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中文論文名稱 重組芋頭半乳糖水解酵素之表現與定性
英文論文名稱 Expression and Characterization of Recombinant α-Galactosidase from Taro
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 101
學期 2
出版年 102
研究生中文姓名 陳逸書
研究生英文姓名 I-Su Chen
學號 698160511
學位類別 碩士
語文別 中文
口試日期 2013-07-18
論文頁數 117頁
口試委員 指導教授-簡素芳
委員-陳銘凱
委員-官宜靜
中文關鍵字 芋頭  α-半乳糖水解酵素  基因表現  酵素純化  紅血球轉型 
英文關鍵字 Taro  α-galactosidase  gene expression  enzyme purification  blood conversion 
學科別分類 學科別自然科學化學
中文摘要 α-半乳糖水解酵素(α-galactosidase)是一種可以將非還原端半乳糖水解的酵素,在各種生物體中都存在,並且擁有許多不同的生物功能,例如在植物中α-半乳糖水解酵素參與醣類的代謝和運輸。在應用方面,α-半乳糖水解酵素可以使B型紅血球表面抗原上的半乳糖水解而變成O型紅血球,因此藉由基因工程的方法取得基因並利用 Pichia pastoris 的蛋白質表現系統來產生大量的芋頭α-半乳糖水解酵素以進行血型轉換的測試和應用。
本研究將已轉殖入α-半乳糖水解酵素基因的酵母菌大量培養,並進行誘導一天產生蛋白質,細胞內酵素活性單位最高達到3.6 units/mL。利用水解酵母菌細胞壁的方式,將酵母菌打破取出α-半乳糖水解酵素。大量的萃取液經過濃縮之後,分別進行四步純化步驟,依序是分子篩 SephadexTM G-100管柱、陰離子交換樹脂Q SepharoseTM Fast flow管柱、疏水性吸附 HiTrap Phenyl FF (high sub), 1mL管柱,分子篩SepharoseTM 6 10/300 GL。純化後的酵素樣品經過十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳與基質輔助雷射脫附離子化-飛行時間質譜儀的分析,確認是α-半乳糖水解酵素。進行一系列酵素的定性分析,包含紅血球轉型實驗,2 units的純化酵素可於2小時將實驗中總體積為120 μL的B型紅血球懸浮液轉型為O型紅血球,其轉型百分比約為80%。
英文摘要 α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological functions such as metabolism and transportation of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to fastly produce large amount of taro α-galactosidase to proceed the seroconversion of erythrocyte.
In this work, the Pichia patoris cells harboring the taro α-galactosidase gene on the expression vector were grown to large scale. After the recombinant gene had been induced for one day, the highest intracellular enzyme activities were 3.6 units/mL. Lysis of yeasts was achieved by using Lyticase to extract the crude taro α-galactosidase.
From the crude extractsα-galactosidase was then purified to homogeneity by using four different types of column chromatography, include SephadexTM G-100, Q SepharoseTM Fast flow, HiTrap Phenyl FF (high sub), and SepharoseTM 6 10/300 GL. The purified enzyme showed single band on SDS-PAGE, and then identified by by MALDI-TOF MS analysis.
The purified α-galactosidase was characterized in terms of blood conversion, and a conversion rate of 80% type B red blood cell into type O with use 2 units of purified α-galactosidase in 2h was achieved.
論文目次 目錄

第一章:序論
1.1. 前言 1
1.2. GH27家族α-半乳糖水解酶 3
1.2.1. 性質與分類 3
1.2.2. 催化機制 5
1.2.3. 生物功能 8
1.3. α-半乳糖水解酵素的應用 13
1.3.1. 食品添加應用 13
1.3.2. 製糖工業應用 13
1.3.3. 疾病醫療應用 14
1.4. 人類血型系統的介紹與應用 14
1.4.1. 紅血球的血型分類 14
1.4.2. ABO血型系統 15
1.4.3. 紅血球的血型轉換 17
1.5. 嗜甲基酵母菌(P.pastoris) 20
1.5.1. P. pastoris蛋白質表現系統 20
1.5.2. P. pastoris的甲醇代謝路徑 21
1.5.3. 酒精氧化酵素之啟動子(AOX promoter) 23
1.5.4. P. pastoris表現異源蛋白質 23
1.5.5. 使用P. pastoris表現系統的優點 24






























第二章:實驗材料與儀器設備
2.1. 物種與菌種 26
2.2. 培養基製備 28
2.3. 實驗耗材 30
2.4. 化學藥品 31
2.5. 儀器設備 35












第三章: 實驗方法與步驟
3.1. 蛋白質實驗 37
3.1.1. 蛋白質的定量:Bradford method 37
3.1.2. 酵素活性測定 38
