系統識別號 | U0002-1608200711495100 |
---|---|
DOI | 10.6846/TKU.2007.00467 |
論文名稱(中文) | 重組稻米半乳糖水解酶之表現與鑑定 |
論文名稱(英文) | Expression and Characterization of Recombinant α-Galactosidase from Rice |
第三語言論文名稱 | |
校院名稱 | 淡江大學 |
系所名稱(中文) | 生命科學研究所碩士班 |
系所名稱(英文) | Graduate Institute of Life Sciences |
外國學位學校名稱 | |
外國學位學院名稱 | |
外國學位研究所名稱 | |
學年度 | 95 |
學期 | 2 |
出版年 | 96 |
研究生(中文) | 陳詩慧 |
研究生(英文) | Shi-Hui Chen |
學號 | 694290197 |
學位類別 | 碩士 |
語言別 | 繁體中文 |
第二語言別 | |
口試日期 | 2007-07-17 |
論文頁數 | 80頁 |
口試委員 |
指導教授
-
簡素芳
委員 - 張可中 委員 - 莊子超 |
關鍵字(中) |
Pichia pastoris α-galactosidase |
關鍵字(英) |
Pichia pastoris α-galactosidase |
第三語言關鍵字 | |
學科別分類 | |
中文摘要 |
半乳糖水解酶可分解多醣,水解B型紅血球表面抗原的半乳糖基,使B型紅血球轉換成O型紅血球,可用於臨床輸血。本研究將稻米半乳糖水解酶基因整合到pPIC9K質體,在轉殖到重組酵母菌(Pichia pastoris)的染色體DAN上,並以4 mg/ml G418篩選出具有multicopy的菌株,以表現稻米半乳糖水解酶。重組酵母菌之培養條件測試,分別針對培養基種類、誘導溫度、誘導起始A600值、每日甲醇量及時間間隔、誘導培養基添加額外碳源檢測其對產量的影響,得到最佳表現量,細胞內酵素單位為5.124 units/ml,細胞外酵素單位為1.127 units/ml。大量培養之細胞內及分泌酵素,經由陰離子交換樹脂(DEAE-Sepharose FF)與疏水性管柱層析(Phenyl-Sepharose FF)分離純化,純化倍率分別為74及94。純化後重組半乳糖水解酶經SDS-PAGE分析,有2種主要蛋白質大小分別為40及60 kDa。重組半乳糖水解酶特性分析,最適反應溫度為37℃、熱穩定性為50℃以下、最適反應pH為4、pH穩定性為3.0~5.0。以HPLC分析重組半乳糖水解酶水解α1→6連結的多糖特異性,Melibiose>Raffinose>Stachyose。在實驗特定條件下,以1.5 units的純化酵素在2小時內可將約50%的B型紅血球轉為O型紅血球。 |
英文摘要 |
α-Galactosidase hydrolyzed the terminal galactosyl residues from oligosaccharides including blood group B substance on the B red blood cell surface. For the blood conversion purpose, We had cloned the rice-α-galactosidase in pPIC9K vector and transformed into Pichia pastoris SMD1168. We intended to find a optimal culture condition for α-galactosidase to be expressed. We had tested the type of the medium , the start pH of the medium, the induce temperature, the starting A600 of the culture, the concentration of methanol to induce and the effects of carbon source concentration for the expression on α-galactosidase. The enzyme activity inside the cells was 5.124 units/ml; in the medium was 1.127 units/ml. Both ion exchange(DEAE-Sepharose) and hydrophobic interaction (Phenyl-Sepharose) column chromatographies were used to purify intracellular and secreted protein. They were purified 74 and 94 fold, respectively. The purified enzyme showed two major band on SDS-PAGE. The molecular weight of recombinant α-Galactosidase was estimated about 60 and 40 kDa. The maximum activity occurred at a temperature of 37℃;however, inactivation was observed at the temperature above 50℃.The enzyme showed maximal activity at pH 4.4 and was slowly inactivated above pH 5.0 and below pH 3.0. The substrate specificities of the enzyme for α1→6 linked galactose were investigated by using galactose-containing oligosaccharides: melibiose, raffinose and stachyose. The enzyme specificity of these oligosaccharides was in the decreasing order: melibiose> raffinose> stachyose. For the blood conversion test, in the experiment condition, 1.5 units of purified enzyme converted 50% B RBC into O RBC in 2 hours. |
第三語言摘要 | |
論文目次 |
中文摘要 Ⅰ 英文摘要 Ⅱ 目錄 Ⅲ 圖表目錄 Ⅴ 第一章 緒論 第一節 前言 1 第二節 α-半乳糖水解酶 3 第三節 嗜甲基酵母菌(P. pastoris) 7 第二章 實驗材料、設備 第一節 實驗材料 17 第二節 實驗設備 20 第三節 培養基配置 22 第三章 實驗方法 第一節 測試培養條件 25 第二節 400ml大量培養 29 第三節 酵素的萃取與純化 29 第四節 蛋白質的鑑定 32 第四章 結果與討論 第一節 不同培養條件對表現重組半乳糖水解酶的影響 40 第二節 400ml大量培養 45 第三節 酵素的萃取與純化 46 第四節 重組半乳糖水解酶的鑑定 48 第五章 結論與未來展望 73 第六章 參考文獻 76 圖表目錄 表1 利用P. pastoris 表現的異源蛋白質 16 表2 細胞內重組半乳糖水解酶純化表 63 表3 分泌重組半乳糖水解酶純化表 63 表4 以HPLC 分析重組稻米α-半乳糖水解酶水解Melibiose、 Raffinose 與Stachyose 的能力 70 圖1 α-半乳糖水解酶催化作用 12 圖2 稻米α-半乳糖水解酶結構 13 圖3 P. pastoris 利用甲醇的代謝路徑 14 圖4 異源蛋白質分泌至胞外示意圖 15 圖5 培養基種類測試 53 圖6 誘導溫度測試 54 圖7 誘導起始菌液濃度測試 55 圖8 誘導添加甲醇量測試 56 圖9 誘導添加碳源種類測試 57 圖10 誘導添加碳源量測試 58 圖11 誘導添加甲醇時間間隔測試 59 圖12 400ml 大量培養菌數及活性變化圖 60 圖13 FPLC DEAE-Sepharose Fast Flow 純化流程圖 61 圖14 FPLC Phenyl-Sepharose Fast Flow 純化流程圖 62 圖15 誘導後細胞及培養基蛋白質以SDS-PAGE 分析含量變化 64 圖16 酵素純化結果 65 圖17 酵素最適pH 值與最穩定pH 值 66 圖18 酵素熱穩定性 67 圖19 酵素產物p-nitrophenol 標準線 68 圖20 酵素動力學測試 69 圖21 測試血球所需凝血時間及量 71 圖22 B 型紅血球轉成O 型紅血球能力測試 72 |
