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

淡江大學機構典藏 > 理學院 > 化學學系暨研究所 > 學位論文 > Item 987654321/87414

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题名:	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
其它题名:	I. study of a putative methyltransferase YBR271Wp from saccharomyces cerevisiae : protein expression, purification and methylation activity;II. improving ethanol and high-glucose tolerance of saccharomyces cerevisiae by random mutagenesis 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 Study of a putative methyltransferase YBR271Wp from saccharomyces cerevisiae : protein expression, purification and methylation activity Improving ethanol and high-glucose tolerance of saccharomyces cerevisiae by random mutagenesis
作者:	林欣怡;Lin, Hsin-Yi
贡献者:	淡江大學化學學系碩士班陳銘凱
关键词:	甲基轉移酶;甲基化;酒精耐受性;葡萄糖耐受性;methyltransferase;methylation;ethanol tolerance;glucose tolerance
日期:	2012
上传时间:	2013-04-13 11:03:57 (UTC+8)
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。 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.
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