跳到主要內容

臺灣博碩士論文加值系統

(54.91.62.236) 您好!臺灣時間:2022/01/18 00:50
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林雅慧
研究生(外文):Ya Huei Lin
論文名稱:Arthrobacterglobiformis胺類氧化酵素的表現,定點突變及特性之研究
論文名稱(外文):Expression, Mutagenesis and Characterization of Arthrobacter globiformis Histamine Oxidase
指導教授:袁俊傑袁俊傑引用關係
指導教授(外文):Chiun-Jye Yuan
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:110
中文關鍵詞:胺類氧化酵素含銅胺類氧化酵素
外文關鍵詞:Histamine Oxidaseamine oxidasecopper amine oxidase
相關次數:
  • 被引用被引用:0
  • 點閱點閱:240
  • 評分評分:
  • 下載下載:38
  • 收藏至我的研究室書目清單書目收藏:0
含銅胺類氧化酵素 [EC 1.4.3.6]普遍存於原核及真核生物,此酵素作用為催化生物胺類化合物之氧化而產生醛(aldehydes)、氨(ammonia)及過氧化氫(hydrogen peroxide)。 在原核生物中胺類氧化酵素提供了氮源及碳源的來源 ; 而在真核生物中則與細胞分化、細胞生長、去毒素作用、傷口的處理、細胞的訊號有關,甚至可能對於細胞死亡亦有關聯。
大體而言,含銅胺類氧化酵素以同源雙體 (homodimer) 的形式存在,依照來源其每一單元體分子量為70,000至105,000 daltons之間,每一單元含有一個銅離子及以胺基酸tyrosine經過後轉譯修飾作用所產生的共價結合的TPQ輔因子。從革蘭氏陽性菌Arthrobacter globiformis分離出來的含銅胺類氧化酵素中,依照胺基酸的相似性及受質專一性的不同劃分為兩類,其一為以histamine為受質,命名為histaminase 或 AGHO,另外一種則為以 phenylethylamine為主要受質的,命名為phenylethylamine oxidase 或 AGPEO。
在本研究中,我們將帶有AGHO的全長基因架至pET30b(+) 上,建構成一個帶有His6-Tag及AGHO全長的融合蛋白質。我們將已經將pET30b(+)/AGHO轉殖成功送入大腸桿菌BL21(DE3)中表現,首先以37 ℃培養後,加入新鮮的LB 培養液,添加50 μM 及0.05 mM IPTG 後在25 ℃誘導8小時,純化方式則選用硫胺沉澱及親和性管柱層析法,在1 L的培養液中,所得到的重組AGHO酵素產率為34 mg。
我們發現重組AGHO酵素對於高於溫度30 ℃的環境敏感,將重組酵素分別置於45 ℃、60 ℃ 30分鐘後,比起30 ℃作用的酵素活性各減少了10 %與40 %。在AGHO/HRP耦合酵素活性裡,當我們投以histamine 受質時,反應最適當的pH值為 8.0至pH 9.0之間。經過酵素動力學研究後,所得到的結果以phenylethylamine為受質時Km為134 μM, Vmax 為8.66 nmole/min/μg protein,而以histamine為受質時Vmax 為0.02 nmole/min/μg protein,Km為10.94 μM。
此外,本研究的另一個方向為此酵素及突變株的受質專一性探討,胺基酸Y316位置位於受質通道的閘道處,因此對於不同種的胺類受質具有選擇性,而此位置亦在反應中與TPQ輔因子的定位及去質子作用有關,因此我們將此胺基酸以點突變的方式改變成A、E、F、H、W。當我們投以不同種類的受質如 histamine, phenylethylamine, tyramine及 tryptamine ,結果顯示Y316位置的突變株具有比較低甚至沒有酵素的活性,這樣低的酵素活性據推測有可能與TPQ的形成有關。

Copper-containing amine oxidases [EC 1.4.3.6] present in both prokaryotes and eukaryotes. The enzymes use primary amines as substrates to catalyze the oxidative deamination and produce aldehydes, ammonia, and hydrogen peroxide. In prokaryotes, amine oxidases provide the source of carbon and nitrogen but in eukaryotes they are suggested to be involved in cell differentiation, cell growth, detoxification, wound healing, cell signaling, and possibly in cell death.
