Cloning, Expression And Characterization Of A Gene Of Don Catabolic Enzymes From NJA-1

Feed are contaminated by mycotoxins which produced by molds during them growth. In China, it is common that feed and food are contaminated by mycotoxins which include deoxynivalenol (DON), fumigatus and zearalenone. DON belongs to trichothecene B, and it is produced by fusarium. DON is widespread in nature and contaminates cereals and their products.Mycotoxin detoxification methods can be divided into three categories:physical, chemical and biological detoxification. Physical detoxification and chemical detoxification are the traditonal detoxification method and are the most widely used method. But these methods have some disadvantages that are loss of feed nutrients and toxins can not be eradicated. Biological detoxification method have several advantages that fungal toxins can be degradaed in gentle condition without using harmful chemicals and it does not affect the palatability and nutritional value without material loss.Recent studies have found that some microorganisms of rumen and soil could produce epoxide hydrolase. These epoxide hydrolase can removal the epoxy ring to decrease the toxic effect of trichothecene. One strain of Aspergillus tubingensis was isolated from soil that it can transform DON. The strain was named NJA-1. The aim of the experiment was to amplify the epoxide hydrolase gene of NJA-1 and to recombine the PCR product and pET-32a (+). And BL21 will be transformed by recombinant plasmid pET-EHse. Study the activity of fussion protein and the correlated conditions which affect enzyme activity, to gain optimal reaction conditions.Test I:Cloning of EH gene in NJA-1 strains and constructing prokaryotic expression vectorNJA-1 was isolated from soil and kept in our laboratory. The purpose of the test was to extract total RNA of the strain and to get the product of EH gene by RT-PCR. Use the product to construct prokaryotic expression vector pET-EHse. E.coli BL21 was be transform by the recombinant plasmid. The results showed that total RNA of NJA-1 by the method of RNAisoTM Plus homogenization had good integrity and no effect on downstream tests. The EH gene was be amplified specifily by a pair of primer which was named EHal and EHa2. Got the E.coli BL21 containing the recombinant plasmid pET-EHse by double digestion, connection and transformation.Test II:The expression, analysis and purification of fusion proteins sEHThe aim of the test was that E.coli BL21 containing recombinant plasmid pET-EHse expressed fusion proteins sEH by IPTG induction. To analyze the protein content of total cellular protein, medium, periplasm, cell soluble fraction and insoluble fraction of cell fusion protein by SDS-PAGE gel electrophoresis to determine the expression position of fusion protein. And through the Ni affinity chromatography method, the fusion protein was purified. The results showed that the cell periplasm contained most part of fusion protein. When the final concentration of IPTG was 0.4 mol·L-1, at 25 ℃ for 16h, it was available to get more soluble protein, and protein expression was up to 2.3g·L-1. Soluble protein was purified by using Ni column and got high purity fusion protein.Test III:Study on the nature of fusion protein sEHThe aim of the tes was to extract fusion protein sEH by freezing and muramidase treatment, purify fusion protein by Ni column and study the nature of fusion protein sEH. The results show that sEH optimum pH is 7.0, the enzyme was stable in pH6.5～7.5 range; optimal temperature was 35℃, and sEH had good thermal stability at 4℃～30℃; the activity of sEH was promote by acetonitrile; Cu2+ could significantly promote the activity and be used as an activator of the enzyme; Mn2+ could also promote the activity.