Mutational Modification?Secretion Expression And Application Of Zearalenone Degrading Enzyme Zhd101

Zearalenone?ZEN?is a non-steroidal estrogen mycotoxin produced by Fusarium.Because of its estrogen toxicity,it can cause reproductive disorders in animals and humans,and induce various types of cancer.ZEN can bring huge losses to the breeding industry and have a great threat on human health,Therefore,it is of great significance for human health and food industry to study the degradation of zearalenone.The zearalenone degrading enzyme Zhd101 can cleave the lactone bond of ZEN to achieve the purpose of detoxification.This paper aims to improve the degradation activity of Zhd101 on ZEN and achieve the secretory expression of mutant enzymes.Firstly,based on the wild-type zearalenone degrading enzyme expression vector pET28a-zhd101,the primers were designed according to the degenerate codon NDT principle for site-directed mutagenesis,the mutant Zhd101?V153H-V158F?with improved enzyme activity was screened and the mutant was named Zhd101.1.Compared with the wild type enzyme Zhd101,the ZEN degradation rate of the mutant enzyme Zhd101.1 was increased by5.79%,and the enzyme activity was 1.10 times that of the wild type enzyme Zhd101.Secondly,the enzymatic properties of the mutant enzyme Zhd101.1 were studied.The optimum reaction temperature of the enzyme was 40°C,and its thermal stability was maintained at 40°C for 3 h.The degradation rate of ZEN can be kept above 70.00%,Its optimum pH 8.0,and pH stability is maintained in the pH 8.0 Tris-HCl buffer solution for 3 h,the degradation rate of ZEN can be maintained above 70.00%,metal ions Na⁺?Mn²⁺?Ca²⁺?Li⁺?Mg²⁺and K⁺have different activation effects on the enzyme activity of the enzyme Zhd101.1.The activation of Li⁺and Ca²⁺is relatively large and the degradation rate of ZEN is increased by 8.66%and 7.92%respectively under the system of 5.0 mmol/L.Thirdiy,in order to achieve the exocrine expression of the mutant enzyme Zhd101.1,an attempt was made in Bacillus subtilis WB800,and twelve different signal peptide coding sequences were introduced,but the secreted expression of the mutant enzyme Zhd101.1 was not achieved.Further attempted to secrete and express the mutant enzyme Zhd101.1 in K.lactis,and constructed the recombinant K.lactis GG799?pKLAC1-zhd101.1?,which fermented the supernatant mutant enzyme Zhd101.1.The activity reached 84.68 U/mg,and the secreted expression of the enzyme in K.lactis was successfully achieved.Fourthly,the conditions for inducing expression of recombinant K.lactis GG799?pKLAC1-zhd101.1?and the conditions for degradation of ZEN in fermentation supernatant were optimized.The optimal induction conditions were:induction time was 84 h,induction temperature was 28°C,The induction medium pH was 7.5,and the galactose concentration of the induction medium system was 60.0 g/L.Under this condition,the degradation rate of ZEN reached 61.45%,an increase of 18.54%.The degradation conditions of ZEN were optimized under the optimal induction conditions.In the pH 8.0 buffer,the degradation rate of ZEN reached 96.98%and increased by 35.23%after 30 min at 35°C and 230 rpm.Finally,the recombinant K.lactis GG799?pKLAC1-zhd101.1?fermentation supernatant was applied to the degradation of ZEN in the feed supplemented with ZEN standard.The concentration of the mutant enzyme Zhd101.1 in the reaction system was 49.34 mg/L,and the reaction at 40°C for 12 h.The degradation rate of ZEN in the fermentation supernatant was74.60%.The results showed that the recombinant strain K.lactis GG799?pKLAC1-zhd101.1?can be used to degrade the actual product.