Copper-containing amine oxidases are homodimers with molecular weight or single subunit ranging from 70,000 to 105,000 daltons depending on the sources. Each subunit of this type of enzymes contains a single copper ion and a covalently bound cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), formed by the post-translational modified tyrosine side chain. Two kinds of amine oxidases from gram positive bacterium Atrhrobacter globiformis have been identified based on their amino acid similarity and substrate specificity. One of two isoforms, termed histaminase or AGHO, prefers histamine as the substrate, while the other, phenylethylamine oxidase or AGPEO, prefers phenylethylamine as the major substrate.
The AGHO gene encoding for histaminase has been subcloned from Atrhrobacter globiformis and inserted into an expression vector pET30(+) to give a fusion protein of full length AGHO and His6-tag. The His6-tagged protein was expressed in Escherichia coli strain BL21(DE3). The E. coli BL21(DE3) harboring pET30b(+)/ AGHO were grown at 37℃ in LB medium. Then, transfer to the fresh medium containing 50 μM CuSO4 and 0.05 mM IPTG at 25℃ for 8 hours. The purification was done by ammonium sulfate precipitation and affinity chromatography. The yield of recombinant AGHO was 33.75 mg/L cultural medium.
The recombinant AGHO was found to be sensitive to temperature higher than 30 ºC. The enzyme activity decreased about 10 % and 40 %, when recombinant AGHO was kept at 45 ºC and 60 ºC, respectively, for 30 min. The optimal pH range for the AGHO/HRP coupled-enzyme activity assay in the presence of histamine and ABTS is between pH 8.0 and 9.0. The kinetic study showed that the Km for PEA is 134.9 μM and Vmaxis 8.66 nmole/min/μg protein.. The Km for histamine is 10.9 μM and Vmaxis 0.02 nmole/min/μg protein.
Part of the goal of this research is to study the substrate specificities of wild type as well as AGHO mutants. The residue Y316 is found to locate at the gate of substrate channel and is postulated to play a role in recognition of various substrates. Y316 is also the residue that was shown to control the orientation of TPQ cofactor and to facilitate the deprotonation of TPQ during reaction. We have changed this residue into different amino acids, A, E, F, H, and W. We found that Y316 mutants showed low or no enzymic activity toward various substrates, including histamine, phenylethylamine, tyramine, and tryptamine. Interestingly, the low enzymic activity is due to the lack of the formation of TPQ in these Y316 mutants.

目 次 表
中文摘要 i
英文摘要 iii
致謝 v
目次表 vi
圖目錄 vii
表目錄 viii
附圖及附表目錄 ix
縮寫表 x
一、緒論 1
二、材料與方法 7
三、結果 20
四、討論與結論 26
五、參考文獻 30
圖 目 錄
圖一、pET-30a-c (+)圖譜 35
圖二、pET-30b (+) /AGHO圖譜 36
圖三、在37℃以不同濃度之IPTG誘導8小時表現AGHO的情形
37
圖四、在37℃表現以不同濃度之IPTG誘導後之山葵過氧化酵素耦合反應活性測試 38
圖五、AGHO在不同溫度及不同IPTG濃度誘導下所表現的結果
39
圖六、以硫銨沈澱法對可溶性蛋白質進行前處理的結果 40
圖七、以硫銨沈澱法(10%、50%)對可溶性蛋白質進行前處理的結果
41
圖八、親和性管柱層析法純化的結果 42
圖九、含醌蛋白質的轉漬及呈色法 43
圖十、不同濃度之AGHO的活性測試 44
圖十一、AGHO與不同濃度HRP的活性測試 45
圖十二、AGHO在不同濃度的Histamine活性測試 46
圖十三、AGHO在不同溫度下的活性測試 47
圖十四、AGHO在不同pH值下的活性測試 48
圖十五、山葵過氧化酵素反應活性測試的相關曲線 49
圖十六、突變株的轉漬及呈色法 50
表 目 錄
表一、純化產率 50
表二、酵素動力學的計算結果 50
表三、有關胺類氧化酵素之活性測試的相關文獻 51
表四、有關輔因子 (quinone、Cu) 之活性測試的相關文獻 52
附 圖 及 附 表 目 錄
附 圖
附圖一、AGPEO 結構 54
附圖二、Y316位置在AGHO 所扮演的角色 55
附圖三、AGHO從分子表面至活性區的通道 56
附 表
附表一、AGHO胺基酸序列 57
附表二、不同來源的胺類氧化酵素胺基酸序列的比對 58
附表三、本實驗中所使用引子序列 60
附表四、本實驗中所建構的質體 61
附表五、各種常見的胺類結構式 65

[1] Akimoto, K., Shinmen, Y., Sumida, M., Asami, S., Amachi, T., Yoshizumi, H., Saeki, Y., Shimizu, S., and Yamada, H. Luminol Chemiluminescence Reaction Catalyzed by a Microbial Peroxidase. Analysis Biochemistry 189, 182-185,1990.