3.1.3. 十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(SDS-PAGE) 40
3.1.4. 基質輔助雷射脫附離子化-飛行時間質譜儀 44
3.2. 酵母菌培養條件測試 47
3.2.1. 誘導時間測試 47
3.2.2. 誘導起始菌液濃度測試 48
3.3. 酵母菌大量培養與蛋白質誘導表現 49
3.4. α-半乳糖水解酵素的萃取與純化 50
3.4.1. 酵母菌細胞內外的酵素濃度測試 50
3.4.2. 酵母菌細胞內酵素的萃取 50
3.4.3. 酵素濃縮 52
3.4.4. 酵素的純化方法 53
3.5重組α-半乳糖水解酵素的定性分析 58
3.5.1. 酵素最適pH值與最穩定pH值 58
3.5.2. 酵素熱穩定性 59
3.5.3. 酵素動力學測試 60
3.5.4. 酵素水解Melibiose、Raffinose 與Stachyose 的能力 61
3.5.5. 人類紅血球轉型測試 63





















第四章:實驗結果與討論
4.1. 不同培養條件對表現重組α-半乳糖水解酵素的影響 65
4.2. 酵母菌大量培養與蛋白質誘導表現 66
4.3. α-半乳糖水解酶的萃取與純化 67
4.4. 純化後重組α-半乳糖水解酵素的電泳與質譜分析 73
4.5. 重組α-半乳糖水解酵素的定性分析結果 75
















第五章:結論與未來展望
5.1. 表現重組芋頭α-半乳糖水解酵素的培養條件修正 102
5.2. 酵母菌大量培養與蛋白質誘導表現的產量 103
5.3. α-半乳糖水解酵素的萃取方法選擇與純化結果分析 104
5.4. 純化後重組α-半乳糖水解酵素的電泳與質譜分析 105
5.5. 重組α-半乳糖水解酵素的定性分析結果 106
5.6. 未來展望 107


第六章:參考文獻 109
參考文獻 [1] Dey PM, Pridham JB. Biochemistry of α-galactosidases. Advances in
Enzymology and Related Areas of Molecular Biology. 1972;36:91–130
[2] Schaefer, R. M., Tylki-Szymanska, A., Hilz, M. J. (2009) Enzyme
replacement therapy for Fabry disease: a systematic review of available
evidence. Drugs 69, 2179-2205
[3] Springer, G. F., Nickols, J. M., Callahan, H. J. (1964) Galactosidase
action on human blood group B active Escherichia coli and Ox Red
cell substances. Science 146, 946-947
[4] Alex, Z., Lin, L., Catherine, M., Zhanfan, Z., Rosa, H., Leslie, L.,
Jack, G. (1996) Characterization of recombinant α-galactosidase for
use in seroconversion from blood group B to O of human erythrocytes.
Arch. Biochem. Biophys. 327, 324-329
[5] Wook-Dong, K., Osamu, K., Satoshi, K., Yoshikiyo, S., Gwi-Gun, P.,
Isao, K., Hideo, T., Hideyuki, K. (2002) α-Galactosidase from cultured
rice (Oryza sativa L. var. Nipponbare) cells. Phytochemistry 61, 621-
630
[6] Su-Fang, C., Marie L. C. (1991) The conversion of group B red blood
cells into group O by an α-D-galactosidase from taro (Colocasia
esculenta). Carbohyd. Res. 217, 191-200
[7] Ming-Kai Chern , Huang-YiLi, Po-FanChen, Su-FangChien(2012)
Taro α-galactosidase:A new gene product for blood conversion.
Biocatalysis and Agricultural Biotechnology 1 (2012) 135–139
[8] Bernard, H. (1991) A classification of glycosyl hydrolases based on
amino acid sequence similarities. Biochem. J. 280, 309-316
[9] Bernard, H., Gideon, D. (1997) Structural and sequence-based
classification of glycoside hydrolases. Curr. Opin. Struc. Biol. 7, 637-
644
[10] Zui, F., Osamu, K., Satoshi, K., Mintsuru, M. Hideyuki, K., Hiroshi,
M. (2002) Crystallization and preliminary X-ray crystallographic
studies of rice α-galactosidase. Acta Crystallogr. D 58, 1378-1375
[11] Zui, F., Wook-Dong, K., Satoshi, K., Gwi-Gun, P., Mitsuru, M.,
Hideyuki, K., Hiroshi, M. (2003) Crystallization and preliminary X-ray
crystallographic studies of α-galactosidase I from Mortierella vinacea.
Acta Crystallogr. D 59, 2289-2291
[12] Golubev, A. M., Nagem, R. A. P., Brandao Neto, J. R., Neustroev, K.
