Study On Construction And Application Of Recombinant Yeast For Simultaneous Degradation Of Aflatoxin B₁ And Zearalenone

Mycotoxin contamination in food is a global threat to human health and safety.Among them aflatoxin B₁(AFB₁)and zearalenone(ZEN)are two predominant mycotoxins ubiquitously found in corn,peanuts,and other grains,which cause a variety of diseases such as liver cancer,growth retardation,reproductive system damage,posing a great threat to the health of humans and livestock.Although various methods are available for the degradation of these two mycotoxins,there is a lack of research on the simultaneous degradation of these two mycotoxins by biological method.Due to the prevalence of AFB₁ and ZEN contamination in cereals,there is an urgent need for a method that can achieve the simultaneous degradation of both toxins in a gentle and efficient manner.Zearalenone hydrolase ZHD101.1 was obtained by mutation of ZHD101,with a degradation ratio of 42.91%for ZEN before optimization.The manganese peroxidase Phc Mnp was able to detoxify AFB₁ by breaking the double bond at the8,9 position on the furan ring,with a degradation ratio of 50.52%before optimization.Both enzymes were secreted in the food-grade yeast Kluyveromyces lactis.Therefore,in this paper,a fusion enzyme of ZHD101.1 and Phc Mnp was constructed using the peptide of(GGGGS)_n(n=1?4),and the fusion enzyme was expressed by Kluyveromyces lactis.The fusion enzyme was then studied and optimized for the simultaneous degradation of both toxins by the fusion enzyme expression product.The specific studies were as follows.1.Multiple units of flexible linker peptide(GGGGS)_n(n=1?4)were attempted to link zearalenone hydrolase ZHD101.1 and manganese peroxidase Phc Mnp in this study.A fusion enzyme ZPF1 constructed with one unit of GGGGS and a food-grade recombinant Kluyveromyces lactis GG799(p KLAC1-ZPF1)were successfully obtained.The degradation ratios of AFB₁ and ZEN by the fermentation broth of this recombinant strain were 46.46%and35.38%,respectively.2.The induction expression conditions of recombinant GG799(p KLAC1-ZPF1)were optimized and the optimal conditions were:inducing at 25?for 72 h,YEPG medium containing 40.0 g/L galactose,0.5 mmol/L Mn SO₄,and 0.2 mmol/L hemin.3.The degradation efficiency of AFB₁ and ZEN by fusion enzyme ZPF1 in two reaction systems was investigated,and the reaction conditions were optimized.(1)The optimal conditions for degradation of both mycotoxins by the ZPF1 in reaction system 1(Phc Mnp reaction system)were:malonic acid buffer(p H 4.0)containing 0.2 mmol/L Mn SO₄ and 1.0 mmol/L H₂O₂,added with 2.0 mg/m L expressed proteins,reacted at 40?for 8h.Under optimized conditions,the degradation ratios of AFB₁ and ZEN both reached the maximum data of 64.11±2.93%and 46.21±3.17%respectively,an increase of 17.65%and7.45%,respectively.(2)The fusion enzyme ZPF1 in reaction system 2(ZHD101.1 reaction system)had no degradation effect on AFB₁,and the optimal reaction conditions for the degradation of ZEN were as follows:a Tris-HCl(p H 7.5)buffer containing 2.0 mg/m L of fermentation broth protein was reacted at 40°C for 30 min,and the degradation rate was 41.45±2.24%,an increase of6.07%.4.In order to obtain a huge number of yeast preparation,the medium compositions and culture conditions for the culture of recombinant GG799(p KLAC1-ZPF1)were optimized to increase the biomass.The optimal medium compositions were:20.409 g/L glucose,30.455 g/L peptone,24.725 g/L yeast extract,4.477 g/L KH₂PO₄,0.646 g/L Mg SO₄.The optimal culture conditions were:26?,220 rpm,and initial p H 4.5.The culture with a 5 L fermenter was able to reach a biomass of 34.05±3.34 g/L,which is 3.95 times higher than the normal shake flask.5.The preparation technological conditions(centrifugation conditions,ratio of protectant,drying method and storage method)of yeast preparation were optimized.The optimal preparation processes were:centrifugation at 6000 rpm for 15 min to collect the yeast,using spray drying method with the ratio of protective agent to yeast cells at 3:1,and stored at-20?.Degradation tests were performed after three months of storage,and the degradation ratios for AFB₁ and ZEN in reaction system 1 were 48.18±3.23%and 34.83±2.82%,respectively.The degradation ratio of ZEN in reaction system 2 was 30.1±2.73%.