1. 毕业设计（论文）的内容和要求

论文内容 青霉素酰化酶（pga）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，但野生型青霉素酰化酶的性能仍然难以满足需求，我们需要深入研究青霉素酰化酶结构，从而可以为高性能酶的改造与进一步开发奠定基础，这对酶法合成β-内酰胺类抗生素具有重大意义。

本文从实验室自行筛选的achromobacter xylosoxidans来源的青霉素酰化酶（axpga）出发，对其关键位点进行分析及饱和突变，筛选得到高效合成氨苄西林的突变子，并结合动力学常数进行分析。

2. 参考文献

[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.

3. 毕业设计（论文）进程安排

起讫日期 设计（论文）各阶段工作内容 备 注 2018.12.28～2019.1.18 查阅、收集文献资料 2019.3.1～2019.3.15 理论学习及总体方案规划 2019.3.16～2019.4.2 对AxPGA的三个位点进行饱和突变 2019.4.3～2019.4.15 筛选在氨苄西林合成中效果较好的突变子 2019.4.16～2019.4.30 结合动力学常数进行分析 2019.5.4～2019.530 论文撰写