抗生素合成中关键酶的分离纯化任务书
2020-05-26 20:46:54
1. 毕业设计（论文）的内容和要求
青霉素酰化酶具有良好的催化酰化/去酰化特性，因此在医药工业中发挥着重要的作用，主要应用于半合成青霉素和头孢菌素类的工业生产，可以催化制备高效、广谱、适用于不同用途的新型β-内酰胺抗生素。尽管青霉素酰化酶的研究以开展多年并取得重要进展，但基础性的深入研究和大规模工业生产应用所需的优良酶类仍然缺乏，因此筛选性能更优越的青霉素酰化酶产生菌对酶法合成β-内酰胺类抗生素具有重要意义。
本课题主要研究内容：
1. 构建蛋白c端含6个组氨酸标签的青霉素酰化酶表达载体 his-pet28a-px02。
2. 参考文献
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[1] Sakaguchi K, Murao S. A new enzyme,penicillin-amidase [J]. J Agril Chem Soc Japan, 1950, 23: 411-416. [2] Rolisonm G N. Formation of 6-aminopenicillin acid fron penicillin by enzymatic hydrolysis [J]. Nature, 1960, 187: 236-237. [3] Vadamme E J. Enzyme involved in β-lactam antibiotic biosynthesis [J]. Adv Appl Microbiol, 1977, 21: 89-123. [4] Vadamme E J, Votes J P. Microbial penicillin acylase [J]. Adv Appl Microbiol, 1974, 17: 311-369. [5] Martin L, M.A.Prieto, E.Cortes, et al. Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945 [J].FEMS Microbiol Lett, 1995, 125: 2879-2923. [6] Ohashi H, Katsuta Y, Hashizaume T, et al. Molecular cloning of the penicillin G acylase gene from Arthrobater viscosus [J]. Appl Environ Microbiol, 1988, 54(11):2 603-26063. [7] Schumacher G, D Sizmann,H Haug, P Buckel, et al. Penicillin acylase from E.coli:unique gene-protein relation [J]. Nucleic Acids Res, 1986, 14: 5713-5727. [8] Verhaert R M, Riemens A M, van der Laan J M, et al. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis [J].Appl Microbiol, 2003, 62: 507-516. [9] Plhackova K, Becka S, Skrob F, et al. Isolation and chataterization of a new strain of Achromobacter sp. with β-lactam antibiotic acylase activity [J]. Appl Environ Microbiol, 1997, 63(9): 3412-3418. [10] Barbero J L, Buesa J M, de Buitrago GG, et al. Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila [J]. Gene, 1986, 49: 69-80. [11] Ljubijankic G M, Konstantinovic, and Glisin V. The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases [J]. DNA Seq, 1992, 3: 195-200. [12] A Matsuda, K I Komatsu. Molecular cloning and structure of the gene for 7-beta-(4-carboxybutanamido) cephalosporanic acid acylase from a Pseudomonas strain [J]. J.Bacteriol, 1985, 163(3): 1222-1228. [13] Be#269;ka S, #352;těp#225;nek V, Vyasarayani R W, et al. Penicillin G acylase from Achromobacter sp. CCM 4824[J]. Applied microbiology and biotechnology, 2014, 98(3): 1195-1203. |
3. 毕业设计（论文）进程安排
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2015.12～2016.1 |
查阅、收集文献资料 |
阅读文献综述、开题报告。 |
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2016.3～2016.3 |
理论学习及总体方案规划 |
了解实验的目的，规划最佳实验方案。 |
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2016.3～2016.4 |
青霉素酰化酶连接6个组氨酸标签构建 |
构建青霉素酰化酶基因重组质粒，导入大肠杆菌宿主内高效表达。 |
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2016.4～2016.4 |
发酵 |
发酵通过破碎得到青霉素酰化酶。 |
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2016.4～2016.5 |
青霉素酰化酶的蛋白纯化 |
摸索出Ni纯化的最佳条件，对青霉素酰化酶进行蛋白纯化。 |
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2016.5～2016.5 |
论文撰写 |
完成论文。 |
