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。

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.

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

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