N., Eneyskaya, E. V., Kulminskaya, A. A., Shabalin, K. A., Savel’ev, A.
N., Polikarpov, I. (2004) Crystal structure of α-galactosidase from
Trichoderma reesei and its complex with galactose: Implications for
catalytic mechanism. J. Mol. Boil. 339, 413-422
[13] Esther, M. T. L., Thomas, S., Felix, K. (2007) The C-terminal
sequence from common bugle leaf galactan:galactan
galactosyltransferase is a non-sequence-specific vacuolar sorting
determinant. FEBS Lett. 581, 1811-1818
[14] Daniel, O. H., Shouming, H., Calvin, J. C., Stephen, G. W., Paul, F.
G. S., Michael, L. S., Harry, B. (2000) Identification of Asp-130 as the
catalytic nucleophile in the main α-galactosidase from Phanerochaete
chrysosporium, a family 27 glycosyl hydrolase. Biochemistry 39, 9826
-9836
[15] Hoa, D.L., Steven, H., Kelly, S., Shouming, H., Alex, Z., Stephen, G.
W. (2000) The synthesis, testing and use of 5-fluoro-α-D-galactosyl
fluoride to trap an intermediate on green coffee bean α-galactosidase
and identify the catalytic nucleophile. Carbohyd. Res. 329, 539-547
[16] Zui, F., Satoshi, K., Mitsuru, M., Hideyuki, K., Hiroshi, M. (2003)
Crystal structure of rice α-galactosidase complexed with D-galactose.
J. Biol. Chem. 278, 20313-20318
[17] Keller, F., Matile, P. (1985) The role of the vacuole in storage and
mobilization of stachyose in tubers of Stachys sieboldii. J. Plant
Physiol. 119, 369-380
[18] Carlos, C., Serge, D., Hernani, G. (2008) Physiological, biochemical
and molecular changes occurring during olive development and
repening. J. Plant Physiol. 165, 1545-1562
[19] Chin-Pin, S., Zainon, M. A., Hamid, L. (2006) Characterisation of an
α-galactosidase with potential relevance to ripening related texture
changes. Phytochemistry 67, 242-254
[20] Bozena, C., Uener, K., Burkhard, S., Karin, K. (2007) An
α-galactosidase with an essential function during leaf development.
Planta 225, 311-320
[21] Glena, A. G., Clyde, W., Monica, A. M. (1997) Root-zone salinity
alters raffinose oligosaccharide metabolism and transport in coleus.
Plant Physiol. 115, 1267-1276
[22] Graciela, L.S., Horacio, G. P. (1989) Raffinose synthesis in Chlorella
vulgaris cultures after a cold shock. Plant Physiol. 89, 648-651
[23] Joyce, C. P., Michelle, L. J., Ceil, S. (2003) Down-regulating
α-galactosidases enhances freezing tolerance in transgenic petunia.
Plant Physiol. 133, 901-909
[24] Tian-Yong, Z., J. Willis, C. III, Jeffrey, M., Robert, B. M., Timothy,
H., David, M., Bruce, D. (2006) An alkaline α-galactosidase transcript
is present in maize seeds and cultured embryo cells, and accumulates
during stress. Seed Sci. Res. 16, 107-121
[25] Mulimani, V. H., Ramalingam. (1995) Enzymic hydrolysis of
raffinose and stachyose in soymilk by alpha-galactosidase from
Gibberella fujikuroi. Biochem. Mol. Biol. Int. 36, 897-90
[26] Thippeswamy, S., Mulimani, V. H. (2002) Enzymic degradation of
raffinose family oligosaccharides in soymilk by immobilized
α-galactosidase from Gibberella fujikuroi. Process Biochem. 38, 635-
640
[27] Linden, J. C. (1982) Immobilized α-D-galactosidase in the sugar beet
industry. Enzyme Microb. Tech. 4, 130-136
[28] Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan
Johnson, Maryanna Quon Warner, David LaHart, Jill D. Wright.
Human Biology and Health. Englewood Cliffs, New Jersey, USA:
Prentice Hall. 1993. ISBN 0-13-981176-1
[29] Table of blood group systems. International Society of Blood
Transfusion. 2008.8 [2008-12-24].
[30] Landsteiner K. Zur Kenntnis der antifermentativen, lytischen und
agglutinierenden Wirkungen des Blutserums und der Lymphe. Zbl
Bakt.1900, 27: 357–62
[31] Landsteiner K, Levin P. A new agglutinable factor differentiating
individual human bloods. Proc Soc Exp Biol Med. 1927, 24: 600–2.
[32] Landsteiner K, Levin P. Further observations on individual
differences of human blood. Proc Soc Exp Biol Med. 1927, 24: 941–2.
