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系統識別號 U0002-2008201213461000
中文論文名稱 Ι. 啤酒酵母菌假設性甲基轉移酶YBR271Wp蛋白質表現、純化及甲基化活性之研究 ΙΙ. 利用隨機突變方法改良啤酒酵母菌之酒精與高葡萄糖耐受性
英文論文名稱 Ι. Study of a putative methyltransferase YBR271Wp from Saccharomyces cerevisiae: protein expression, purification and methylation activity. ΙΙ. Improving ethanol and high-glucose tolerance of Saccharomyces cerevisiae by random mutagenesis.
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 100
學期 2
出版年 101
研究生中文姓名 林欣怡
研究生英文姓名 Hsin-Yi Lin
學號 699160239
學位類別 碩士
語文別 中文
口試日期 2012-07-20
論文頁數 75頁
口試委員 指導教授-陳銘凱
委員-林賜恩
委員-官宜靜
委員-陳銘凱
中文關鍵字 甲基轉移酶  甲基化  酒精耐受性  葡萄糖耐受性 
英文關鍵字 Methyltransferase  methylation  ethanol tolerance  glucose tolerance 
學科別分類 學科別自然科學化學
中文摘要 Part Ι
常見的蛋白質轉譯後修飾作用有:乙醯化、泛素化、醣化、磷酸化、甲基化等。在其他研究發現生物體中有許多甲基化生理反應,但是大部分相關甲基轉移酶卻還未被發現。
本研究利用啤酒酵母菌進行甲基轉移酶探討。根據 Steven Clarke 所提出甲基轉移酶在motif Ι、ΙΙ、ΙΙΙ、post-Ι位置具有高保留性,並比對啤酒酵母菌基因找出假設性甲基轉移酶。在此針對假設性甲基轉移酶YBR271Wp探討是否具有甲基化活性。以E. coli大量表現重組蛋白質,使用His-tag column純化,最後將YBR271Wp.與ΔYBR271W和Hot-SAM反應。壓片結果顯示在pI 5-6、分子量110 kDa位置左右有受質訊號。利用基質輔助雷射脫附游離質譜儀分析受質,判定該受質為 Elongation factor 2 (EF 2),由此實驗證實YBR271Wp為甲基轉移酶。

Part ΙΙ
現今大部分生質酒精是由一般製酒工業常用的啤酒酵母菌所產生。但是當培養環境中酒精或葡萄糖濃度過高會降低啤酒酵母菌的存活率,使酒精的總產率受限。
本研究針對此問題,使用EMS 化學藥劑、UV兩種方法分別將啤酒酵母菌隨機突變,再藉由高濃度葡萄糖與不同濃度酒精的培養條件,把隨機突變之菌株進行篩選,最後篩選出EMS突變菌株有較高酒精耐受性。
英文摘要 Part Ι
Common protein post-translational modifications include: acetylation, ubiquitination, glycosylated, phosphorylation, methylation. Physiological responses of methylation have been found in some studies, but most of the methyltransferases are still undiscovered.
In this study, the Saccharomyces cerevisiae methyltransferase was investigated. Accroding to Steven Clarke’s study, the highly conserved methyltransferase motif Ι, ΙΙ, ΙΙΙ, post-Ι position, were aligned to identify the hypothetical methyltransferase in Saccharomyces cerevisiae. The putative methyltransferase YBR271Wp was investigated to determine whether it has methylation activity. Using the E. coli host to express the recombinant proteins, the recombinant protein was purified by the His-tag affinity column. The YBR271Wp reacted with ΔYBR271W and Hot-SAM. The X-ray autoradiography showed substrate signal in the pI 5-6, molecular weight of around 100 kDa position. By utilizing MALDI mass spectrometry to identify the substrate, finally we found the substrate is Elongation factor 2 (EF 2), and demonstrated YBR271Wp is a methyltransferase.
Part ΙΙ
Today most of the ethanol is generated by the liquor industry Saccharomyces cerevisiae. But when the culture contains high ethanol or glucose concentrations in the environment, the survival rate of Saccharomyces cerevisiae will be redueced, so that the total yield of alcohol is limited.
To counter this problem, EMS chemical agents and UV are two ways to generate random mutations in Saccharomyces cerevisiae. By the selective conditions of high concentrations of glucose and different concentrations of ethanol in the culture, the random mutation strains were screened. Finally, the EMS mutation strains with a higher alcohol tolerance were screened out.
