請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32967完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張文章 | |
| dc.contributor.author | Wei-Ling Lin | en |
| dc.contributor.author | 林韋伶 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:20:27Z | - |
| dc.date.available | 2009-07-28 | |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-22 | |
| dc.identifier.citation | 參考文獻
Ames, B. N. (1995) Understanding the causes of aging and cancer. Microbiologia. 11: 305-308 Bai, J., Rodriguez, A. M., Melendez, J. A. and Cederbaum, A. I. (1999) Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J. Biol. Chem. 274(37): 26217-26224 Battista, J. R., Earl, A. M. and White, O. (2000) The stress responses of Deinococcus radiodurans, p. 383-391. In Storz, G. and Hengge-Aronis, R. (ed.), Bacterial Stress Responses. Washington, D.C. Berthet, S., Nykyri, L., Bravo, J., Maté, M. J., Berthet-Colominas, C., Alzari, P. M. et al. (1997) Crystallization and preliminary structural analysis of catalase-A from Saccharomyces cerevisiae. Protein Sci. 6: 481–483 Bravo, J., Verdaguer, N., Tormo, J., Betzel, C., Switala, J., Loewen, P. C. et al. (1995) Crystal structure of catalase HPII from Escherichia coli. Structure. 3: 491–502 Bravo, J., Maté, M. J., Schneider, T., Switala, J., Wilson, K., Loewen, P. C. et al. (1999) Structure of catalase HPII from Escherichia coli at 1.9 Å resolution. Proteins. 34: 155–166 Carpena, X., Perez, R., Ochoa, W. F., Verdaguer, N., Klotz, M. G., Switala, J. et al. (2001) Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri. Acta Crystallograph. D57: 1184–1186 Carpena, X., Soriano, M., Klotz, M. G., Duckworth, H. W., Donald, L. J., Melik-Adamyan, W. et al. (2003) Structure of the clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 Å resolution. Proteins. 50: 423–436 Chelikani, P., Fita, I. and Loewen, P. C. (2004) Diversity of structures and properties among catalases. Cell. Mol. Life Sci. 61: 192-208 Chou, F. I. and Tan, S. T. (1990) Manganese(II) induces cell division and increases in superoxide dismutase and catalase activities in an aging deinococcal culture. J. Bacteriol. 172: 2029-2035 Elder, G. H. (1994) Heme synthesis and the porphyrias. In Williams, D. L. and Marks, V. (2nd ed.), Scientific Foundations of Biochemistry in Clinical Practice. Butterworth-Heinemann. Fita, I., Silva, A. M., Murthy, M. R. N. and Rossmann, M. G. (1986) The refined structure of beef liver catalase at 2.5 A resolution. Acta Crystallogr. B42: 497–515 Gouet, P., Jouve, H. M. and Dideberg, O. (1995) Crystal structure of Proteus mirabilis PR catalase with and without bound NADPH. J. Mol. Biol. 249: 933–954 Klotz, M. G. and Loewent, P. C. (2003) The molecular evolution of catalatic hydroperoxidases: evidence for multiple lateral transfer of genes between prokaryota and from bacteria into eukaryota. Mol. Biol. Evol. 20(7): 1098-1112 Lombardo, M. E., Araujo, L. S., Ciccarelli, A. B. and Batlle, A. (2005) A spectrophotometric method for estimating hemin in biological systems. Anal. Biochem. 341: 199-203 Marcel, Z., Jana, G., Juraj, G., Franz, K. and Bystrík. P. (2004) Expression, purifcation, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H. Prot. Exp. Purif. 36: 115–123 Markillie, L. M., Varnum, S. M., Hradecky, P. and Wong, K. K. (1999) Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants. J. Bacteriol. 