簡易檢索 / 詳目顯示

研究生: 陳筱婷
Shiao-ting Chen
論文名稱: 透明顫菌血紅蛋白表現對木質醋酸菌生長及細菌纖維素生產之效應
Effect of Vitreoscilla hemoglobin expression on the growth of Acetobacter xylinum and its production of bacterial cellulose
指導教授: 李振綱
Cheng-Kang Lee
口試委員: 陳秀美
Hsiu-Mei Chen
陳志成
C -Will Chen
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2006
畢業學年度: 94
語文別: 中文
論文頁數: 101
中文關鍵詞: 透明顫菌血紅蛋白木質醋酸菌細菌纖維素
外文關鍵詞: Vitreoscilla hemoglobin, Acetobacter xylinum, bacterial cellulose
相關次數: 點閱:189下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

本研究方向主要區分為兩大部分,第一部份主要是探討能提高菌體氧氣利用能力之透明顫菌血紅蛋白(Vitreoscilla hemoglobin;VHb)對木質醋酸菌 ( Acetobacter xylinum;A. xylinum)轉型株之影響。首先以pBBR122 載體進行A. xylinum電轉殖與化學轉殖之測試,結果電轉殖法有較高的轉殖效率。在轉殖方法成功建立後,我們將VHb基因轉殖入 A. xylinum 菌體中,並開始探討A. xylinum轉型株在充氧和微氧的條件下,菌體生長及生產細菌纖維素之研究。實驗結果顯示VHb基因表現可幫助 A. xylinum 菌體的生長,在充氧的條件下,含VHb基因表現的重組菌株比生長速率為宿主細胞的3倍;在微氧的條件下含VHb基因表現的重組菌株比生長速率則和宿主細胞相當。在探討細菌纖維素的研究方面,在充氧的條件下含VHb基因表現的重組菌株細菌纖維素的生產量為宿主細胞的2倍,在微氧的條件下含VHb基因表現的重組菌株細菌纖維素的生產量為宿主細胞的3倍。

第二部分研究是延續第一部分的結果以A. xylinum菌體為主軸,探討是否能夠藉由在A. xylinum中本身就有UDP-Glucose dehydrogenase (aceM) 和UDP-Glucose pyrophorylase (hasC),我們再將hyaluronic acid synthase (hasA)基因轉殖入 A. xylinum 菌體中,希望能夠利用A. xylinum合成透明質酸。結果顯示我們成功的將hyaluronic acid synthase (hasA)基因轉殖入 A. xylinum 菌體中進行表現,但是由於hyaluronic acid synthase(hasA)是膜蛋白,表現量相當的少,所以我們只針對透明質酸產量做初步檢測,結果發現利用 A. xylinum合成透明質酸是可行的。


Bacterial cellulose can be produced by Gram-negative bacterium,such as rod-shaped Acetobacter xylinum. As the organism is obligate aerobic, bacterial cellulose is always produced at the air/liquid interface. In a static culture, the oxygen supply is considered as the limiting factor for cell growth and cellulose production. Vitreoscilla, a strict aerobic bacterium, synthesizes bacterial hemoglobin (VHb) to survive and grow in an O2-poor environment.VHb plays the role in facilitating oxygen transfer to the respiratory chain in cell membrane. In this work, the constitutive bla-promoter was employed to drive the expression of VHb in cellulose producing bacteria, Acetobacter xylinum BCRC12334.The feasibilities of using bla-promoter in Acetobacter xylinum to drive expression of heterologous proteins as well as the function of expressed VHb to enhance the cell growth and cellulose productivity at microaerobic and anaerobic condition were studied. The results show that VHb did improve cellulose yield during the static flask cultivation at microaerobic and anaerobic condition. The growth rate of VHb expressing strain VHb+ was 3 fold higher than that of host strain at microaerobic condition. At anaerobic condition, the growth rate of VHb+ strain also similar to that of host strain. The amount of bacterial cellulose producted by VHb+ strain was 2 fold higher than that of host strain under the microaerobic condition. At anaerobic condition, the produced amount of bacterial cellulose by VHb+ strain was 3 fold of that produced by host strain.