[33] Springer, G. F., Feeny, K. (1956) Inhibition of blood-group
agglutinins by substances occurring in plants. J. Immunol. 76, 399-
407
[34] Yatziv, S., Flowers, H. M., (1971) Action of α-galactosidase on
glycoprotein from human β-erythrocyte. Biochem. Bioph. Res. Co. 45,
514-518
[35] Harpaz, N., Flowers, H.M., Sharon, N. (1975) Studies on
B-antigenic sites of human erythrocytes by use of coffee bean
α-galactosidase. Arch. Biochem. Biophys. 170, 676-683
[36] Dybus, S., Aminoff, D. (1983) Action of α-galactosidase from
Clostridium sporogenes and coffee beans on blood group B antigen of
erythrocytes. The effect on the viability of erythrocytes in circulation.
Transfusion 23, 244-247
[37] Russell, P. (1984) Tomato α-galactosidases: conversion of human
type b erythrocyte to type o. Phytochemistry 23, 55-58
[38] Hobbs, L., Mitra, M., Phillips, R., Haibach, H., Smith, D. (1995)
Deantigenation of human type B erythrocytes with Glycin max
α-D-galactosidase. Biomed. Parmacother 5, 244-250
[39] Larissa, A. B., Irina, Y. B. et at. (2010) Molecular characterization
and therapeutic potential of marine bacteria Pseudialteromonas sp.
KMM 701 α-galactosidase. Mar. Biotechnol. 12, 111-120
[40] Alex, Z., Lin, L., Catherine, M., Zhanfan, Z., Rosa, H., Leslie, L.,
Jack, G. (1996) Characterization of recombinant α-galactosidase for
use in seroconversion from blood group B to O of hman erythrocytes.
Arch. Biochem. Biophys. 327, 324-329
[41] Davis, M. O., Hata, D. J., Johnson, S. A., Smith, D. S. (1996)
Cloning, expression and characterization of a blood group B active
recombinant alpha-D-galactosidase from soybean (Glycine max).
Biochem. Mol. Biol. Int. 39, 471-485
[42] Davis, M. O., Hata, D. J., Johnson, S. A., Jones, D. E., Harmata, M.
A., Evans, M. L., Walker, J. C. Smith, D. S. (1997) Cloning, sequence,
and expression of a blood group B active recombinant
alpha-D-galactosidase from pinto bean (Phaseolus vulgaris). Biochem.
Mol. Biol. Int. 42, 453-467
[43] Su-Fang, C., Shi-Hui, C., Ming-Yang, C. (2008) Cloning,
expression,and characterization of rice α-galactosidase. Plant Mol.
Biol.Rep. 26, 213-224
[44]Gellissen, G. (2000). Heterologous protein production in
methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741-750.
[45] 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.
[46] 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.
[47] 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.
[48] 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
[49] 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.
[50] 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
[51] 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.
[52] Buckholz, R. G., and Gleeson, M. A. (1991). Yeast systems for the
commercial production of heterologous proteins. Biotechnology
(N.Y.)9, 1067-1072.
[53] Cleveland, D.W., Fischer, S.G., Kirschner, M.W. & Laemmli, U.K.
Peptide mapping by limited proteolysis in sodium dodecyl sulfate and
analysis by gel electrophoresis. J. Biol. Chem. 252:1102-6 (1977).
[54] Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C.
& Watanabe, C. Identifyingproteins from two-dimensional gels by
molecular mass searching of peptide fragments in proteinsequence
atabases. Proc. Natl. Acad. Sci. USA 90:5011-5 (1993).
[55] JANET H. SCOTTt AND RANDY SCHEKMAN*(1980) Lyticase:
Endoglucanase and Protease Activities That Act Together in Yeast
Cell Lysis. JOURNAL OF BACTERIOLOGY, May 1980,p.414-423
[56] MARIA V. FLORES,g RODOLFO J. J. ERTOLA, AND CLAUDIO
E. VOGET* (1996). Characterization of a Glutaraldehyde Stabilized
Yeast Cell Biocatalyst with ,&Galactosidase Activity? JOURNALO P
FERMENTATIOANN D BIOENGINEERING Vol. 81, No. 6,
524-529. 1996
[57] Water Quality and Solid Waste Management, Universita Stuttgart,
Determination of estrogenic activity by LYES-assay(yeast estrogen
screen-assay assisted by enzymatic digestion with lyticase),
Chemosphere 57 (2004) 1649–1655
[58] Kitae Baek, Bo-Kyong Kim, Hyun-Jeong Cho, Ji-Won Yang∗
Removal characteristics of anionic metals by micellar-enhanced
ultrafiltration, Journal of Hazardous Materials B99 (2003) 303–311
[59]淡江大學 生命科學研究所碩士論文 李皇毅 撰(2010)
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