抗生素合成中关键酶的高效表达任务书
2020-05-22 21:11:00
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，探讨青霉素酰化酶的高效表达策略，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. 将实验前期获得的一段编码青霉素酰化酶的基因进行改造，替换原有信号肽为大肠杆菌常用信号肽pelb, 并构建表达载体pet22b/pelb-pgapx04，将其导入大肠杆菌宿主bl21中。
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] Kameda Y, Y Kimura, E Toyoura, et al. A method for isolating bacteria capable of producing 6-amino-penicillinic acid from benzylpenicillin [J]. Nature(London), 1961, 191: 1122-1123. [14] 王祯祥, 韩文珍, 门大鹏, 等. 胞外青霉素酰化酶产生菌的选择 [J]. 微生物学报, 1992, 32(2): 99-194. [15]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.
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3. 毕业设计（论文）进程安排
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起讫日期 |
设计（论文）各阶段工作内容 |
备 注 |
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2015.12～2016.1 |
查阅、收集文献资料 |
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2016.3～2016.3 |
理论学习及总体方案规划 |
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2016.3～2016.4 |
克隆去除信号肽的目的基因，构建表达载体pET22b/pelB-pgapx04 |
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2016.4～2016.4 |
将表达载体导入到宿主大肠杆菌BL21中 |
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2016.4～2016.5 |
优化重组菌的诱导表达条件 |
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2016.5～2016.5 |
论文撰写 |