參考文獻 |
Baez, J., Olsen, D., and Polarek, J. W. (2005). Recombinant microbial systems for the production of human collagen and gelatin. Appl. Microbiol. Biotechnol. 69, 245-252. Bobrowicz, P., Davidson, R. C., Li, H., Potgieter, T. I., Nett, J. H., Hamilton, S. R., Stadheim, T. A., Miele, R. G., Bobrowicz, B., Mitchell, T., et al. (2004). Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14, 757-766. Buckholz, R. G., and Gleeson, M. A. (1991). Yeast systems for the commercial production of heterologous proteins. Biotechnology (N.Y.) 9, 1067-1072. Chien, S. F., and Lin-Chu, M. (1991). The conversion of group B red blood cells into group O by an alpha-D-galactosidase from taro (Colocasia esculenta). Carbohydr. Res. 217, 191-200. Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989). Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316-1323. Desnick, R. J., Dean, K. J., Grabowski, G., Bishop, D. F., and Sweeley, C. C. (1979). Enzyme therapy in Fabry disease: differential in vivo plasma clearance and metabolic effectiveness of plasma and splenic alpha-galactosidase A isozymes. Proc. Natl. Acad. Sci. U.S.A. 76, 5326-5330. Desnick, R. J., Ioannou, Y. A., Fairley, J. L., and Eng, C. M. (1995). α-Galactosidase A Deficiency: Fabry Disease (New York: McGraw-Hill). Dey, P. M., Patel, S., and Brownleader, M. D. (1993). Induction of alpha-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum. Biotechnol. Appl. Biochem. 17 (Pt 3), 361-371. Fujimoto, Z., Kaneko, S., Momma, M., Kobayashi, H., and Mizuno, H. (2003). Crystal structure of rice alpha-galactosidase complexed with D-galactose. J. Biol. Chem. 278, 20313-20318. Ge, T., Fu, S. H., Xu, L. H., Tang, Q., Wang, H. Y., Guan, K. P., and Liang, G. D. (2007). High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris. Protein Expr. Purif. 52, 1-7. Gellissen, G. (2000). Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741-750. Gemmill, T. R., and Trimble, R. B. (1999). Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426, 227-237. Hart, D. O., He, S., Chany, C. J., 2nd, Withers, S. G., Sims, P. F., Sinnott, M. L., and Brumer, H., 3rd (2000). Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase. Biochemistry 39, 9826-9836. Hartner, F. S., and Glieder, A. (2006). Regulation of methanol utilisation pathway genes in yeasts. Microb. Cell Fact. 5, 39. Henrissat, B., and Davies, G. (1997). Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637-644. Houard, S., Heinderyckx, M., and Bollen, A. (2002). Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera. Biochimie 84, 1089-1093. Ioannou, Y. A., Zeidner, K. M., Gordon, R. E., and Desnick, R. J. (2001). Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice. Am. J. Hum. Genet. 68, 14-25. Johnson, M. A., Waterham, H. R., Ksheminska, G. P., Fayura, L. R., Cereghino, J. L., Stasyk, O. V., Veenhuis, M., Kulachkovsky, A. R., Sibirny, A. A., and Cregg, J. M. (1999). Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris. Genetics 151, 1379-1391. Jungo, C., Marison, I., and von Stockar, U. (2007). Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures. J. Biotechnol. 128, 824-837. Kaneko, R., Kusakabe, I., Sakai, Y., and Murakami, K. (1990). Substrate Specificity of α-Galactosidase from Mortierella vinacea. Agric. Biol. Chem. 54, 237-238. Kim, W. D., Kobayashi, O., Kaneko, S., Sakakibara, Y., Park, G. G., Kusakabe, I., Tanaka, H., and Kobayashi, H. (2002). alpha-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells. Phytochemistry 61, 621-630. Koutz, P., Davis, G. R., Stillman, C., Barringer, K., Cregg, J., and Thill, G. (1989). Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5, 167-177. Lee, B., Yurimoto, H., Sakai, Y., and Kato, N. (2002). Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii. Microbiology 148, 2697-2704. Lee, J. D., and Komagata, K. (1980). Taxonomic study of methanol- assimilating yeasts. J. Gen. Appl. Microbiol. 