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System No. U0002-3108201311112200
Title (in Chinese) 重組芋頭半乳糖水解酵素之表現與定性
Title (in English) Expression and Characterization of Recombinant α-Galactosidase from Taro
Other Title
Institution 淡江大學
Department (in Chinese) 化學學系碩士班
Department (in English) Department of Chemistry
Other Division
Other Division Name
Other Department/Institution
Academic Year 101
Semester 2
PublicationYear 102
Author's name (in Chinese) 陳逸書
Author's name(in English) I-Su Chen
Student ID 698160511
Degree 碩士
Language Traditional Chinese
Other Language
Date of Oral Defense 2013-07-18
Pagination 117page
Committee Member advisor - Su-Fang Chien
co-chair - Ming-Kai Chern
co-chair - I-Ching Kuan
Keyword (inChinese) 芋頭
Keyword (in English) Taro
gene expression
enzyme purification
blood conversion
Other Keywords
Abstract (in Chinese)
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%。
Abstract (in English)
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.
Other Abstract
Table of Content (with Page Number)

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

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

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

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
[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-
[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-
[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
[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-
[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-
[34] Yatziv, S., Flowers, H. M., (1971) Action of α-galactosidase on 
  glycoprotein from human β-erythrocyte. Biochem. Bioph. Res. Co. 45, 
[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
  E. VOGET* (1996). Characterization of a Glutaraldehyde Stabilized  
  Yeast Cell Biocatalyst with ,&Galactosidase Activity? JOURNALO P  
  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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