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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林俊宏(Chun-Hung Lin) | |
dc.contributor.author | Chia-Feng Hsieh | en |
dc.contributor.author | 謝嘉峰 | zh_TW |
dc.date.accessioned | 2021-06-13T00:42:37Z | - |
dc.date.available | 2007-09-29 | |
dc.date.copyright | 2007-07-31 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-25 | |
dc.identifier.citation | 伍. 參考文獻
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(1997) Presence of UDP-N-acetylmuramylhexapeptides and heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin. J. Bacteriol. 179, 4684–4688. 27. Brandish, P. E., Burnham, M. K., Lonsdale, J. T., Southgate, R., Inukai, M., and Bugg, T.D.H. (1996a) Slow binding inhibition of phospho-N-acetylmuramyl- pentapeptide-translocase (Escherichia coli) by mureidomycin A. J. Biol. Chem. 271, 7609-7614. 28. Stachyra, T., Dini, C., Ferrari, P., Bouhss, A., van Heijenoort, J., Mengin-Lecreulx, D., Blanot, D., Biton, J., and Le Beller, D. (2004) Fluorescence detection-based functional assay for high-throughput screening for MraY. Antimicrob. Agents Chemother. 48, 897-902. 29. Breukink, E., van Heusden, H. E., Vollmerhaus, P. J., Swiezewska, E., Brunner, L., Walker, S., Herk, A. J., de Kruijff, B. (2003) Lipid II is an intrinsic component of the pore induced by nisin in bacteria membrane. J. Biol. Chem. 278, 19898-19903. 30. Anderson, J. 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Auger, G., Crouvoisier, M., Caroff, M., van Heijenoort, J., Blanot, D. (1997) Synthesis of an analogue of the lipoglycopeptide membrane intermediate I of peptidoglycan biosynthesis. Lett. Peptide Sci. 4, 371-376. 35. Men, H., Park, P., Ge, M., Walker, S. (1998) Substrate Synthesis and Activity Assay for MurG. J. Am. Chem. Soc. 120, 2484-2485. 36. Ye, X.-Y., Lo, M.-C. Brunner, L., Walker, D. Kahne, D. Walker, S. (2001) Better substrates for bacterial transglycosylaes. J. Am. Chem. Soc. 123, 3155-3156. 37. Van Nieuwenhze, M. S., Mauldin S. C., Zia-Ebrahimi M., Aikins J. A., Blaszczak L. C. (2001) The total synthesis of Lipid I. J. Am. Chem. Soc. 123, 6983-6988. 38. Ha, S., Chang, E., Lo, M.-C., Men, H., Park, P., Ge, M., Walker, S. (1999) The Kinetic Characterization of Escherichia coli MurG Using Synthetic Substrate Analogues. J. Am. Chem. Soc. 121, 8415-8426. 39. Schwartz, B., Markwalder, J. A., Wang, Y. (2001) Lipid II total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Eschericia coli. J. Am. Chem. Soc. 123, 11638-11643. 40. Van Nieuwenhze, M. S., Mauldin, S. C., Zia-Ebrahimi, M., Winger, B. E., Hornback, W. J. , Saha, S. L., Aikins, J. A., Blaszczak L. C. (2002) The first total synthesis of lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis. J. Am. Chem. Soc. 124, 3656-3660. 41. Breukink, E., van Heusden, H. E., Vollmerhaus, P. J., Swiezewska, E., Brunner, L., Walker, S., Herk, A. J., de Kruijff, B. (2003) Lipid II is an intrinsic component of the pore induced by nisin in bacteria membrane. J. Biol. Chem. 278, 19898-19903. 42. Eefjan Breukink, Hester E. van Heusden, Pauline J. Vollmerhaus, Ewa Swiezewska ,Livia Brunner, Suzanne Walker, Albert J. R. Heck, and Ben de Kruijff.(2003) Lipid II Is an Intrinsic Component of the Pore Induced by Nisin in Bacterial Membranes. J. Biol. Chem. 278, 19898–19903. 43. Reiko Sadamoto, Kenichi Niikura, Pamela S. Sears, Haitian Liu, Chi-Huey Wong,Akarat Suksomcheep, Fusao Tomita, Kenji Monde,Shin-Ichiro Nishimura. (2002) Cell-Wall Engineering of Living Bacteria. J. A. Chem. Soc. 124, 9018-9019. 