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標題: 嗜鹽甲烷菌之相容質甜菜鹼生合成酵素Sarcosine Dimethylglycine N-methyltransferase的純化及活性分析
Purification and Activity assay of Osmolyte Betaine Synthesizing Enzyme-Sarcosine Dimethylglycine N-methyltransferase from Methanohalophilus portucalensis strain FDF1
作者: 李宇堅
Lee, Yu-Chien
關鍵字: archaea;古生菌;glycine betaine;methanogen;methyltransferase;osmotic stress;甜菜鹼;甲烷菌;甲基轉移酵素;滲透壓逆境
出版社: 生命科學系
生長在高鹽環境的嗜鹽甲烷古生菌Methanohalophilus portucalensis FDF1會在胞內累積大量甜菜鹼(glycine betaine)作為相容質(compatible solutes),以對抗胞外的高滲透壓逆境。經由13C-NMR分析及in vivo和 in vitro甜菜鹼生合成實驗,證實能以S-adenosyl-L-methionine (SAM)提供甲基,glycine作為受質進行三次甲基化作用產生甜菜鹼。經由DEAE-sephacel陰離子交換樹脂配合階梯0、0.1、0.2、0.3、0.4、0.5 M KCl梯度分離細胞萃取液,於0.2 M KCl梯度淋洗出的蛋白在800 mM高鉀離子濃度下具有明顯轉移SAM上的甲基至sarcosine及dimethylglycine形成甜菜鹼產物的sarcosine dimethylglycine N-methyltransferase (SDMT)的活性。其中以sarcosine為基質的sarcosine N-methyltransferase (SNMT)比活性為2.49 nmol/μg•hr,而以dimethylglycine為基質的dimethylglycine N-methyltransferase (DNMT)比活性為9.69 nmol/μg•hr。進一步以50 kDa分子量切斷分離及Sephadex G-100膠體層析管柱,可得到一由23 kDa和33 kDa胜肽組成具有SDMT活性的peak S2D-2蛋白,其中23 kDa胜肽在二維電泳上可再被分成兩個pI值為4.8及5.2的胜肽。部分純化的此SDMT蛋白以sarcosine為基質之SNMT比活性增加為5.33 nmol/μg•hr,DNMT比活性增加為21.49 nmol/μg•hr。生長於12 % NaCl環境時M. portucalensis FDF1的世代時間約為13小時,會在胞內累積0.73 M KCl、0.31 M 甜菜鹼、0.46 M Nε-acetyl-β-lysine及0.17 M β-glutamine作為相容質,本研究結果顯示在800 mM KCl及足夠sarcosine基質條件下,SDMT能於36分鐘內迅速生合成累積此高鹽甲烷菌於12 % NaCl 環境所需的3.1 M甜菜鹼。

Methanohalophilus portucalensis strain FDF1 can de novo synthesize glycine betaine as compatible solute to encounter the osmotic stress. The 13C-NMR and radiometric studies in the extract of strain FDF1 proved that glycine betaine can de novo synthesize by transferring threefold methyl groups from S-adenosyl-L-met- hionine (SAM) to glycine. After DEAE-sephacel column separation the crude extract of strain FDF1 with step 0, 0.1, 0.2, 0.3, 0.4, 0.5 M KCl gradient, the sarcosine dimethylglycine N-methyltransferase (SDMT) that could transfer methyl group from SAM to sarcosine (SNMT) and dimethylglycine (DNMT) was detected at peak S2 eluted from 0.2 M KCl step gradient. Protein from the peak S2 showed SDMT activity at 800 mM KCl environment, the specific activity of SNMT was 2.49 nmol/μg•hr and the specific activity of DNMT was 9.69 nmol/μg•hr. Further purification the peak S2 with 50 kDa molecular weight cut off and sephadex G-100 column, significant SDMT activity was obtained at peak S2D-2 which composed by 23 kDa and 33 kDa polypeptide. The 23 kDa polypeptide can further separate into two polypeptides with pI value of 4.8 and 5.2 respectively by 2-D gel electrophoresis. The partial purified SDMT activities of peak S2D-2 was 5.33 nmol/μg•hr and 21.49 nmol/μg•hr for SNMT and DNMT, respectively. While grown at 12 % NaCl environment, cells of strain FDF1 accumulated 0.73 M KCl、0.31 M glycine betaine、0.46 M Nε-acetyl-β-lysine and 0.17 M β-glutamine as compatible solutes and the generation time was 13 hr. Base on the SDMT activity investigated in this study, under the condition of 800 mM KCl and sufficient substrate sarcosine, cells could synthesize 3.1 M required glycine betaine in 36 minutes.
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