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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
dc.contributor.author | Yi-Fan Chen | en |
dc.contributor.author | 陳怡帆 | zh_TW |
dc.date.accessioned | 2021-06-13T07:58:38Z | - |
dc.date.available | 2005-07-27 | |
dc.date.copyright | 2005-07-27 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-22 | |
dc.identifier.citation | 1. 李俊蔚. 慈濟大學醫學檢驗生物技術學系專題研究報告 (2003).
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Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum. J Agric Food Chem 50, 5676-82 (2002). 24. Kim, C. S., Ji, E. S. & Oh, D. K. Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima. J Appl Microbiol 97, 1006-14 (2004). 25. Shaikh, S. A., Khire, J. M. & Khan, M. I. Characterization of a thermostable extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. Biochim Biophys Acta 1472, 314-22 (1999). 26. Zeikus, J. G., Vieille, C. & Savchenko, A. Thermozymes: biotechnology and structure-function relationships. Extremophiles 2, 179-83 (1998). 27. Rastall, R. A. & Bucke, C. Enzymatic synthesis of oligosaccharides. Biotechnol Genet Eng Rev 10, 253-81 (1992). 28. Ichikawa, Y., Look, G. C. & Wong, C. H. Enzyme-catalyzed oligosaccharide synthesis. Anal Biochem 202, 215-38 (1992). 29. Hindsgaul, O. K. a. O. Synthesis of oligosaccharides, glycolipids and glycopeptides. 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A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-54 (1976). 36. Felsenstein. J. PHYLIP--Phylogeny Inference Package(Version 3.2). Cladistics 5, 164-166 (1989). 37. Higgins, D. G., Bleasby, A. J. & Fuchs, R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8, 189-91 (1992). 38. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-80 (1994). 39. Pieper, U. et al. MODBASE, a database of annotated comparative protein structure models, and associated resources. Nucleic Acids Res 32, D217-22 (2004). 40. Ohlsson, J. & Magnusson, G. Galabiosyl donors; efficient synthesis from 1,2,3,4, 6-penta-O-acetyl-beta-D-galactopyranose. Carbohydr Res 329, 49-55 (2000). 41. La Cara, F. et al. Different effects of microwave energy and conventional heat on the activity of a thermophilic beta-galactosidase from Bacillus acidocaldarius. Bioelectromagnetics 20, 172-6 (1999). 42. Henrissat, B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280 (Pt 2), 309-16 (1991). 43. Henrissat, B. & Bairoch, A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293 (Pt 3), 781-8 (1993). 44. Hidaka, M. et al. Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol 322, 79-91 (2002). 45. Gutshall, K. R., Trimbur, D. E., Kasmir, J. J. & Brenchley, J. E. Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate. J Bacteriol 177, 1981-8 (1995). 46. Moore, J. B., Markiewicz, P. & Miller, J. H. Identification and sequencing of the Thermotoga maritima lacZ gene, part of a divergently transcribed operon. Gene 147, 101-6 (1994). 47. Holmes, M. L. et al. Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337, 276-86 (1997). 48. Henrissat, B. et al. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A 92, 7090-4 (1995). 49. Jenkins, J., Lo Leggio, L., Harris, G. & Pickersgill, R. Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven. FEBS Lett 362, 281-5 (1995). 50. Juers, D. H., Huber, R. E. & Matthews, B. W. Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases. Protein Sci 8, 122-36 (1999). 51. Brock, T. D. & Freeze, H. Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J Bacteriol 98, 289-97 (1969). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36374 | - |
