High Level Secretory Expression And Characterization Of ZEN Degradation Enzyme

Zearalenone(ZEN) is a kind of secondary metabolites which is teratogenic carcinogenic mainly produced by Fusarium graminearum, can contaminate many economically important crops, resulting in serious food and feed safety problems all over the world and bring significant economic losses every year. How to perform a safe and effective detoxification treatment of contaminated food or feed has always been a difficult problem. Traditional physical and chemical methods of detoxification are operability poor, easy to destroy the food nutrients, but also easy to introduce the secondary pollution.Biodegradation are favored because it has the advantages of mild reaction conditions and no harmful reagents residue. In this study, we took the ZEN degradation enzyme gene zlhy-6 as the target gene. In order to get efficient secretion engineering bacteria, the exogenous gene zlhy-6 and expression vector p AO815 were reformed on the basis of previous studies. Then the Characteristics of the recombinant zearalenone degradation enzyme was also studied. The main conclusions are listed as follows:1. Zearalenone degradation enzyme encoded by zlhy-6 was optimized according to codon preference of Pichia pastoris and synthesized along with alpha(α) signal peptide gene, later was cloned into p AO815. Recombinant plasmids containing a series of different copies zlhy-6 were transformed into E.coli TOP10, positive clones were identified by resistance screening. Restriction endonucloase analysis was used to proved that recombinant plasmid was constructed successfully.After restriction enzyme digestion, the recombinant plasmids containing one, two, four, six copies of zlhy-6 gene were generated.2. Rrecombinant vectors were linearized and transformed into Pichia pastoris GS115. Positive transformats were identified through histidine-deficient plate and SDS-PAGE. The protein level of different transformants were compared by SDS-PAGE. The effect of inducing p H, methanol and inoculation amount on reconstruction protein expression were investigated Among these transformants, clones carrying four copies of zlhy-6(p AO815-(α-zlhy-6)4/GS115) showed the highest protein level, which with 10 U/m L in the culture supernatant.3. The activity of recombinant zearalenone degradation enzyme was tested with supernatant from p AO815-(α-zlhy-6)4 transformat. The degradation rate of 99% was achieved in the aqueous solution after reaction 1 h. In the corn ballast(containing 2 mg/kg ZEN) detoxification test, ZEN degradation of about 75.51% was reached when the enzyme dosage was 0.5 m L/g(V/W). When the ZEN contaminated corn gluten meal was deal with enzyme, 92.4% ZEN was degradation at the enzyme dosage of 2 m L/g(V/W).4. Further, the effects of temperature, p H, metal ions on enzyme activity were studied. The optimum temperature and p H of recombinant ZLHY-6 for ZEN degradation were 37 ℃ and 8.0 respectively.Ions Li+ and Mg2+ were activators for ZEN-degradation, however, ions Cu2+, Mn2+, Co2+, Ca2+,Zn2+, Ni2+, Fe3+ and EDTA were strong inhibitors.5. The method of chemical modification was used to explore the functional groups of ZEN degradation enzyme. The results showed that the enzyme active center is closely related to serine and lysine.The results of this study gave a support to the industrialized production of ZEN degradation enzyme, reducing the harm of ZEN and also ensuring the safety of food or feed.