[2] Aygün, O., Schneider, E., Scheuer, R., Usleber, E., Gareis, M., and Märtlbauer, E. Comparison of ELISA and HPLC for the Determination of Histamine in Cheese. Journal of Agriculture Food Chemistry 47, 1961-1964. ,1999.
[3] Bracing, C. E., Smoot, J. C., Findlay, R. H. and Actis, L. A. Plasmid-Mediated Histamine Biosynthesis in the Bacterial Fish Pathogen Vibrio anguillarum. Plasmid 39, 235-244,1998.
[4] Berry M. D., Juorio A. V., and Paterson I. A. The function role of monoamine oxidase —A and —B in the mammalian central nervous system. Progress in Neurobiology 42, 375-391, 1994.
[5] Blum, L. J. Chemiluminescence Flow Injection Analysis of Glucose in Drinks with a Bienzyme Fiberoptic Biosensor. Enzyme Microbiological Technology 15, 407-411,1993.
[6] Canizares F., Salinas J., de las Heras M., Diaz J., Tovar I., Martinez P., Penafiel R. Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters. Clinical Cancer Research 5, 2035-2041., 1999.
[7] Choi, Y. H., Matsuzaki, R., Fukui, T., Shimizu,E., Yorifuji, T., Sato,H., Ozaki, Y. and Katsuyuki Tanizawa. Cooper/Topa Quinone-containing Histamine Oxidase from Arthrobacter globiformis. The Journal of Biological Chemistry 270, 4712-4720,1995.
[8] Choi, Y. H., Matsuzaki, R., Suzuki, S. and Tanizawa, K. Role of Conserved Asn-Tyr-Asp-Tyr Sequence in Bacterial Copper/ 2,4,5- Trihydroxy- phenylalanyl Quinone-containing Histamine Oxidase. The Journal of Biological Chemistry 271, 22598-22603, 1996.
[9] Conyers, S. M. and Kidwell, D. A. Chromogenic Substrate for Horseradish Peroxidase. Analytical Biochemistry 192, 207-211, 1991.
[10] Cooper, R. A., Knowles, P. F., Brown, D. E., Mcgulrl, M. A. and Dooley D.M.. Evidence for cooper and 3,4,6-TPQ cofacters in an amine oxidase from the Gram-negative bacterium Escherichia coli K-12. Biochemical Journal 288,337-340., 1992.
[11] Draisci, R., Giannetti, L., Boria, P., Lucentini, L., Palleschi, L. and Cavalli, S.. Improved Ion Chematography-interated Pulsed Amperometric Detection Method for the evaluation of biogenic Amines in Food of Vegetable or Animal Origin and in Fermented Foods. Journal of Chromatography A 798, 109-116, 1998.
[12] Federico, R., Angelini, R., Ercolini, L., Venturini, G., Mattevi, A. and Ascenzi, Paolo. Competitive Inhibition of Swine Kiney Copper Amine Oxidase by Drugs: Amiloride, Clonidine, and Gabexate Mesylate. Biochemical and Biophysical Research Communications 240, 150-152, 1997.
[13] Freeman, T. M. and Seitz, W. R.. Chemiluminescence Fiber Optic probe for Hydrogen Peroxide Based on the Luminol Reaction. Analysis Chemistry 50, 1242-1246, 1978.
[14] Fernandez C., Sharrard R.M., Talbot M., Reed B.D., Monks N. Evaluation of the significance of polyamines and their oxidases in the aetiology of human cervical carcinoma. British Journal of Cancer 72(5), 1194-1199, 1995.