181: 666-669 Maté, M. J., Zamocky, M., Nykyri, L. M., Herzog, C., Alzari, P. M., Betzel, C. et al. (1999) Structure of catalase-A from Saccharomyces cerevisiae. J. Mol. Biol. 286: 135–139 Murshudov, G. N., Melik-Adamyan, W. R., Grebenko, A. I., Barynin, V. V., Vagin, A. A., Vainshtein, B. K. et al. (1982) Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5Å resolution. FEBS Lett. 312: 127–131 Murthy, M. R. N., Reid, T. J., Sicignano, A., Tanaka, N. and Rossmann, M.G. (1981) Structure of beef liver catalase. J. Mol. Biol. 152: 465–499 Pal, J. K. and Joshi-Purandare, M. (2001) Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro. J. Biosci. 26: 225-231 Putnam, C. D., Arvai, A. S., Bourne, Y. and Tainer, J.A. (1999) Active and inhibited human catalase sturctures: ligand and NADPH binding and catalytic mechanism. J. Mol. Biol. 296: 295–309 Seaver, L. C. and Imlay, J. A. (2001a) Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183: 7173-7181 Seaver, L. C. and Imlay, J. A. (2001b) Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J. Bacteriol. 183: 7182-7189 Shih, Y. P., Kung, W. M., Chen, J. C., Yen, C. H., Wang, A. H. J. and Wang, T. F. (2002) High-throughput screening of soluble recombinant proteins. Protein Sci. 11: 1714-1719 Storz, G. and Imlay, J. A. (1999) Oxidative stress. Curr. Opin. Microbiol. 2: 188-194 Supinski, G. S. and Callahan, L. A. (2006) Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis. Free Radic. Biol. Med. 40: 127-137 Thomas, B., Nigel, A. J. E., Jamie, N. J., Jesmin, Judit, M. N., Brigitte, Jamart-Gre´goire., Emma, L. R. and Katherine, A. B. (2004) Crystal Structure of Mycobacterium tuberculosis Catalase-Peroxidase. J. Biol. Chem. 279 (37): 38991-38999 Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A. and Grebenko, A. I. (1981) Three-dimensional structure of the enzyme catalase. Nature. 293: 411–412 Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A., Grebenko, A. I., Borisov, V. V. et al. (1986) Threedimensional structure of catalase from Penicillium vitale, at 2.0 Å resolution. J. Mol. Biol. 188: 49–61 Vladimir, V. B., Mei, M. W., Svetlana, V. A., Victor, S. L., Pauline, M. H., Peter, J. A. and James, W. W. (2001) Crystal Structure of Manganese Catalase from Lactobacillus plantarum. Structure. 9: 725–738 Wang, P. and Schellhorn, H. E. (1995) Induction of resistance to hydrogen peroxide and radiation in Deinococcus radiodurans. Can. J. Microbiol. 41: 170-176 White, O., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., Nelson, W. C., Richardson, D. L., Moffat, K. S., Qin, H., Jiang, L., Pamphile, W., Crosby, M., Shen,M., Vamathevan, J. J., Lam, P., McDonald, L., Utterback, T., Zalewski, C., Makarova, K. S., Aravind, L., Daly, M. J., Minton, K. W., Fleischmann, R. D., Ketchum, K. A., Nelson, K. E., Salzberg, S., Smith, H. O., Venter, J. C. and Fraser, C. M. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science. 286: 1571-1577 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32967 | - |
| dc.description.abstract | 中文摘要