dc.description.abstract | Deinococccus sp. NTU-1079是由台東縣的栗松溫泉分離和鑑定的一株嗜熱性兼具抗輻射性的細菌。選殖它的β-半乳糖水解酶基因,並在大腸桿菌中讓它大量表現,再以鎳離子的親合性管柱純化之。同時,也依序利用DEAE sepharose、Superose 6 HR和HiTrap Q層析管柱,純化出它外泌性的α-半乳糖水解酶。
定性的結果顯示:此二酵素可穩定的存在於50℃的環境中;於60 ~ 65℃及中性pH值下有最好的活性,而且具有良好的輻射抗性。在以X射線,2.58 KGy劑量下照射達8小時,α-和β-半乳糖水解酶仍各別存留25%及90%的催化活性。在實驗中,我們也求得此二酵素對於不同受質的Km和Vmax值。更近一步,根據一級序列分析的結果,發現β-半乳糖水解酶屬於GH-42 family;二級和三級結構的預測和分析結果說明β-半乳糖水解酶屬於4/7 superfamily;以來自於Thermus sp. A4的β-半乳糖水解酶作為模版來做三級結構的電腦模擬,說明酵素的熱穩定性可能來自於次單元間氫鍵的作用力。利用膠體過濾層析和沉降平衡法來決定β-半乳糖水解酶的四級結構,分別得到二單元體及三單元體的結果。 在研究中,亦嘗試利用半乳糖水解酶的轉醣活性,將之應用於合成寡糖衍生物 ─ 凝集素的探針上。利用嗜熱性菌酵素之耐熱性、對有機溶劑的抗性、輻射抗性和酵素催化之專一性,省略傳統有機化學合成法過於繁複的醣化合成步驟,已達成快速建立凝集素的探針資料庫的目的。 | zh_TW |
dc.description.abstract | Deinococccus sp. NTU-1079 is isolated from Zhi-Sung hot spring in Taitung and found thermophilic as well as resistant to ionizing radiation. The gene of β-galactosidase from Deinococccus sp. NTU-1079 is cloned and the recombinant β-galactosidase is overexpressed in Escherichia coli, then simply purified by Ni-NTA column. The extracellular α-galactosidase is purified sequentially by DEAE sepharose, Superose 6 HR and HiTrap Q chromatography to homogeneity
Characterization experiments show that the two enzymes are stable under 50℃ for 3 h, and have the highest activities at neutral pH around 60 ~ 65℃. They are highly resistant to X-ray, under exposure to 2.58 KGy for 8 h, showing that the α-galactosidase and the β-galactosidase have remaining activity about 25% and 90%, respectively. Their Km and Vmax values for different substrates are also obtained. Furthermore, we find that the β-galactosidase belongs to glycoside hydrolase family 42 based on primary sequence alignment, and possesses similar structure to 4/7 superfamily enzymes according to the secondary and tertiary structure predictions. The tertiary structure of the β-galactosidase is built by computer modeling based on the known structure of β-galactosidase from Thermus sp. A4 as a template and the thermostability of the β-galactosidase may come from the formation of hydrogen bonds between subunits. The quaternary structure of the β-galactosidase is examined by gel filtration and sedimentation equilibrium and shows as dimer and trimer, respectively. The transgalactosyl properties of these enzymes are applied to synthesize oligosaccharide derivatives as lectin-ligand probes. Using characteristics of thermostability, organic solvent-resistance, radiation resistance and specific catalytic mechanism of the enzymes, lectin-ligand probes can be established rapidly by the chemoenzymatic method. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T07:58:38Z (GMT). No. of bitstreams: 1 ntu-94-R92b46025-1.pdf: 1841443 bytes, checksum: d1da2284098e227e67bd22e158d1c91e (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 目錄
中文摘要…………………………………………………………………………………………... Ⅵ 英文摘要………………………………………………………………………………………..…. Ⅶ 目錄……………………………………………………………………………….....Ⅰ 圖表目錄……………………………………………………………………………..........…......... Ⅲ 縮寫表……………………………………………………………………………….……………...Ⅴ 第一章 前言 1-1 嗜熱性細菌及其熱穩定性酵素之應用和發展………………………………. 1 1-2 抗輻射及嗜熱性菌Deinococcus (奇異球菌)………………………………… 2 1-3 熱穩定性之Alpha-Galactosidase和Beta-Galactosidase…………………….. 3 1-4 以酵素合成醣類衍生物………………………………………………………. 4 1-5 凝集素探針 (Lectin-Ligand probes)..………………...………………………. 5 1-6 研究目的………………………………………………………………………. 6 1-7 實驗流程圖……………………………………………………………………. 8 第二章 材料與方法 2-1材料 2-1-1 菌種與質體……………………………………………………………….. 9 2-1-2 藥品與酵素……………………………………………………………….. 9 2-1-3 培養基………………………………………………………………… … 10 2-1-4 儀器………………………………………………………………………. 11 2-2方法 2-2-1在載體上Cloning和表現NTU1079-β-Gal……………………………... 12 2-2-2培養 Deinococcus sp. NTU1079………………………………………… 14 2-2-3純化外泌性的NTU1079-α-Gal………………………………………….. 15 2-2-4 測定α- and β-Galactosidase的活性…………………...……………...… 16 2-2-5 決定蛋白質的含量…………………………………………………...