中文摘要 ……………I 英文摘要 ……………..III 誌謝 ……………..V 目錄 ……………..VI 圖目錄 ……………..X 表目錄 ……………..XII 第一章緒論 1 1.1 研究背景及目的 1 1.2 研究內容簡介 2 第二章文獻回顧 4 2.1 木質醋酸菌之簡介及其應用 4 2.1.1 木質醋酸菌之特性 4 2.1.2 木質醋酸菌產生細菌纖維素構造及合成 6 2.1.3 木質醋酸菌生產細菌纖維素之方法與應用 13 2.1.4 木質醋酸菌轉殖的方法與研究 17 2.1.5 氯化鈣轉殖法及電轉殖法之簡介 19 2.1.6 轉殖質體穩定度 20 2.2 氧氣對菌體生長之影響 21 2.3 透明顫菌血紅蛋白(VHb)之特性 22 2.3.1 VHb表現對胞內溶氧之影響 24 2.3.2 表現對菌體生長及蛋白質產率之影響 25 2.4 透明質酸簡介 26 2.4.1 透明質酸的由來與結構 27 2.4.2 透明質酸的生化合成 27 第三章實驗流程、材料與方法 31 3.1實驗流程 31 3.2實驗材料 33 3.2.1菌株 33 3.2.2質體 33 3.2.3引子primers 34 3.2.4 DNA純化套件組 35 3.2.5標準分子量溶液 35 3.2.6抗體 35 3.3 實驗藥品 35 3.4 實驗設備 38 3.5 實驗步驟 39 3.5.1 菌株活化及培養 39 3.5.1.1 A. xylinum的活化 39 3.5.1.2 A. xylinum的培養 39 3.5.1.2.1 搖瓶震盪培養 40 3.5.1.2.2 cuvette震盪培養 40 3.5.1.2.3 靜置培養 41 3.5.2質體轉殖入宿主細胞 41 3.5.2.1 木質醋酸菌電轉殖方法步驟 41 3.5.2.2 木質醋酸菌氯化鈣轉殖法的方法步驟 43 3.5.3 質體的複製 44 3.5.3.1 轉殖到E. coli Top10中複製 44 3.5.3.2質體DNA的萃取 45 3.5.4 各表現載體之轉殖菌株篩選 46 3.5.4.1 抗生素篩選 46 3.5.4.2 PCR快速篩選 46 3.5.5 重組菌株之培養及蛋白表現 49 3.5.5.1菌體之培養 49 3.5.5.2重組蛋白之純化 49 3.5.6 VHb 活性分析 50 3.5.7 VHb 細菌纖維素生長速率測量與包覆菌體之VHb基因檢測 50 3.5.7.1細菌纖維素生長速率測量 50 3.5.7.2細菌纖維素中包覆菌體之VHb基因檢測 51 3.5.8 透明質酸之純化與呈色分析 52 3.5.8.1透明質酸之純化 53 3.5.8.2 透明質酸呈色分析 53 3.5.9 質體懷有率測定 53 3.5.10質體懷有率測定 54 3.5.11西方墨點分析(Western blot) 56 第四章結果與討論 58 4.1 培養基對木質醋酸菌菌體生長及細菌纖維素之影響 59 4.2 VHb表現載體之轉殖菌株篩選 60 4.2.1 電轉殖法和化學轉殖法對醋酸菌轉殖之比較 61 4.2.2 表現質體菌株快速篩選(Colony PCR) 62 4.2.3 質體往返A. xylinum 和E-coli Top-10 64 4.3 基因重組菌株(VHb+)之蛋白質表現 68 4.4 VHb表現對於基因重組菌株之效應 69 4.4.1 VHb表現對於A. xylinum生長情形之影響 69 4.4.2VHb表現對於A. xylinum之細菌纖維素產能及外觀之影響 72 4.4.3 VHb基因於細菌纖維素中穩定行性測試 76 4.5 質體穩定度測試 78 4.6 VHb之活性分析探討 79 4.7 hyaluronic acid synthase 表現載體之轉殖菌株篩選 82 4.7.1聚合酶連鎖反應儀(PCR)快速篩選 83 4.8 A. xylinum合成透明質酸之初步測試 84 第五章結論 85 第六章建議 88 參考文獻 89 附錄 96

戶田登也、桑名好惠、桐山修八。1994。ナタデココの新機能-血中コレステロール低下作用と應用開發食品と開發 29(3):353-362.