耐輻射奇異球菌對於環境中的離子輻射、過氧化氫、紫外線及乾燥的抗性極高。耐輻射奇異球菌的細胞萃取中，已經發現有很強的過氧化氫酶活性，其活性的強度，會受到細胞中過氧化氫濃度而影響。另外，在培養耐輻射奇異球菌時，加入過氧化氫可以誘導過氧化氫酶的產生。所以將耐輻射奇異球菌的過氧化氫酶基因放大後選殖到載體pET-28a，利用大腸桿菌的表現系統大量表現。經過鎳親和性層析法純化後，獲得的重組蛋白質是在N端帶有His-tag的過氧化氫酶，DrCatA (D. radiodurans Catalase A)。得到約2 mg有活性的DrCatA，比活性約為83.97 U/mg。再以DEAE離子交換樹脂純化得到約0.4 mg有活性的DrCatA，比活性約為128.5 U/mg。最後以西方墨點法，確定DrCatA為帶有His-tag的重組蛋白質。再經由高效能液相層析法及質譜分析確定其分子量，質譜分析的實驗值比理論值少193 Da是目前還不知道的原因。 然而apo-DrCatA是需要輔酶hemin才能形成具有活性的holo-DrCatA。另外，在實驗上也發現hemin有過氧化氫酶的催化活性，具有分解過氧化氫的能力。在重組過氧化氫酶的酵素動力學研究中，它的最大催化速率為6.7 (μmol/ml min)；Km是10.1 (mM)；而其kcat (turnover number) 的值為42402.3 (min-1)，所以kcat/Km為2.53 × 108 (M-1sec-1)，其比活性則是250.9 (U/mg) 或15180.1 (U/μmol)。另外，在血基質的酵素動力學中，它的最大速率為2.7 (μmol/ml min)；Km是18.1 (mM)；而其kcat的值為54.6 (min-1)，所以kcat/Km為1.813 × 105 (M-1sec-1)，其比活性則是27.4 (U/mg) 或17.3 (U/μmol)。由以上實驗的Km值我們可以得知，重組過氧化氫酶與受質結合的親和力比血基質好；而從kcat/Km值顯示，在受質濃度飽和狀態下重組過氧化氫酶的催化效率比血基質多出一千倍。 | zh_TW |
| dc.description.abstract | Abstract
Deinococcus radiodurans R1 is well-known for its extraordinary resistance against ionizing radiation, oxidative stress, UV light, heat, and desiccation. Strong catalase activities are found in D. radiodurans cell extracts, and the level of activity can be influenced by exposing cells to a sublethal dose of H2O2. The enzyme of catalase is induced when D. radiodurans cultures are pretreated with H2O2. The gene encoding catalase A from D. radiodurans was amplified and cloned into the vector pET-28a and expressed in E. coli BL21. The expressed DrCatA (Catalase A from D. radiodurans) was purified to apparent homogeneity by affinity chromatography on Ni-NTA column, obtaining 2 mg of purified protein with specific activity of 83.97 U/mg. And 0.4 mg protein is obtained with specific activity of 128.5 U/mg after DEAE ion exchange chromatography. We have used the western blotting to confirm that DrCatA is a His-tag recombinant protein. The molecular mass was analysed by mass spectrum after desalting with reverse-phase HPLC through a C8 column. However, the reason of the experimental molecular mass being less than the theoretical molecular mass of 193 Dalton still remains unknown. DrCatA, with its coenzyme hemin, is able to transform from an inactive apo-DrCatA into an active holo-DrCatA. Hemin by itself also has the catalatic activity, and it can dismutate the hydrogen peroxide into water and oxygen. In the recombinant DrCatA kinetics study, the Vmax is 6.7 μmol/ml min with Km and its turnover number, kcat, of 10.1 mM and 42402.3 min-1 respectively. And the kcat/Km is 2.53 × 108 M-1sec-1 with specific activity of 250.9 U/mg or 15180.1 U/μmol. On the part of hemin kinetics study, the Vmax is 2.7 μmol/ml min with Km and its turnover number, kcat, of 18.1 mM and 54.6 min-1 respectively. And the kcat/Km is 1.813 × 105 M-1sec-1 with specific activity of 27.4 U/mg or 17.3 U/μmol. DrCatA had a greater affinity for the substrate than hemin as determined by their Michaelis constant, Km. As a result from their kcat/Km values, the catalytic efficiency of DrCatA is approximately 103 folds greater than that of hemin operating at substrate concentrations substantially below saturation amounts. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:20:27Z (GMT). No. of bitstreams: 1 ntu-95-R93b46033-1.pdf: 1149066 bytes, checksum: b477de2153aeeeae84f9a4fc674c3d4f (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄
中文摘要 vi Abstract vii 縮寫表 ix 第一章 緒論 (Introduction) 1 第一節 耐輻射奇異球菌 (Deinococcus radiodurans R1) 1 一、 前言 1 二、 耐幅射奇異球菌的遺傳物質 1 三、 耐輻射奇異球菌的抗輻射機制 2 四、 耐輻射奇異球菌過氧化氫酶 3 第二節 過氧化氫酶 (Catalase) 4 一、 前言 4 二、 生物體中的活性氧 4 三、 過氧化氫酶的分類 5 四、 過氧化氫酶的作用機制 6 五、 過氧化氫酶的結構 7 第三節 血基質 (Heme) 8 一、 細胞中血基質的合成 8 二、 血晶質生理上所扮演的角色 9 第四節 研究目的 10 第二章 材料與方法 (Materials and Methods) 11 第一節 實驗材料與使用儀器 11 一、 材料 11 二、 儀器 12 第二節 實驗方法 13 一、 製備大腸桿菌表現載體 (Preparation of expression vectors) 13 二、 設計引子 (Primers design) 14 三、 基因片段的增幅 (Gene amplification using sticky-end PCR) 15 四、 檢查PCR產物 17 五、 將PCR產物磷酸化 18 六、 將PCR產物接入選定之載體 (ligation) 18 七、 培養基 19 八、 轉型 (transformation) 至JM109 (DE3) 勝任細胞 19 九、 利用限制酶檢查殖株之基因 19 十、 將選定之殖株作DNA定序 20 十一、 轉型至宿主細胞ECOS 21 20 十二、 小量表現 21 十三、 蛋白質可溶性的測試 22 十四、 大量表現以及利用sonication的方式破菌 23 十五、 SDS聚丙烯醯胺膠體電泳 24 十六、 Coomassie Blue 染色 25 十七、 蛋白質定量分析 26 十八、 鎳親和性管柱層析法 (Ni-NTA affinity chromatography) 26 十九、 離子交換層析法 (Ion exchange chromatography) 27 二十、 膠體過濾層析法 (Gel filtration chromatography) 28 二十一、 西方墨點法 (Western blotting) 30 二十二、 高效能液相層析法 (HPLC) 與質譜分析 31 二十三、 過氧化氫酶的活性測試 31 二十四、 熱穩定性測試 32 二十五、 最適酸鹼值範圍 (pH optimum) 的測試 33 二十六、 酵素動力學的測試 33 第三章 結果與討論 (Results and Discussion) 34 第一節 重組過氧化氫酶的基因建構 34 第二節 重組過氧化氫酶的大量表現與純化 35 一、 重組過氧化氫酶的大量表現 35 二、 重組過氧化氫酶加入其所需之輔酶的試驗 35 三、 重組過氧化氫酶的純化 36 第三節 重組過氧化氫酶的活性及其分析 38 ㄧ、 重組過氧化氫酶活性的差異分析 38 二、 重組過氧化氫酶與血基質酵素動力學的分析 39 第四節 重組過氧化氫酶的生化研究 40 一、 重組過氧化氫酶四級結構的分析 40 二、 重組過氧化氫酶儲存條件、酸鹼值以及熱穩定性的分析 41 第四章 結論與未來展望 43 參考文獻 68 表目錄 Table 1. Purification of catalase A from D. radiodurans R1 66 Table 2. Comparison with kinetics of DrCatA and hemin 67 圖目錄 Figure 1. Simplified scheme showing the reaction catalysed by monofunctional, heme-containing catalases 45 Figure 2. Stereo views of CatF to illustrate the association of subunits 46 Figure 3. Construction of recombinant plasmid pET28a-catA 47 Figure 4. Agarose gel electrophoresis of the PCR product 48 Figure 5. Restriction enzyme analysis of the recombinant vectors 49 Figure 6. Nucleotide sequence of recombinant D. radiodurans R1 catalase A gene and deduced amino acid sequence of DrCatA protein 50 Figure 7. Time course study for expression of DrCatA 51 Figure 8. Purification of DrCatA using His-tag affinity column chromatography 52 Figure 9. Chromatogram showing elution profile of DrCatA on DEAE sepharose cation exchange column 53 Figure 10. Ultraviolet/visible spectra of purified catalase A from D. radiodurans R1 54 Figure 11. HPLC analysis of inactive and active DrCatA 55 Figure 12. Mass spectrometric analysis of DrCatA 56 Figure 13. Elution volumes versus Mr (logarithmic scale) on a gel filtration column 57 Figure 14. Gel filtration chromatography 58 Figure 15. Kinetic analysis for DrCatA and Hemin 60 Figure 16. Storage test of DrCatA 62 Figure 17. pH optimum of the purified catalase A from D. radiodurans R1 and hemin 63 Figure 18. Thermostability of DrCatA and hemin for 20, 40, and 80 minutes 64 | |
| dc.language.iso | zh-TW | |
| dc.subject | 過氧化氫酶 | zh_TW |
| dc.subject | 耐輻射奇異球菌 | zh_TW |
| dc.subject | catalase | en |
| dc.subject | Deinococcus radiodurans R1 | en |
| dc.title | 耐輻射奇異球菌過氧化氫酶之選殖表現及生化研究 | zh_TW |
| dc.title | Cloning, expression, and biochemical studies of the catalase from Deinococcus radiodurans R1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡珊珊,吳世雄 | |
| dc.subject.keyword | 耐輻射奇異球菌,過氧化氫酶, | zh_TW |
| dc.subject.keyword | Deinococcus radiodurans R1,catalase, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-24 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 未授權公開取用 | 1.12 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。