26, 133-158. Lenny, L., Hurst, R., Goldstein, J., and Galbraith, R. A. (1994). Transfusions to group O subjects of 2 unitsnits of red cells enzymatically converted from group B to group O. Transfusion 34, 209-214. Li, Z., Xiong, F., Lin, Q., d'Anjou, M., Daugulis, A. J., Yang, D. S., and Hew, C. L. (2001). Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr. Purif. 21, 438-445. Ly, H. D., Howard, S., Shum, K., He, S., Zhu, A., and Withers, S. G. (2000). The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean alpha-galactosidase and identify the catalytic nucleophile. Carbohydr. Res. 329, 539-547. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249-270. Merritt, E. A., and Bacon, D. J. (1997). Raster3D: photorealistic molecular graphics. Meth. Enzymol. 277, 505-524. Ostergaard, S., Olsson, L., and Nielsen, J. (2000). Metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 64, 34-50. Reid, J. S., Edwards, M. E., Gidley, M. J., and Clark, A. H. (1992). Mechanism and regulation of galactomannan biosynthesis in developing leguminous seeds. Biochem. Soc. Trans. 20, 23-26. Rossanese, O. W., Soderholm, J., Bevis, B. J., Sears, I. B., O'Connor, J., Williamson, E. K., and Glick, B. S. (1999). Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J. Cell Biol. 145, 69-81. Sakai, Y., Tani, Y., and Kato, N. (1999). Biotechnological application of cellular functions of the methylotrophic yeast. J. Mol. Catal., B Enzym. 6, 161-173. Shibuya, H., Kobayashi, H., Sato, T., Kim, W. S., Yoshida, S., Kaneko, S., Kasamo, K., and Kusakabe, I. (1997). Purification, characterization, and cDNA cloning of a novel alpha-galactosidase from Mortierella vinacea. Biosci. Biotechnol. Biochem. 61, 592-598. Stratton, J., Chiruvolu, V., and Meagher, M. (1998). High cell-density fermentation. Methods Mol. Biol. 103, 107-120. Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A., and Gingeras, T. R. (1987). Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 15, 3859-3876. Vance, D. E., Krivit, W., and Sweeley, C. C. (1969). Concentrations of glycosyl ceramides in plasma and red cells in Fabry's disease, a glycolipid lipidosis. J. Lipid Res. 10, 188-192. Vedvick, T., Buckholz, R. G., Engel, M., Urcan, M., Kinney, J., Provow, S., Siegel, R. S., and Thill, G. P. (1991). High-level secretion of biologically active aprotinin from the yeast Pichia pastoris. J. Ind. Microbiol. 7, 197-201. Von Stockar, U., Gustafsson, L., Larsson, C., Marison, I., Tissot, P., and Gnaiger, E. (1993). Thermodynamic considerations in constructing energy balances for cellular growth. Biochim. Biophys. Acta 1183, 221-240. Woo, J. H., Liu, Y. Y., Mathias, A., Stavrou, S., Wang, Z., Thompson, J., and Neville, D. M., Jr. (2002). Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expr. Purif. 25, 270-282. Xiong, A. S., Yao, Q. H., Peng, R. H., Zhang, Z., Xu, F., Liu, J. G., Han, P. L., and Chen, J. M. (2006). High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Appl. Microbiol. Biotechnol. 72, 1039-1047. Yamane, T. (1971). Decomposition of raffinose by α-galactosidase. An enzymatic reaction applied in the factory-process in Japanese beet sugar factories. Sucr. Belge/Sugar Ind. Abstr. 90, 345-348. Zhang, W., Hywood Potter, K. J., Plantz, B. A., Schlegel, V. L., Smith, L. A., and Meagher, M. M. (2003). Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. J. Ind. Microbiol. Biotechnol. 30, 210-215. Zhang, Y., and Yang, B. (2006). In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning. Anal. Biochem. 357, 232-239. Zsebo, K. M., Lu, H. S., Fieschko, J. C., Goldstein, L., Davis, J., Duker, K., Suggs, S. V., Lai, P. H., and Bitter, G. A. (1986). Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region. J. Biol. Chem. 261, 5858-5865. |
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