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標題: | 酵素反應製造細菌細胞壁生合成的中間產物以及細菌表面的螢光表現 Enzymatic Synthesis of the key Intermediates in Peptidoglycan Biosynthesis and the Cell Wall Engineering in Living Bacteria |
作者: | Chia-Feng Hsieh 謝嘉峰 |
指導教授: | 林俊宏(Chun-Hung Lin) |
關鍵字: | 細胞壁,抗生素, peptidoglycan,cell wall,antibiotics, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 在過去數十年間,抗生素被用做治療用途,而抗生素抗藥性的問題也逐漸浮現,因此迫切地找尋新的且可以廣泛地使用的抗生素是相當重要的,抗生素作用的目標相當多,而我們是著重在研究細菌細胞壁生合成的酵素上,UDP-MurNAc-pentapeptide、Lipid I 和 Lipid II是肽聚醣合成的重要中間物質,我們必須能夠找到方法來做有效的製備。細胞壁的生合成有十個步驟,其中包括有兩的酵素是存在於細胞膜上並且作用在細胞膜內側,是為MraY 和 MurG,UDP-MurNAc-pentapeptide 和Lipid I是它們分別的受質,再此我們建立一個方法來大量純化自然界存在的DAP和Lys型的UDP-MurNAc-pentapeptide,並以化學的方式製備了它們的螢光衍生物,如dansyl-UDP-MurNAc-pentapeptide和FITC-UDP-MurNAc-pentapeptide,再以酵素反應與另一個受質,即具不同鏈長的單磷酸脂質長鏈反應合成出Lipid I和Lipid II及其類似物。
另外,我們試著以化學工程的方式在原核細胞表面表現帶有螢光標定的細胞壁生合成先趨物質FITC-UDP-MurNAc-pentapeptide,並且可以被明顯觀察到,用化學合成的方式作修飾讓菌體表面可以表現的分子具有多樣性,這有助於針對細菌作用的藥物及疫苗上的研究與開發,具有很大的應用性。 Abstract In the past few decades when antibiotics have been extensively in therapeutic use, the antibiotics resistances have emerged. It is important to find out new and broad-range antibiotics substitutes. There are many targets for antibiotics to work and we lay a special emphasis on the enzymes involved in bacteria cell wall biosynthesis. UDP-MurNAc-pentapeptide, Lipid I and Lipid II are the key intermediates in peptidoglycan biosynthesis, are the targets of our large scale synthesis. There are ten enzymes in this process including MraY and MurG that are located in the membrane. UDP-MurNAc-pentapeptide and Lipid I are their substrates respectively. We establish an efficient method to prepare UDP-MurNAc-pentapeptide including DAP and Lys form, Lipid I and their derivatives with a fluorescent group like dansyl and FITC. Furthermore, enzymatic methods were then studied to incorperate lipid chains of different length for preparing Lipid I and Lipid II analogues. Furthermore, we use the fluorescent group labeled peptidoglycan precursors FITC-UDP-MurNAc-pentapeptide to develop a method for the chemical engineering of the bacterial cell surface. It will allow a large variety of molecular specimens to be displayed, leading to a wide range of applications, such as in discovery of novel drugs or vaccines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29142 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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