Fermentation Optimization And Application Of The Zearalenone-degrading Engineering Strain

Zearalenone (ZEN), a estrogen Fusarium toxic produced by some fungal species of the genera Fusarium, is a secondary metabolite. ZEN causes harmful effects on animal and human health because of its highly toxic properties. The food security was threatened by the serious pollution of ZEN which often result in the economic losses significantly, such as the feed and livestock breeding, as well as international trade disputes. Traditional physical and chemical methods were poor operation, incomplete detoxication food nutrient destruction and easy to lead to secondary pollution. Biodegradation has become the technology way to solve food pollution by ZEN for the non-toxic products and environmental protection. The study based on preliminary work included optimization and transformation of the gene engineered strain, optimization of fermentation conditions, application of the degrading enzymes.The main results are as follows:In order to improve the expression of ZEN degrading enzyme further, further molecular transformation and induced expression of engineering E. coliZPR6and P. pastoris NZPP11wered maked. The further improve of expression level of degrading enzyme was proved by the SDS-PAGE protein electrophoresis and quantification. Meanwhile, the mass spectrum analysis of degradation product of the purified enzyme solution prove that degrading enzyme has the same degradation mechanisms with the gene encoding strain.In order to research the characteristic of ZEN degrading enzyme, using high-performance liquid chromatography(HPLC)method to detect the degradation rate of ZEN influenced by temperature, pH and metal ions. The results indicated that the optimum reaction temperature of degrading enzyme was37℃and a better thermal stability below50℃. The optimum pH for enzymatic hydrolysis reaction was8.5,and acertain tolerate in acidic environment.Mg2+can activate the enzyme, Cu2+, Mn2+, Co2+, Ca2+, Zn2+, K+has an inhibitory effect on the enzyme.The screening of the fermentation medium, a cost-effective medium for P.pastoris NZPP11was obtained. Considering the elements of yeast growth and the fermentation medium common used, a optimized medium formulation was determined by the response surface method. The summary of formulation is that3.5%glycerol,0.65%yeast extract,10%phosphate buffer,1.25%ammonia sulfate,0.45%magnesium sulfate,0.04%calcium sulfate. We have the same density of bacteria between the optimized medium and BMGY medium after3days.Studies show that the ZEN degrading enzyme has a significant degradation on the DDGS which is the byproduct of the fuel ethanol production.The experiment results showed that the degradation rate of ZEN is72.70%with adding the enzyme into the corn mash and degradation rate of ZEN is94.8%when added the enzyme after the alcoholic fermentation. Furthermore, adding the degrading enzyme into the corn mash emulsified, the ZEN degradation rate reached95.40%.All the results above provide technical support for the industrialization of the ZEN degrading enzymes, which reduce the damage caused by ZEN and protect the reasonable utilizationin maize grain polluted by ZEN.