吳東和、林慶福。1994。“Nata— 被遺忘的機能性食品” 食品工業 26(6):42-47.

Alaban, C A. (1967) Studies on the optimum conditions for“ Nata de coco” bacterium or “Nata” formation in coconut water. Phil. Agric. 45 : 490-515

Angelis, P. L., Weigel, P. H.(1993) “Isolation of a Streptococcus pyogenes gene locus that directs hyalyronan biosynthesis in acapsular mutants and in heterologous bacteria ” J. biol. Chem. 268 : 14568- 14571

Annette M. G. , Kirstin J. E., Victor J. M., Michael J. G.(1997) “Genetic analysis of acetan biosynthesis in Acetobacter xylinum: DNA sequence analysis of the aceM gene encoding an UDP-glucose dehydrogenase” Biotechnol. lett, 19, 469–474

Ashbaugh, C. D., Sebastian, A. (1998) “Molecular analysis of the capsule gene region of group A Streptococcus : the hasAB genes are sufficient for capsule expression” J. Bacteriol., Sept : 4955-4959

Armstrong, D. L., Cooney, M. J., John, M. R.(1997) “Growth and amino acid requirements of hyaluronic acid-producting Strepococci” Appl. microbiol. Biotechnol.,47:309-312

Bailey, J. E., Khosla, C. (1988) “The Vitreoscilla hemoglobin gene : molecular cloning, nucleotide sequence and genetic expression in Escherichia coli” Mol. Genet., 214 : 158-161

Bailey, J. E., Khosla, C. (1989) “Characterization of oxygen-dependent promoter of the Vitreoscilla Hemoglobin gene in Escherichia coli” J. Bacterial., 171 : 5995-6004

Bailey, J. E., Kallio, P. T., Tsai, P. S. (1996) “Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin expression for enhancing Escherichia coli growth in a microaerobic bioreactor” Biotechnol. Prog., 12 : 751-757

Bailey, J. E., Lilius, G., Holmberg, N., Bülow, L. (1999) “The metabolic effects of native and transgenic hemoglobins on plants” Tibtech. January., 17 : 21-24

Balazs, E. A., Laurent, T. C., Jeanloz, R. W.(1986) “Nomenclature of hyaluronic acid” Biochem. J. 235 : 903

Banzon, J. A., Gonzalez, O.N., Dedeon, S. Y. and Sanchez, P. C. 1990. Chapter XIV Nata de coconut as food. In “Philippine coconut research and development foundation” Inc (PCRDF), Philippines.

Blackwell, J. 1982. The macromolecular organization of cellulose and chitin. In Brown, R. M., Jr. (ed.). “Cellulose and other nature polymer systems” Plenum Publishing Corp., New York. pp.327-329.

Cannon, R. E. and Anderson, S. M. 1991. “Biogenesis of bacterial cellulose ” Crit. Rev. Microbiol. 17: 415-447

Colquhoun, I. J., Defernez, M. and Morris, V. J. 1995. “NMR studies of acetan and the related bacterial polysaccharide, CR 1/4, produced by a mutant strain of Acetobacter xylinum ” Carbohydrate Research. 269:319-331.

DeAngelis, P. L., Weigel, P. H. (1994) “Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme” Biochemistry, 33(31): 9033-9039.

Dinene, L.C.r ,Ivo Van de Rijn (1995) “Hyaluronic acid synthesis operon (has) Expression in Group A Streptococci” J. biol. chem. 270 :18452-18458

Dougherty, B. A., Rijn, Ivo van de, (1992) “Molecular characterization of a locus required for hyaluronic acid capsule production in group A Streptococci” J. exp. Med 175 :1291-1299

Dougherty, B. A., Rijn, Ivo van de, (1994) “Molecular characterization of hasA from an operon required for hyaluronic acid synthesis in group A Streptococci” J. biol. chem.269:169-175

Dougherty, B. A., Rijn, Ivo van de, (1993) “Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A Streptococci” J. biol. chem.268:7188-7124

Dougherty, B. A., Rijn, Ivo van de, (1995) “Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A Streptococci” J. biol. chem.270:28676-28680

Dudman, W. F. 1959 a. “Cellulose production by Acetobacter acetigenum in defined medium ” J. Gen. Microbiol. 21 : 312-326

Emubuscado, M. E., Marks, J. S. and Bemiller, J. N. 1994. “Bacterial cellulose. Ⅰ. Factors affection the production of cellulose by Acetobacter xylinum” Food Hydrocoll. 8 (5): 419-430

Fujiwara, M., Fukushi, K., Takai, M., Hayashi, J., Fukaya, M., Okumura, H. and Kawamura, Y. 1992. “Construction of shuttle vectors derived from Acetobacter xylinum for cellulose-producing bacterium Acetobacter xylinum ” Biotechology Letters 14(7): 539-542.

