw value of the enzyme was 0.11 μM and the Vmax value of the enzyme was 0.87 μM/(mg min).The fumonisin B1(FBI)degradation enzyme gene ypfd have bee inserted into the modified shuttle vector pSWB980 by restriction endonuclease sites and the recombinant plasmid pSWB980-ypfd was then transformed into competent cells of E.coli DH5α.Nucleotide-sequencing analysis was confirmed that the cloned DNA fragment contained the target gene sequence of 1673 nucleotides.The recombinant pSWB980-ypfd-Bs 168 was obtained by electrotransformation of pSWB980-ypfd into Bs 168.The positive transformant pSWB980-ypfd-Bs 168 was grown on Bacillus subtilis common fermentation medium and target protein was secreted into the medium.The activity was detected after 3 h of fermentation.Itreached the highest level at 12 h of fermentation with the activity of degrading enzyme of 19.15 U/mL.The residual rate of FB1 was up to 20.43%and 39.71%respectively when the fermentation supernatant of engineering bacteriumpSWB980-ypfd-Bs 168 at the optimal enzyme activity work together with waste mash(the content of FB,was 900 μg/kg)and DDGS(the content of FB1 was 4700 μg/kg)under condition of 15 h.After treated with degradation enzyme,the toxin content of the sample was significantly reduced.In summary,the results of this study gave a support to the industrialized production of FB1 degradation enzyme,developed technology of reducing the harm of FB1,and also ensured the safety of food or feed.