[15] Glória, M. B. A. and Izquierdo-Pulido, M. Levels and Significance of Biogenic Amines in Brazilian Beers. Journal of Food Composition and Analysis 12, 129-136, 1999.
[16] Göpel, W. and Heiduschka, P. Interface Analysis in Biosensor Design. Biosensor and Bioelectronics 10, 853-883, 1995.
[17] Ha, H. C., Woster, P. M., Yager, J. D. and Casero, R. A.. The Role of Polyamine Catabolism in Polyamine Analogue-Induced Programmed Cell Death. Proceedings of the National Academy of Sciences 94, 11557-11562, 1997.
[18] Hevel, J. M., Mills , S. A., and Klinman J. P.. Mutation of a Strictly Conserved, Active- Site Residue Alters Substrate Specificity and Cofactor Biogenesis in a Copper Amine Oxidase. Biochemistry 38, 3683-3693, 1999.
[19] Holgate, S.T.. The Role of Mast Cells and Basophils in Inflammation. Clinical and Experimental Allergy 30, 28-32, 2000.
[20] Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M. and Klinman, J. P.. Identification of Topaquinone and Its Consensus Sequence in Copper Amine Oxidases. Biochemistry 31, 12147-12154, 1992.
[21] Kjelke, M., Andersen, M. B., Scjneider, P., Christensen, B., Schülein, M. and Welinder, K. G.. Comparison of Structure and Activities of Peroxidase from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus. Biochemica et Biophysica Acta. 1120, 248-256, 1992.
[22] Knoll, J.. (-)Deprenyl(Selegiline), a catecholaminergic activity enhancer (CAE) substance acting in the brain. Pharmacol. Toxicol. 82, 57-66, 1998.
[23] Klinman, J.P. and Mu, D.. Quinoenzymes in biology. Annual Review Biochemistry 63, 299—344, 1994.
[24] Kumar, V., Dooley D. M., Freeman H.C., Guss J.M., Harvey, I., McGuirl, M. A., Wilce, M.C.J. and ZuBak V. M. Structure 4, 943-955, 1996.
[25] Loughran, M. G., Hall, J. M., Turner, A. P. F. and Davidson, V. L. Amperometric Detection of histamine at a Quinoprotein Dehydrogenase Enzyme Electrode. Biosensors and Bioelectronics 10, 569-576, 1995.
[26] Mclntire W. S. and Hartmann C. Copper-containing amine oxidases. Principles and Applications of Quinoproteins 97-171, 1993.
[27] Nothen, M. M., Erdmann, J., Shimronabarbanell, D. and Propping, P. Identification of Genetic Variation in the Human Serotonin 1D Receptor Gene. Biochemical and Biophysical Research Communications 205, 1194-1200, 1994.
[28] Ortiz, J., Gómez, J., Torrent, A., Aldavert, M. and Blanco, I. Quantitative Radioisotopic Determination of Histidine Decarboxylase using High-Performance Liquid Chromatography. Analytical Biochemistry 280, 111-117, 2000.
[29] Paz, M. P., Fluckiger, R., Boak, A., Kagan, H. M. and Gallop, P. M.. Specific Detection of Quinoproteins by Redox-cycling Staining. The Journal of Biological Chemistry 266, 689-692., 1991.
[30] Parsons, M.R., Convery, M. A., Wilmot, C. M., Yadav, K. D. S., Blakeley, V., Corner, A. S., Phillips, S. E. V., Mcpherson, M. J. and Knowles, P. F.. Crystal Structure of a Quinoenzyme: Copper Amine Oxidase of Escherichia coli at 2 Å Resolution. Structure 3, 1171-1184, 1995.
[31] Plastino, J., Green, E. L., Sanders-Loehr, J., and Klinman J. P.. An Unexpected Role for the Active Site Base in Cofactor Orientation and Flexibility in the Copper Amine Oxidase from Hansenula polymorpha. Biochemistry 38, 8204-8216, 1999.
[32] Schmidt, T. G. M. and Skerra, A.. Use of the Strep-Tag and Streptavidin for Detection and Purification of Recombinant Protein. Protein Engineering 6(109),271-305, 1993.
[33] Schwartz, B., Green, E. L., Sanders-Loehr, J., and Klinman, J. P.. Relationship between Conserved Consensus Site Residues and the Productive Conformation for the TPQ Cofactor in a Copper-Containing Amine Oxidase from Yeast.. Biochemistry 37, 16591-16600, 1998.