44. Sarah J. Luchansky, Kevin J. Yarema, Saori Takahashi, and Carolyn R. Bertozzi. (2002) GlcNAc 2-Epimerase Can Serve a Catabolic Role in Sialic Acid Metabolism. J. Biol. Chem. 278, 8035-8042. 45. Lara K. Mahal, Kevin J. Yarema, Carolyn R. Bertozzi. (1997) Engineering Chemical Reactivity on Cell Surfaces Through Oligosaccharide Biosynthesis. Science. 276, 1125 - 1128. 46. Stefan Ståhl and Mathias Uhlén. (1997) Bacterial surface display: trends and progress. Trends Biotechol.15, 185-192. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29142 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T00:42:37Z (GMT). No. of bitstreams: 1 ntu-96-R94b46036-1.pdf: 1925815 bytes, checksum: ca58c7520cf75ef07e15df0713cc04b5 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 目 錄
目錄 I 表次 V 圖次 VI 縮寫表 VIII 中文摘要 IX 英文摘要 X 壹. 序論 1 一、抗生素的簡介 1 1. 抗生素的歷史與發展 1 2. 抗生素的定義與分類 1 3. 抗生素作用機制 2 4. 抗生素抗藥性的產生與機轉 4 二、抗生素在細菌細胞壁生合成的作用 6 1. 細菌細胞壁生合成 6 2. 抗生素研發的標的 7 三、MraY和MurG 8 四、合成Lipid I和Lipid II 9 五、細菌細胞壁結構的改造 10 貳. 材料與方法 11 一、實驗材料 11 1. 一般化學藥品、限制酶及一般酵素 11 2. 菌株及載體 11 二、實驗儀器設備 11 三、實驗方法 12 1. UDP-MurNAc pentapeptide (DAP form) 的純化 12 1.1 菌種的培養 12 1.2 破菌與前處理 12 1.3 C18 open column的初步純化 13 1.4 以逆向層析高效液態層析儀 (Reversed phase HPLC) 純化分析 14 1.5 高效液態層析儀除鹽 (Desalting) 14 2. UDP-MurNAc pentapeptide (Lys form) 的純化 15 2.1 菌種的培養 15 2.2 破菌與前處理 16 2.3 以逆向層析高效液態層析儀 (Reversed phase HPLC) 純化分析 16 3. 製備Dansyl chloride螢光基團標定的UDP-MurNAc pentapeptide 17 3.1 DAP form UDP-MurNAc pentapeptide的標定 17 3.2 Lys form UDP-MurNAc pentapeptide的標定 17 4. 製備FITC (Fluorescein Iso-Thiocyanate) 螢光基團標定的UDP-MurNAc pentapeptide 17 4.1 DAP form UDP-MurNAc pentapeptide的標定 17 4.2 Lys form UDP-MurNAc pentapeptide的標定 18 5. 以酵素反應製造Lipid I和Lipid I 18 5.1製備分離Micrococcus luteus 菌種的細胞膜成分 18 5.1.1 法一 18 5.1.2 法二 19 5.2 酵素反應合成細胞壁重要中間物質Lipid I和Lipid II 19 5.3 酵素反應的分析與純化 21 5.3.1 Lipid I、II (C55p) 21 5.3.2 FITC- Lipid I、II (C55p) 21 5.3.3 Dansyl- Lipid I、II (C55p) 21 5.4 Nucleotide pyrophosphatase酵素作用 22 6. 活體細菌細胞壁的螢光表現 22 6.1 Staphylococcus simulans菌體的螢光表現 22 6.2 Staphylococcus simulans破菌後的螢光顯微觀測 23 6.3高解析螢光顯微鏡觀察Staphylococcus simulans與E. coli (BL21) 細胞 壁的螢光表現 23 6.3.1 細胞壁FITC的螢光表現 23 6.3.2 Vancomycin 處理後菌體細胞壁的螢光表現 25 參. 實驗結果 26 一、DAP form UDP-MurNAc pentapeptide 的純化 26 二、Lys form UDP-MurNAc pentapeptide 的純化 27 三、製備螢光基團標定的UDP-MurNAc pentapeptide 29 1. DAP form Dansyl-UDP-MurNAc pentapeptide的製備與純化 29 2. Lys form Dansyl-UDP-MurNAc pentapeptide的製備與純化 29 3. DAP form FITC-UDP-MurNAc pentapeptide的製備與純化 29 4. Lys form FITC-UDP-MurNAc pentapeptide的製備與純化 29 四、以酵素製備Lipid I和Lipid II 30 1. TLC分析酵素反應的進行 30 2.高效液態層析儀的分析與純化 33 2.1 Lys form UDP-MurNAc pentapeptide經酵素反應後產物之純化 33 2.2 Lys form FITC-UDP-MurNAc pentapeptide經酵素反應後產物之純化 33 2.3 Lys form dansyl-UDP-MurNAc pentapeptide經酵素反應後產物之純化 33 3. Nucleotide pyrophosphatase酵素作用 34 五、活體細菌細胞壁的螢光表現 34 1. Staphylococcus simulans菌體的螢光表現 34 2. 高解析螢光顯微鏡觀察Staphylococcus simulans與E. Coli (BL21) 細胞壁構 造的螢光表現 35 肆. 結果與討論 36 一、DAP form UDP-MurNAc pentapeptide 的純化 36 二、 Lys form UDP-MurNAc pentapeptide 的純化 37 三、螢光標定UDP-MurNAc pentapeptide 41 四、酵素合成Lipid I 及Lipid II 41 五、細菌細胞壁之螢光表現 43 伍. 參考文獻 46 圖. 52 | |
dc.language.iso | zh-TW | |
dc.title | 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 | zh_TW |
dc.title | Enzymatic Synthesis of the key Intermediates in Peptidoglycan Biosynthesis and the Cell Wall Engineering in Living Bacteria | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林俊成(Chun-Cheng Lin),楊文彬(Wen-Bin Yang),馬徹(Che Ma) | |
dc.subject.keyword | 細胞壁,抗生素, | zh_TW |
dc.subject.keyword | peptidoglycan,cell wall,antibiotics, | en |
dc.relation.page | 84 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-25 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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