Haigler, C. H. 1985. “The functions and biogenesis of native cellulose, In R. P. Nevell and S. H. Zeronian (ed.), Cellulose chemistry and itsapplications ” Ellis Horwood Ltd., Chichester, England. p. 30-83.

Hall, P. E.anderson, S. M., Johnston, D. M. and Cannon, R. E. 1992. “Transformation of Acetobacter xylinum with plasmid DNA by electroporation ” Plasmid 28:194-200.

Hestrin, S. and Schramm, M. 1954. “Synthesis of cellulose by Acetobacter xylinum. Ⅱ. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose ” Biochem. J. 58 : 345-352

Kallio, P. T., Kim, D. J., Tasi, P. S., Vailey, J. E. (1994) “Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions” Eur. J. Biochem., 219 : 201-208

Kamide, K., Matsuda, Y.,Iijima, H. and Okajima, K. 1990. “Dffect of culture condition of acetic acid bacteria on cellulose biosynthesis ” Br. Polym. J. 22 : 167-171

Kent, R. A., Stephens, R. S. and Westland, T. A. 1991. “Bacterial cellulose fiber orovides an alternative for thickening and coating ” Food Technol. 45(6) : 108-118

Kozak, M. 1983. “Comparison of initiation of protein synthesis in prokaryotes, eucaryotes, and organelles ” Microbiol. Rev. 47:1-45.

Krieg, N. 1984. “Bergey’s Manual of Systematic Vacteriology Vol. 1 ”The Williams and Wilkins Co., Baltimore, MD. Pp. 267-274

Kumar P.K.R., Maschke H.E., Friehs K., Schugerl K.1991. “Strategies for improving plasmid stability in genetically modified bacteria in bioreactors ” Tibtech. 9:279-284

Lapuz, M. M., Gallardo, E. G. and Dalo, M. A. 1967. “The Nata organism-Cultural requirement, characteristics and identify ” Philip. J. Sci. 96(2) : 91-96

Masaoka, S., Ohe, T. and Sakota, N. 1993. “Production of cellulose form glucose by Acetobacter xylinum ” J. Fer. Bioeng. 75(1) : 18-22

Meyer, K. ,Weissmann, B.(1954) “The structure of hyaluronic acid and hyaluronic acid from umbilical cord. ” J. Am. Chem. Soc.; 76 :1753-1757

Nilsson, M., Kallio, P. T., Bailey, J. E., Bülow, L., Wahlund, K. G. (1999) “Expression of Vitreoscilla hemoglobin in Escherichia coli enhances ribosome and Trna levels : A flow field-flow fractionation study” Biotechnol. Prog., 15 : 158-163

Oikawa, T., Morino, T. and Ameyama, M. 1995 a. “Production of cellulose from D-Arabitol by Acetobacter xylinum Ku-1 Biosci ” Biotech. Biochem. 59(8) :1564-1565

Oikawa, T., Ohtori, T. and Ameyama, M. 1995 b. “Production of cellulose from E-Mannitol by Acetobacter xylinum Ku-1 Biosci ” Biotech. Biochem. 59(2) : 331-332

Okiyama, A., Motaki, M. and Yamanaka, S. 1992 a. “Bacterial cellulose Ⅱ. Processing of the gelatinous cellulose for food materials ” Food-Hydrocoll. 6 (5):479-487

Okiyama, A., Shirae, H., Kano, H. and Yamanaka, S. 1992. “Bacterial cellulose I. Two-stage fermentation process for cellulose production by Acetobacter acet. ” Food-Hydrocoll. 6 (5):471-477.

Ramandeep, A., Wikstrom, M. (2001) “The heme groups of cytochrome o from Escherichia coli” Proc. Natl. Acad. Sci., 88 : 6122-6126

Ross, P., Mayer, R. and Benziman, M. 1991. “Cellulose biosynthesis and function in bacteria” Microbiol Rev. 55 (1) :35-58.

Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger-Ohana, P.,Mayer, R., Braun, S., Vroom, E.d., Marel, G. A. v.d., Boom, J.H.v. and Benziman, M. 1987. “Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid ” Nature 325:279-281.

Saturnino-Dimaguila, L. A. 1967. “ The Nata de coco I. Characterization
and identity of the causal organism ” Phil. Agric. 51:462-474.

Shine, J. and Dalgarno, L., 1974. “The 3’-terminal sequence of Escherichia coli 16S ribosome RNA: complementarity to nonsense triplets and ribosome binding sites ” Proc. Natl. Acad. Sci. 71:1342-1346.

Sievers, M., Ludwig, W. and Teuber, M. 1994. “ Phylogenetic positioning of Acetobacter, Gluconobacter, Rhodopila and Acidiphilium species as a branch of acidophilic bacteria in the a-subclass of Proteobacteria based on 16S ribosomal DNA sequences ” System. Appl. Microbiol. 17:189-196.

Stormo, G. D., Schneider, T. D. and Gold, L. M. 1982. “Characterization of translational initiation sites in Escherichia coli. Nucl. Acids ” Res. 10:2871-2996.

Takemura, H., Tsuchida, T., Yoshinaga, F., Matsushita, K. and Adachi, O. 1994. “ Prosthetic group of aldehyde dehydrogenase in acetic acid bacteria not pyrroloquinoline quinone. Biosci ” Biotech. Biochem. 58: 2082-2083.

Tarr, H L. A. and Hibbert, H. 1931. “Polysacharide synthesis by the action of Acetobacter xylinum on carbohydrates and related compounds ” Canad. J. Res. 4 : 372-388

Tonouchi, N., Tsuchida, T., Yoshinaga, F., Horinouchi, S. and Beppu, T., 1994. “A host-vector system system for a cellulose-producing Acetobacter strain ” Biosci. Biotech. Biochem. 58:1899-1901
.
Toyosaki, H. Nacitomi, T., Seto, A., Matsuoka, M., Tsuchida, T. and Yoshinaga, F. 1995. “Screening of bacterial cellulose-producing Acetobacter strains suitable for agitated culture. Biosci. Biotech. Biochem ” 59 (8) : 1498-1502

Valla, P. G. and Kiosbakken, J. 1982. “Cellulose-negative mutants of Acetobacter xylinum ” J. Gen. Microbiol. 128 : 1401-1408

Volpi, N, “Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine ” J. Biol. Chem.,277 : 19-24

Webster, D. A., Hackett, D. P. (1966) “ The purification and Properties of cytochrome o from Vitreoscilla” J. Biol. Chem.,241 : 3308-3315

Webster, D. A., Wakabayashi, S., Mastubara, H. (1986) “Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla” Nature, 322 : 481-4823

Webster, D. A., Dikshit, K. L. (1988) “Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli” Gene, 70 : 377-386

Weinhouse, H., Sapir, S., Amikam, D., Shilo, Y., Volman, G., Ohana, P. and Benziman, M. 1997. “ C-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum ” FEBS Letters 416:207-211.

Williams, W. S. and Cannon, R. E. 1989. “Alternative environmental roles for cellulose produced by Acetobacter xylinum ” Appl. Environ. Microbiol. 55 (10):2448-2452

Wong, H. C., Fear, A. L., Calhoon, R. D., Eichinger, G. H., Mayer, R., Amikam, D., Benziman, M., Gelfand, D. H., Meade, J. H., Emerick, A. W., Bruner, R., Ben-Bassat, A. and Rony, T. 1990. “Genetic organization of the cellulose synthease operon in Acetobacter xylinum ” Proc. Natl. Acad. Sci. USA.87:8130-8134.

Yamada, Y., Hoshino, K. and Ishikawa, T. 1997. “The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to the generic level ”

Yamanaka, S., Watanabe, K. and Kitamura, N. 1989. “The structure and mechanical properties of sheets prepared from bacterial cellulose ” J. Mater. Sci. 24 : 3141-3145.

無法下載圖示
全文公開日期 本全文未授權公開 (校外網路)
全文公開日期 本全文未授權公開 (國家圖書館:臺灣博碩士論文系統)
QR CODE