[34] Shalaby, A. R.. Significance of Biogenic Amines to Food Safety and Human Health. Food Research international 29, 675-690, 1996.
[35] Stryer, D.B. and Bero L.A. Drug promotion. The New England Journal of Medicine. 332(15), 1032, 1995.
[36] Szutowicz, A.,Kobes, R. D., and Orsulak, P. J.. Colorimetric Assay for Monoamine Oxidase in Tissues Using Peroxidase and 2,2’-Azinodi (3- ethylbenzthiazoline -6-sulfonic Acid) as Chromogen. Analytical Biochemistry 138, 86-94, 1984.
[37] Tanaka, M., Ishimori, K. and Morishima, I.. Luminol Activity of Horseradish Peroxidase Mutants Mimicking a Proposed Binding Site for Luminol in Arthromyces ramosus Peroxidase. Biochemistry 38, 10463-10473, 1999.
[38] Tanizawa, K., Matsuzaki, R., Shimizu,E., Yorifuji, T. and Fukui, T.. Cloning and Sequence of Phenylethylamine Oxidase from Arthrobacter globiformis and Implication of Tyr-382 as the Precursor to its Covalently Bound Quinone Cofactor. Biochemical and Biophysical Research Communications 199, 1096-1102, 1994.
[39] Wilce, M. C. J., Dooley, D. M., Freeman, H. C., Guss, J. M., Matsunami, H., McIntire, W. S., Ruggiero, C. E., Tanizawa, K. and Hiroshi Yamaguchi, H.. Crystal Structures of the Copper- Containing Amine Oxidase from Arthrobacter globiformis in the Holo and Apo Forms: Implications of the Biogenesis of Topaquinone. Biochemistry 36, 16116-16133, 1997.
[40] Wilmot, C. M., Murray, J. M., Alton, G., Parsons, M. R., Convery, M. A., Blakeley,, V., Corner, A. S., Palcic, M. M., Knowles, P. F., McPherson, M. J., and Phillips, S. E. V.. Catalytic Mechanism of the Quinoenzyme Amine Oxidase from Escherichia coli: Exploring the Reductive Half-Reaction. Biochemistry 36, 1608-1620, 1997.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
1. 4 王伯徹、邱世浩、黃仁彰(1998):食用菇保健食品專輯。食品工業月刊,30(5):1-36。
2. 12 李根永、李孟修(1998):Corynebacterium glutamicum在高濃度鹽份培養基脯胺酸發酵之研究。中國農業化學會誌,36(1):57-64。
3. 16 邱紫文、劉懷勝(1995):生物反應器概述。化工技術,3(3):86-92。
4. 13 李泰興(1998):氧氣傳送介質及消泡劑對模擬醱酵液中氧氣傳送之影響。技術學刊,13(1):103-108。
5. 7 吳文騰(1994):談醱酵控制。化工,41(3):41-43。
6. 1 丁懷謙(2000):食藥用菇多醣體之免疫生理活性。食品工業,32(5):28-42。
7. 11 陳惠英、顏國欽(1998):自由基、抗氧化防禦與人體健康。中華民國營養學會雜誌,21(3):105-121。
8. 10 李俊賢、高寶璧、詹美華、蘇慶華(1992):真菌性中藥材水溶性多糖成分之分析。北醫學報,21(1):25-33。
9. 25 許瑞祥(1998):靈芝的研究現況與發展趨勢。食品市場資訊,8711:26-28。
10. 27 陳勁初(1999):生技食品之生產製程與設備。食品資訊,162:36-38。
11. 40 廖英明(1998):菇類中的許不了∼樟芝。農業世界雜誌,176:76-79。
12. 41 劉永銓(1997):生物反應器之放大。化工,44(1):26-35,53。
13. 42 劉永銓、陳光宇(1999):錐形瓶中氣液質傳係數及好氣培養菌體濃度之估算方法。興大工程學刊,10(2):9-14。
14. 46 蕭世洪(1999):神祕的傳說-靈芝。中化藥訊,41(1):30-35。
15. 47 錢明賽(1998):蔬果中之抗氧化物質。食品工業月刊,30(8):21-34。