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首页> 外文期刊>Saudi Journal of Biological Sciences >Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products
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Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products

机译：黑曲霉N5-5在大肠杆菌中的表达及产物的纯化与鉴定

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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 Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan -pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger 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 pro -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.
机译：由于鞣酸酶具有高度水解的特点，因此被广泛应用于食品，饮料，酿造等领域。然而，生产天然鞣酸酶的高成本使得难以将鞣酸酶大规模地应用于工业。微生物表达系统可用于低成本制备大量酶，因此在本文中，使用大肠杆菌系统表达黑曲霉N5-5。基于黑曲霉N5-5序列N3（GenBank，No。：KP677552）设计特异性引物，并且通过PCR扩增将鞣酸酶基因tan促进携带6个His标签和包含NdeI / HindIII的酶切位点。然后，通过NdeI / HindIII酶切将鞣酸酶基因tan连接到表达载体。以这种方式，形成了重组表达载体tan -pET43.1a。然后，通过NdeI / HindIII酶切割的表达载体pET43.1a被转化到大肠杆菌BL21（DE3）中以诱导黑曲霉N5-5的表达。超声波破坏诱导的真菌后，使用IMAC提取和纯化粗酶，然后测定粗酶和纯酶的活性。根据鞣酸酶活性的测定结果，纯化粗酶后，粗酶的鞣酸酶活性大大提高，其比活度约为粗酶的8倍。对纯酶的SDS-PAGE结果表明，纯酶的分子量约为65？kDa / 64–65？kDa，与预期结果（64.2？kDa）相吻合，可以得出结论。粗酶溶液成功纯化。 Western Blotting鉴定纯酶蛋白质的结果表明，观察到了清晰的pro -3蛋白带。透明蛋白条带pro-3的分子量约为65？kDa，与预期结果一致（64.2？kDa）。可以看出，上述表达蛋白可以与His标签特异性结合。证明表达蛋白是具有6 His标签的重组融合蛋白。

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