| 灰色链霉菌胰蛋白酶在真核系统中的异源表达研究 |
| Alternative Title | STUDY ON HETEROLOGOUS EXPRESSION OF STREPTOMYCES GRISEUS TRYPSIN IN
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| 向泊霖 |
| Subtype | 学士
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| Thesis Advisor | 令桢民
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| 2020-06-05
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 理学学士
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| Degree Discipline | 理学-生物学-微生物学
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| Keyword | 灰色链霉菌
胰蛋白酶
酿酒酵母菌
毕赤酵母菌
异源表达
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| Abstract | ~胰蛋白酶(trypsin，EC 3.4.21.4)是一种可以专一选择切割由羧基和碱性氨基酸形成的酯键或酰胺键的丝氨酸蛋白酶。它在在食品、医药和皮革等工业领域中有着广泛的应用前景。目前绝大部分的胰蛋白酶是从猪、牛、羊的胰脏中获取，但提取胰腺素的传统方法复杂，且胰腺素产品价格昂贵，不适合工业生产。其次，有关链霉菌胰蛋白酶的研究比较详细，与哺乳类的胰蛋白酶相比，其组成单一、水解作用缓和、提取及精制工艺简单，因此在应用酶研究中受到了广泛关注。灰色链霉菌胰蛋白酶存在着表达量低的问题。因此，本研究尝试通过异源表达系统解决上述问题。首先将链霉菌胰蛋白酶酶原基因和成熟酶基因，通过不同的组合方式，分别连接到表达载体后，在酿酒酵母中利用组成型启动子进行异源表达及分析，对链霉菌胰蛋白酶在酿酒酵母系统中的表达进行了原因分析和总结其次将链霉菌胰蛋白酶基因在毕赤酵母菌中进行了表达，并对其在组成型表达和诱导型表达的差别进行比较分析。最终得出结论，酿酒酵母菌的表达产量与酶活均比较低，其最高酶活为0.48U/ml而毕赤酵母相比于前者，有着明显的优势，其最高酶活为7.2U/ml同时，通过对比前导肽对酶活性的影响，证明了其对胰蛋白酶的活性有着抑制作用。 |
| Other Abstract | ~Trypsin (EC 3.4.21.4) is a serine protease that can specifically cleave ester bonds or amide bonds formed by carboxyl groups and basic amino acids. It has wide application prospects in the industrial fields of food, medicine and leather. At present, most of the trypsin is obtained from the pancreas of pigs, cattle, and sheep, but the traditional method of extracting pancreatin is complicated, and the price of pancreatin is expensive, which is not suitable for industrial production. Secondly, the study on trypsin of Streptomyces is more detailed. Compared with mammalian trypsin, its composition is simple, its hydrolysis is mild, and its extraction and purification process is simple. Therefore, it has received extensive attention in the research of applied enzymes. Streptomyces griseus trypsin has the problem of low expression. Therefore, this study attempts to solve the above problems through heterologous expression systems. First, the Streptomyces trypsinogen gene and the mature enzyme gene were connected to the expression vector by different combinations, and the constitutive promoter was used for heterologous expression and analysis in Saccharomyces cerevisiae. The reasons for the expression in the yeast system were analyzed and summarized. Secondly, the trypsin gene of Streptomyces was expressed in Pichia pastoris, and the difference between constitutive expression and inducible expression was compared and analyzed. Finally, it was concluded that the expression yield and enzyme activity of Saccharomyces cerevisiae were relatively low, and the highest enzyme activity was 0.48U / mlPichia pastoris had a clear advantage over the former, and its highest enzyme activity was 7.2U / At the same time, by comparing the effect of the leader peptide on enzyme activity, it was proved that it has an inhibitory effect on trypsin activity. |
| Pages | 30
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| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/466500
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| Collection | 生命科学学院
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| Affiliation | 生命科学学院
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| First Author Affilication | School of Life Sciences
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Recommended Citation
GB/T 7714 |
向泊霖. 灰色链霉菌胰蛋白酶在真核系统中的异源表达研究[D]. 兰州. 兰州大学,2020.
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