Study On Expression Of Manganese Peroxidase And Its Degradation Of Aflatoxin By Recombinant Yeast

Aflatoxin B₁?Aflatoxin B₁,AFB₁?was a natural high-toxic secondary metabolite,widely presented in nature,could easily contaminate peanuts,corn,rice and other grains,and it was classified as a Class?carcinogen by the World Health Organization's Cancer Research Agency?IARC?.AFB₁ posed a great threat to the health and safety of humans and livestock.Therefore,studying the degradation and detoxification of AFB₁ was of great significance to the food and feed processing industry.Manganese peroxidase?MnP?could cleave the double bond at the 8,9 position of the AFB₁ furan ring,oxidize AFB₁ to form AFB₁-8,9 epoxide,and the epoxide produced by MnP oxidation will spontaneously hydrolyze to AFB₁-8,9-dihydrodiol,so as to achieve the purpose of detoxifying AFB₁.In this paper,the secretory expression of manganese peroxidase from three different sources was achieved in food-grade Kluyveromyces lactis,and the induction conditions and reaction conditions of the recombinant bacteria with the best AFB₁degradation effect were optimized,so as to realize the direct use of its fermentation broth to efficiently degrade AFB₁,the specific research is as follows:1.This study realized the secretion and expression of MnP from three different sources in K.lactis,and successfully constructed recombinant yeasts GG799?p KLAC1-Phsmnp?,GG799?p KLAC1-Plomnp?and GG799?pKLAC1-Phcmnp?.It was found that the AFB₁ degradation rates of the three crude recombinant fermentation enzymes that have been successful constructed were 35.55±3.30%,40.02±1.77%,and 50.52±3.69%.Among them,the fermentation broth of recombinant GG799?pKLAC1-Phcmnp?had the best degradation effect on AFB₁,and was significantly different from the other two recombinant bacteria.2.The recombinant GG799?p KLAC1-Phcmnp?induction expression conditions were optimized.The optimal induction expression conditions were:induction temperature 30°C,Hemin concentration 1.0 mmol/L,induction time 96 h,MnSO₄ concentration 1.0 mmol/L,speed 200 rpm,initial medium pH 6.5,galactose concentration 50.0 g/L.Expression was induced under this condition,and the degradation rate of recombinant enzyme GG799?p KLAC1-Phcmnp?fermentation broth towards AFB₁ was 67.40±0.74%.The degradation rate increased by 16.88%compared with that before optimization,.3.The reaction conditions of AFB₁ degradation by recombinant enzyme GG799?p KLAC1-Phcmnp?crude fermentation broth were optimized.The optimal reaction conditions were:pH in the reaction system was 4.5,reaction temperature was 40°C,and MnSO₄concentration was 1.2 mmol/L The glucose content was 2.5 mmol/L,the reaction time was 40h,the protein content in the fermentation broth was 3.0 g/L,and the glucose oxidase content was 1.5 U/mL.The degradation reaction was carried out under this condition,and it was found that the degradation rate of recombinant enzyme GG799?pKLAC1-Phcmnp?fermentation broth towards AFB₁ was 75.71±1.21%.The degradation rate increased by 8.31%compared with that before optimization,.4.The products produced after the degradation of AFB₁ by recombinant GG799?p KLAC1-Phcmnp?fermentation broth were analyzed.A large number of[M+H]⁺ions of m/z346.681 were found.It was speculated that Mn³⁺,peroxy free radicals,formate free radicals and superoxide anion free radicals generated during the self-circulation reaction of Mn P could oxidize the 8,9 vinyl group of AFB₁,and the resulting epoxide would spontaneously hydrolyze to produce AFB₁-8,9 dihydrodiol.5.The degradation effect of recombinant enzyme GG799?p KLAC1-Phcmnp?fermentation broth on the degradation of AFB₁ in contaminated peanut samples was studied,and it was found that the degradation rate of peanut samples contaminated by AFB₁ in different degrees reached more than 74.5%.Therefore,it was considered that the fermentation broth of recombinant bacteria GG799?p KLAC1-Phcmnp?fermentation had potential value for the degradation of AFB₁ in actual products.6.The recombinant strain GG799?pKLAC1-Phcmnp?constructed in this paper was passaged 6 times in succession.There was no significant difference in the degradation effect on AFB₁ between the first 5 generations strain fermentation fermentation broth and the original strain fermentation fermentation broth?p>0.05?,and the degradation rates were all more than72.0%.Therefore,the stability of the genetically engineered bacteria constructed in this paper was good.