1. 研究目的与意义
1.1背景和目的ε-聚赖氨酸(ε-poly-l-lysine，ε-pl) 是由一分子 l-赖氨酸( l-lys) 的ε-氨基与另一分子l-lys 的α-羧基通过肽键连接而成的 l-lys 同聚物。
相对分子量在 3 500～4 500，它的溶解度在水中相对而言很高，等电点值在 9.0 左右。
ε-pl 是一种固体粉末，呈淡黄色，有较强的吸湿性，略苦；极易溶于水、盐酸，不溶于乙醇、乙醚等有机溶剂，热稳定性好，熔点为 250℃，在 100℃及 120℃分别加热处理 30 min和 20 min，仍能保持抑菌能力，因此方便使用。
2. 研究内容和预期目标
2.1 主要研究内容：本课题研究立足于国内外研究现状，目前国内外应用基因工程手段来选育ε-pl生成菌的研究已初步开展，但还未有利用辅酶调控技术来提高其产量的研究。
本研究将为将来通过调节辅酶的供应来对白色链霉菌ε-pl的合成途径进行分子改造提供一定的参考依据，并为其打下良好的基础。
具体研究内容如下：1）菌体培养及感受态细胞的制备和转化：大肠杆菌在lb平板上37℃培养，或在lb液体培养基中37℃，200 rpm培养。
3. 研究的方法与步骤
3.1 主要研究方法：分子改造。
3.2 工艺流程：设计引物、扩增基因→制备感受态、转化→质粒重组、菌株构建→融合蛋白表达纯化→酶活测定→动力学研究→进行参数分析。
3.3 实验方法：（1）atp/nad激酶基因的上下游引物：这是直接购买得到的，并于4℃冰箱保存。
4. 参考文献
[1] 施庆珊, 陈仪本, 欧阳友生. ε-聚赖氨酸的微生物合成与降解[J]. 生物技术, 2004, 14(6): 77-79. [2] Hiraki J, Ichikawa T, Ninomiya S, et al. Use of ADME studies to conrm the safety of ε-polylysine as a preservative in food [J]. Regul Toxicol Pharm, 2003, 37(2): 328-340. [3] 刘慧, 徐红华, 王明丽,等.聚赖氨酸抑菌性能的研究[J].东北农业大学学报,2000,31:294- 298[4] Kawai T, Kubota T, Hiraki J, et al. Biosynthesis of epsilon- poly-L-lysine in a cell-free system of Streptomycesalbulus[J].BiochemicalandBiophysicalResearchCommunications,2003,311(3): 635-640. [5] Takagi H, Hoshino Y, Nakamori S, et al. Isolation and sequence analysis of plasmid pNO33 in the epsilon-poly-L-lysine-producingactinomyceteStreptomycesalbulusIFO14147[J].Journalof Bioscience and Bioengineering, 2000, 89(1): 94-96. [6]HamanoY,NicchuI,HoshinoY,etal.Developmentofgenedeliverysystemsforthe epsilon-poly-L-lysine producer, Streptomyces albulus [J]. J Biosci Bioeng, 2005, 99(6), 636-641. [7] Yamanaka K, Maruyama C, Takagi H, et al. Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase [J]. Nat Chem Biol, 2008, 4(12): 766-772. [8]KitoM,TakimotoR,YoshidaT,etal.Purificationandcharacterizationofanepsilon- poly-L-lysine-degradingenzymefromanepsilon-poly-L-lysine-producingstrainofStreptomyces albulus [J]. Archives Of Microbiology, 2002, 178(5): 325-330. [9] Kito M, Onji Y, Yoshida T, et al. Occurrence of epsilon-poly-L-lysine-degrading enzyme in epsilon-poly-L-lysine-tolerant Sphingobacterium multivorum OJ10: purification and characterization [J]. Fems Microbiology Letters, 2002, 207(2): 147-151. [10] Kito M, Takimoto R, Onji Y, et al. Purification and characterization of an epsilon-poly- L-lysine-degrading enzyme from the epsilon-poly-L-lysine-tolerant Chryseobacterium sp. OJ7 [J]. Journal Of Bioscience and Bioengineering, 2003, 96(1): 92-94. [11] Feng X H, Xu H, Xu X Y, et al. Purification and some properties of ε-poly-L-lysine-degrading enzyme from Kitasatospora sp. CCTCC M205012 [J]. Process Biochemistry, 2008, 43(6): 667-672. [12] Yamanaka K, Kito N, Imokawa Y, et al. Mechanism of ε-poly-L-lysine production and accumulation revealed by identification and analysisof anε-poly-L-lysine-degradingenzyme [J]. Appl Environ Microbiol, 2010, 76 (17): 5669-5675. [13] Hamano Y, YoshidaT, Kito M,et al. Biological function ofthepld gene product that degrades epsilon- poly-L-lysine in Streptomyces albulus [J]. Applied Microbiology and Biotechnology, 2006, 72(1): 173-181. [14] Hamano Y, Nicchu I, Shimizu T, et al. epsilon-Poly-L-lysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase [J]. Applied Microbiology and Biotechnology, 2007, 76 (4): 873-882. [15] Hirohara H, Takehara M, Saimura M, et al. Biosynthesis of poly (ε-L-lysine) s in two newly isolated strains of Streptomyces sp. [J]. Applied Microbiology and Biotechnology, 2006, 73 (11): 321-331. [16] Grose J H, Joss L, Velick S F, et al. Evidencethat feedback inhibition of NAD kinase controls responses to oxidative stress [J]. Proc Natl Acad Sci, 2006, 103(7): 7601-7606. [17] Gao H, Xu X. The cyanobacterial NAD kinase gene sll1415 is required for photoheterotrophic growth and cellular redox homeostasis in Synechocystis sp. strain PCC 6803 [J]. J Bacteriol, 2012, 194(2): 218-24. [18] Takahashi H, Watanabe A, Tanaka A, et al. Chloroplast NAD kinase is essential for energy transduction through the xanthophyll cycle in photosynthesis [J]. Plant Cell Physiol, 2006, 47(12): 1678-82. [19] Ziegler M. New functions of a long-known molecule: Emerging roles of NAD in cellular signaling [J]. Eur J Biochem, 2000, 267(6): 1550-1564. [20] Zhang Q, Piston R H. Goodman. Regulation of corepressor function by nuclear NADH [J]. Science, 2002, 295(5561): 1895-1897. [21] Rutter J M, Reick L C, Wu, et al. Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors [J]. Science, 2001, 293(5529): 510-514. [22] Pain J, Balamurali M M, Daneis A, et al. A mitoehondrial NADH kinase, Pos5P, is required forefficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae [J]. J Biol Chem, 2010, 285(50): 39409-24. [23] Yoshizawa F. New therapeutic strategy for amino acid medicine: notable functions of branched chainamino acids as biological regulators [J]. J Pharmacol Sci, 2012, 118(2): 149-155.[24] Kawai S, Fukuda C, Murata K. Function and structure of NAD kinase: the key enzyme for biosynthesis of NADP (H) [J]. Tanpakushitsu Kakusan Koso, 2007, 52(3): 243-248.[25] Xu DQ, Tan YZ, Huan XJ, et al. Construction of a novel shuttle vector for use in Brevibacteriumflavum, an industrial amino acid producer [J]. J Microbiol Methods, 2010, 80(1): 86-92.
5. 计划与进度安排
1）2022-03-04～2022-03-10：查阅资料撰写开题报告；2）2022-03-11～2022-03-13：菌体培养及感受态细胞的制备和转化；3）2022-03-14～2022-03-21：基因组和质粒的提取；4）2022-03-22～2022-03-28：引物的设计和ATP/NAD激酶基因的扩增、纯化；5）2022-03-29～2022-04-10：质粒和菌株的构建；6）2022-04-11～2022-04-25：融合蛋白的表达和纯化；7）2022-04-26～2022-05-08：NAD 激酶和NADH激酶酶活的测定；8）2022-05-09～2022-05-19：NAD 激酶和NADH激酶酶促反应动力学研究；9）2022-05-20～2022-05-28：补充实验，整理试验数据和结果，撰写论文；10）2022-05-28～：修改补充论文，准备答辩。
