Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21276
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dc.contributor.advisor賴美津zh_TW
dc.contributor.advisorMei-Chin Laien_US
dc.contributor.author吳琰奇zh_TW
dc.contributor.authorWu, Yen-Chien_US
dc.date2001zh_TW
dc.date.accessioned2014-06-06T07:15:32Z-
dc.date.available2014-06-06T07:15:32Z-
dc.identifier.urihttp://hdl.handle.net/11455/21276-
dc.description.abstract嗜鹽甲烷菌Methanohalophilus portucalensis strain FDF1在細胞中累積betaine作為相容質以抵抗外界的高滲透壓。利用14C-NMR的分析及in vivo和in vitro的betaine生合成實驗,已証實嗜鹽甲烷菌以glycine為基質,由S-adenosyl-L-methionine(SAM)提供甲基經三次甲基化生合成betaine。將M. portucalensis strain FDF1的細胞萃取液通過DEAE-Sephacel陰離子交換樹脂,並以含0.1 M、0.2 M、0.3 M、0.4 M及0.5 M鉀離子濃度的階梯方式沖洗管柱,自0.5 M鉀離子濃度的沖洗液中純化出具有以glycine為基質生合成sarcosine活性的glycine N-methyltransferase(GNMT, EC 2.1.1.20)。此GNMT蛋白約占細胞總蛋白量的0.14 %,是由52 kD的蛋白所構成的hexamer(302 kD),其酵素活性為0.56 nmol/mg×hr。此嗜鹽甲烷菌的GNMT除了能利用glycine作為基質外,尚能利用sarcosine和dimethylglycine為基質,具有sarcosine N-methyltransferase和dimethylglycine N-methyl- transferase的活性。此外,反應產物S-adenosylhomocysteine會抑制GNMT的活性。以glycine為基質時,其最適反應條件如下:蛋白濃度為0.4 mg, SAM濃度為1.15 mM, glycine基質濃度為4 mM,鉀離子濃度為400 mM及最適溫度為37℃。zh_TW
dc.description.abstractMethanohalophilus portucalensis strain FDF1 can de novo synthesize betaine from glycine as compatible solutes while cells encounter the salt stress. The compatible solute betaine synthesizing through threefold of methylations from glycine was confirmed. Glycine N-methyltransferase (GNMT, EC 2.1.1.20) which transfer methyl group of S-adenosyl-L-methionine to glycine was purified by DEAE-Sephacel ion-exchange chromatography with the step gradient of potassium chloride and GNMT activity was detected at the elution with 0.5 M potassium. GNMT was a hexamer composed by 52 kD polypeptide. The purified GNMT composed 0.14 % of the total cell protein with specific activity of 0.56 nmol/mg×hr. Except using glycine as substrate, GNMT can also use sarcosine and dimethylglycine as substrates. The optimal assay conditions were performed under 0.4 mg GNMT, 1.15 mM SAM, 4 mM glycine and 400 mM KCl at 37℃. In addition, GNMT was strongly inhibited by the reaction product S-adenosylhomocysteine.en_US
dc.description.tableofcontents壹、前言 1 貳、前人研究 2 一、相容質 2 1. 相容質的種類 2 2. 相容質的累積及滲透壓調控 3 二、嗜鹽甲烷菌的相容質 6 三、相容質Betaine 8 1. Betaine的合成途徑 9 2. Betaine生合成調控因子 11 3. Betaine自體生合成酵素 12 4. 哺乳類動物glycine N-methyltransferase13 四、嗜鹽性蛋白的結構與特性 15 參、材料與方法 17 一、菌種 17 二、無氧培養基與藥品的配製 17 三、接菌及菌體生長 19 四、細胞萃取液的製備 19 五、蛋白質定量 21 六、管柱層析分離GNMT 21 七、濃縮蛋白質 23 八、GNMT的保存 25 九、蛋白質電泳 25 十、GNMT酵素活性分析 28 十一、核酸分析技術 31 十二、聚合 連鎖反應 33 十三、GNMT基因的選殖 35 十四、蛋白質轉印 37 肆、結果與討論 39 Ⅰ. 純化GNMT 39 一、以DEAE-Sephacel陰離子交換樹脂純化GNMT 39 二、以gel filtration(Superose 12 column)純化GNMT42 三、GNMT的蛋白結構 42 Ⅱ. GNMT反應的最適條件 44 一、最適蛋白質反應濃度 44 二、最適glycine濃度 44 三、最適甲基提供者-SAM濃度 45 四、最適鉀離子濃度 46 五、反應最適溫度 48 六、酵素的穩定性 48 Ⅲ. SAH對GNMT活性的抑制 48 Ⅳ. GNMT活性偵測方法的評估 49 Ⅴ. 鉀離子對GNMT蛋白構形的影響 50 Ⅵ. 嗜鹽甲烷菌GNMT的基質 51 Ⅶ. GNMT的基因 53 伍、結論 55 陸、表與圖 56 柒、參考文獻 73zh_TW
dc.language.isoen_USzh_TW
dc.publisher植物學系zh_TW
dc.subject甜菜鹼zh_TW
dc.subjectbetaineen_US
dc.subject滲透壓調控zh_TW
dc.subjectglycine N-methyltransferase (GNMT)en_US
dc.subjectMethanohalophilus portucalensis strain FDF1en_US
dc.subjectosmoregulationen_US
dc.title嗜鹽甲烷古生菌之相容質甜菜鹼生合成酵素glycine N-methyltransferase的特性探討zh_TW
dc.titlePurification and Characterization of Osmolyte Betaine Synthesizing Enzyme-Glycine N-methyltransferase from Methanohalophilus portucalensis strain FDF1en_US
dc.typeThesis and Dissertationzh_TW
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