….. 16 2-2-6 蛋白質電泳……………………...……………………………………….. 16 2-2-7 NTU1079-α-Gal和NTU1079-β-Gal之定性……………………..……... 17 2-2-7 a. 溫度對酵素穩定性的影響………………………………………... 17 2-2-7 b. 溫度對酵素活性的影響………………………………………...… 17 2-2-7 c. 利用Circular Dichlorism spectra (CD) 計算溫度對 NTU1079-β-Gal的二級結構穩定性................................................ 17 2-2-7 d. pH值對酵素穩定性的影響…………………………………...…... 18 2-2-7 e. pH值對酵素活性的影響……………………………………...…... 18 2-2-7 f. NTU1079-α-Gal和NTU1079-β-Gal的輻射抗性……………........ 19 2-2-7 g. 金屬離子對酵素活性的影響………………………………...…… 19 2-2-7 h. 計算Km和Vmax值……………………………………………...… 19 2-2-7 i. NTU1079-β-Gal的序列比對 (alignment) 和家族從屬性………... 19 2-2-7 j. 預測NTU1079-β-Gal的二級和三級結構………………………... 20 2-2-7 k. 決定NTU1079-β-Gal native form 的分子量 …………………… 20 2-2-8 Lectin-Ligand probes (凝集素探針) 之合成…………………………… 22 第三章 結果與討論 3-1表現和純化NTU1079-α-Gal和NTU1079-β-Gal…………………………... 24 3-2溫度對酵素穩定性和活性的影響…………………………………………... 24 3-3利用Circular Dichlorism spectra (CD) 計算溫度對 NTU1079-β-Gal二級結構穩定性的影響…………………………………… 24 3-4 pH值對酵素穩定性和活性的影響……...………………………………….. 24 3-5 NTU1079-α-Gal和NTU1079-β-Gal的輻射抗性…………………………... 25 3-6金屬離子對酵素活性的影響………………………………………………... 25 3-7計算Km和Vmax值………………………………………………………..…. 25 3-8 NTU1079-β-Gal的序列比對和家族從屬性………...……………………… 26 3-9預測NTU1079-β-Gal的二級和三級結構………………………………….. 27 3-10決定NTU1079-β-Gal native form 的分子量………………………...……. 28 3-11 NTU1079-β-Gal的蛋白質結構穩定性.......................................................... 28 3-12 Lectin-Ligand probes (凝集素探針) 之合成……………………...……….. 29 第四章 結論………………………………………………………………………. 30 第五章 參考文獻…………………………………………………………………. 50 附錄 附錄一……………………………………………………………………………. 54 附錄二……………………………………………………………………………. 55 附錄三……………………………………………………………………………. 56 附錄四……………………………………………………………………………. 57 圖表目錄 圖一、NTU1079-β-Gal之一級序列……………………………………………… 31 圖二、Glycosidase的反應機制…………………………………………………... 32 圖三、有機化學合成雙醣的標準步驟…………………………………………... 33 圖四、純化NTU1079-β-Gal……………………………………………………… 34 圖五、NTU1079-β-Gal的質譜圖….……………………………………………... 34 圖六、純化NTU1079-α-Gal……………………………………………………… 35 圖七、溫度對酵素穩定性的影響………………………………………………... 36 圖八、溫度對酵素活性的影響…………………………………………………... 36 圖九、利用Circular Dichlorism spectra (CD) 計算溫度對NTU1079-β-Gal的 二級結構穩定性…………………………………………………………... 37 圖九A、NTU1079-β-Gal的CD全波長掃描圖…………………………………. 37 圖九B、溫度對NTU1079-β-Gal二級結構的影響……………………………... 37 圖十、pH值對酵素穩定性的影響……………………………………………...... 38 圖十一、pH值對酵素活性的影響…………………………………………...…... 38 圖十二、NTU1079-α-Gal和NTU1079-β-Gal的輻射抗性…………………..…. 39 圖十三、金屬離子對酵素活性的影響…………………………………………... 39 圖十四、NTU1079-β-Gal的序列比對和家族從屬性…………………….……... 40 圖十五、NTU1079-β-Gal和其他GH-42 family β-Galactosidase的序列比對.… 41 圖十六、預測NTU1079-β-Gal的二級結構…………………………………....... 42 圖十七、預測NTU1079-β-Gal的三級結構……………………………………... 43 圖十八、利用superimposition預測NTU1079-β-Gal的催化位………………... 44 圖十九、利用Gel-Filtration決定NTU1079-β-Gal native form的分子量……... 45 圖十九A、Gel-Filtration之標準曲線……………………………………………. 45 圖十九B、利用Gel-Filtration決定NTU1079-β-Gal native form的分子量…… 45 圖二十、利用Sedimentation equilibrium決定NTU1079-β-Gal native form M.W... 46 圖二十一、利用NTU1079-β-Gal合成N-Acetyllactosamine之生成物質譜圖…... 47 圖二十二、化合物 (5) 的1H NMR氫核光譜圖…………………………………... 48 表一、Km和Vmax值的計算…………………………………………………………. 49 | |
dc.language.iso | zh-TW | |
dc.title | 台灣嗜熱性奇異球菌NTU-1079之α-和β-半乳糖水解酶的研究及其於寡糖合成之應用 | zh_TW |
dc.title | Characterization of α- and β-Galactosidases from Deinoccocus sp. NTU-1079 and their application in oligosaccharides synthesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡珊珊(San-San Tsay),林俊宏(Chun-Hung Lin),林光慧(Guang-Huey Lin) | |
dc.subject.keyword | 半乳糖水解酶,嗜熱菌,寡糖生合成, | zh_TW |
dc.subject.keyword | alpha-galactosidase,beta-galactosidase,thermophiles,oligosaccharides synthesis, | en |
dc.relation.page | 57 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-23 | |
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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