論文目次 目錄
謝誌………………………………………………………………….…….Ι
中文摘要…………………………………….…………………….…….…Ⅱ
英文摘要…………………………………….………………….…….……Ⅲ
目錄…………………………………………………………….………….Ⅳ
第一部分 啤酒酵母菌假設性甲基轉移酶YBR271Wp蛋白質表現、純化及
甲基化活性之研究
第一章 緒論 ..................................................................................................1
第二章 材料與實驗方法…………………………………………………….………6
1. 實驗材料..…………………………………………………………………..……6
2. 實驗方法……………………………………………………………………..…9
2.1 抽取野生型酵母菌BY4742的genomic DNA…………………….…………..9
2.1.2 引子設計...........................................................................................10
2.1.3 放大複製目標基因…………………………………………………………11
2.1.4 DNA 電泳…………………………………………………………………13
2.1.5 純化PCR產物…………………………………………………………….13
2.1.6 A-tailing (補A)…………………………………………………………14
2.2 TA-Cloning…………………………………………………………….15
2.2.1 酶接反應 (Ligation)……………………………………………………15
2.2.2 轉形 (Transformation)………………………………………………16
2.2.3藍白篩選.......................................................................................16
2.2.4 以限制酶進行酶切反應確認基因片段………………………….………18
2.2.5 定序…………………………………………………………………….18
2.3 將目標基因TA載體轉殖至表現載體pET43.1b……………….……..19
2.4 重組質體的蛋白質表現與純化……………………………………….…..22
2.4.1 誘導目標蛋白質表現………………………………………………….…….23
2.4.2 蛋白質在各個時間點的表現……………………………………..……..23
2.4.3 分析粗抽蛋白質…………………………………………………….…….……24
2.4.4大量表現蛋白質的純化……………………………………………..………26
2.5 甲基化活性測試………………………………………………………..…………28
2.5.1 甲基化受質 (substrate)…………………………………………..………..28
2.5.2 甲基轉移酶甲基化反應…………………………………………………….29
2.5.3 甲基化反應分析 (聚丙烯醯胺膠體電泳,SDS-PAGE)……..29
2.5.4甲基化反應分析 (等電點電泳,IEF)…………………………………30
2.6 甲基轉移酶受質分析……………………………………………………………32
2.6.1 質譜儀樣品製備 ( form SDS-PAGE)……………………..……………32
2.6.2 質譜儀樣品製備 ( form IEF-PAGE)……………………………..……..34
第三章 實驗結果與討論…………………………………………………………….35
圖與表………………………………………………………………………………..39
第二部分 利用隨機突變方法改良啤酒酵母菌之酒精與高葡萄糖耐受性
第一章 緒論………………………………………………………………………49
第二章 材料與實驗方法…………………………………………………… 51
1. 實驗材料………………………………………………………………………51
2. 實驗方法………………………………………………………………………52
2.1 化學突變法………………………………………………………………………..52
2.2 UV突變法…………………………………………………………………………53
2.3 菌株篩選……………………………………………………………………53
2.4 酒精耐受性測試………………………………………………………………54
2.5 糖濃度耐受性測試……………………………………………………………55
2.6 發酵測試……………………………………………………………………55
2.7 酒精濃度測定……………………………………………………………………56
第三章 結果與討論…………………………………………………………………57
圖與表………………………………………………………………………………..61
附錄(一) pGEMR-T Easy Vector Map……………………………………66
附錄(二)pET-43b Vector Map……………………………………………………67
附錄 (三)藥品製備……………………………………………………………………68
參考文獻……………………………………………………………………………….73




參考文獻 參考文獻
1. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, et al. (1996) Life with 6000 Genes. Science 274: 546-567.
2. Mell JC, Burgess SM (2001) Yeast as a Model Genetic Organism. eLS: John Wiley & Sons, Ltd.
3. Karathia H, Vilaprinyo E, Sorribas A, Alves R (2011) Saccharomyces cerevisiae as a Model Organism: A Comparative Study. PLoS ONE 6: e16015.
4. Walsh CT, Garneau-Tsodikova S, Gatto GJ (2005) Protein Posttranslational Modifications: The Chemistry of Proteome Diversifications. Angewandte Chemie International Edition 44: 7342-7372.
5. Lee DY, Teyssier C, Strahl BD, Stallcup MR (2005) Role of protein methylation in regulation of transcription. Endocrine reviews 26: 147-170.
