Expression of N5-5 in and purification and identification of products.

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper N5-5 was expressed using system. Specific primers were designed based on the N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector -pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into BL21 (DE3) to induce expression of N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64-65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme's protein identification by Western Blotting showed that clear protein bands -3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.