Expression Of A Gene Of Don Catabolic Enzyme From NJA-1in Pichia Pastoris And Analysis Of Their Effects On Don Degradation

Deoxynivalenol (DON) is a kind of widely existing Trichothecenes mycotoxin, which is toxic metabolite of Fusarium graminearum. DON is a kind of global cereal pollutants. DON can cause acute poisoning of humans and animals, and cause serious economic losses. Research data show that12,13epoxide ring of Trichothecenes mycotoxin is the main toxicity group. Once the ring is destroyed, the toxicity can be greatly reduced.Epoxide hydrolase shows a high enantio-selectivity in the catalytic reaction, it can be hydrolyzed to generate an optically active epoxide and the corresponding1,2-diol. The studies found that some microorganisms in the rumen and soil can produce epoxide hydrolase. These epoxide hydrolase can decompose the epoxy groups of the Trichothecenes mycotoxin to decrease the toxicity.One strain named NJA-1was isolated from soil that it can hydrolyze the epoxy ring of DON. The results showed that the epoxide hydrolases in NJA-1play a key role in the transforming DON. The aim of the experiment is to amplify the epoxide hydrolase gene of NJA-1and recombine the PCR product and the plasmid pPIC9K to get the eukaryotic expression vector pPIC9K-EHsq. Then recombinant plasmid pPIC9K-EHsq will be transformed into Pichia pastoris GS115, and the recombinant strain will be induced expression. Using the method of high performance liquid chromatography to study the expression products’degradation effect. All these studies lay the foundation for the experiments of enzyme fermentation and conversion of DON in vivo.Test I:Construction of eukaryotic expression vector of EH gene in NJA-1strains and its expression in Pichia pastorisThrough PCR to get the EH gene from the pET32a (+)-EHse/BL21vector. Then the EH gene was spliced into pPIC9K after digested with Not I and EocR I, then transformed into Pichia pastoris GS115by electroporation. The recombinant yeast strains were screened by methanol utilization phenotype first, and then through G418to screen to get high-copy recombinant yeast strains. At last, through PCR to identify the recombinant strains. Recombinant strains with EH gene were acquired and induced with methanol. Analyzing the protein by SDS-PAGE gel electrophoresis to determine the expression position of the protein. The test results show that the amplified EH genes fragment were successfully spliced into pPIC9K, and the eukaryotic expression vector pPIC9K-EHsq was successfully constructed. SDS-PAGE analysis showed that the genes could be expressed in Pichia pastoris and the target protein was mainly existed in the form of inclusion bodies. The molecular weight of the protein was about74.0kDa.Test II:The detection of the effect of pEH transformation of DON and optimization of transformation ConditionsThe aim of the test was to study the expression products have an effect of transforming DON by HPLC. On this basis, by changing the temperature, adding the metal ion and changing a broken manner to get an optimal conversion condition. We study the relations between transformation conditions and transformation effect. The results showed that pEH can effectively transforming DON. The optimal conditions of transforming DON were:27℃, ultrasound broken way and adding Cu2+of final concentration of10mmol/L.