6. Kagan RM, Clarke S (1994) Widespread Occurrence of Three Sequence Motifs in Diverse S-Adenosylmethionine-Dependent Methyltransferases Suggests a Common Structure for These Enzymes. Archives of Biochemistry and Biophysics 310: 417-427.
7. Niewmierzycka A, Clarke S (1999) S-Adenosylmethionine-dependent Methylation in Saccharomyces cerevisiae. Journal of Biological Chemistry 274: 814-824.
8. Martin JL, McMillan FM (2002) SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold. Current Opinion in Structural Biology 12: 783-793.
9. Grillo MA, Colombatto S (2005) S-Adenosylmethionine and protein methylation. Amino Acids 28: 357-362.
10. Petrossian TC, Clarke SG (2009) Multiple Motif Scanning to Identify Methyltransferases from the Yeast Proteome. Molecular & Cellular Proteomics 8: 1516-1526.
11. Wlodarski T, Kutner J, Towpik J, Knizewski L, Rychlewski L, et al. (2011) Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome. PLoS ONE 6: e23168.
12. Structural Genomics Consortium,et al. (2008) Protein production and purification. Nat Meth 5: 135-146.
13. Ploegh HL (2001) One-Dimensional Isoelectric Focusing of Proteins in Slab Gels. Current Protocols in Protein Science: John Wiley & Sons, Inc.
14. Anderson JC, Peck SC (2008) A simple and rapid technique for detecting protein phosphorylation using one-dimensional isoelectric focusing gels and immunoblot analysis. The Plant Journal 55: 881-885.
15. Couttas TA, Raftery MJ, Padula MP, Herbert BR, Wilkins MR (2012) Methylation of translation-associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases. PROTEOMICS 12: 960-972.
16. Gray KA, Zhao L, Emptage M (2006) Bioethanol. Current Opinion in Chemical Biology 10: 141-146.
17. Antoni D, Zverlov V, Schwarz W (2007) Biofuels from microbes. Applied Microbiology and Biotechnology 77: 23-35.
18. Stokstad E (2012) Biofuels. Engineered superbugs boost hopes of turning seaweed into fuel. Science (New York, NY) 335: 273.
19. Balat M, Balat H, Oz C (2008) Progress in bioethanol processing. Progress in Energy and Combustion Science 34: 551-573.
20. You KM, Rosenfield C-L, Knipple DC (2003) Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content. Applied and Environmental Microbiology 69: 1499-1503.
21. Stanley D, Bandara A, Fraser S, Chambers PJ, Stanley GA (2010) The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae. Journal of Applied Microbiology 109: 13-24.
22. Stanley D, Fraser S, Chambers P, Rogers P, Stanley G (2010) Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae. Journal of Industrial Microbiology & Biotechnology 37: 139-149.
23. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science (New York, NY) 314: 1565-1568.
24. Lawrence CW (1991) [18] Classical mutagenesis techniques. In: Christine Guthrie GRF, editor. Methods in Enzymology: Academic Press. pp. 273-281.
25. Pilone GJ (1985) Determination of ethanol in wine by titrimetric and spectrophotometric dichromate methods: collaborative study. Journal - Association of Official Analytical Chemists 68: 188-190.
26. Verbelen P, Saerens S, Van Mulders S, Delvaux F (2009) The role of oxygen in yeast metabolism during high cell density brewery fermentations. Applied Microbiology and Biotechnology 82: 1143-1156.
27. Zanon J, Peres M, Gattas E (2007) Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast. Enzyme and Microbial Technology 40: 466-470.
28. Castellari M, Versari A, Spinabelli U, Galassi S, Amati A (2000) An improved HPLC method for the analysis of organic acids, carbohydrates, and alcohols in grape musts and wines. Journal of liquid chromatography & related technologies 23: 2047-2056.
29. Gray JV, Petsko GA, Johnston GC, Ringe D, Singer RA, et al. (2004) Sleeping Beauty: Quiescence in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 68: 187-206.
30. Galdieri L, Mehrotra S, Yu S, Vancura A (2010) Transcriptional regulation in yeast during diauxic shift and stationary phase. Omics : a journal of integrative biology 14: 629-638.
31. Gerchman Y, Schnitzer A, Gal R, Mirsky N, Chinkov N (2012) A simple rapid gas-chromatography flame-ionization-detector (GC-FID) method for the determination of ethanol from fermentation processes. African Journal of Biotechnology 11: 3612-3616

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