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標題: 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
Purification and Characterization of Osmolyte Betaine Synthesizing Enzyme-Glycine N-methyltransferase from Methanohalophilus portucalensis strain FDF1
作者: 吳琰奇
Wu, Yen-Chi
關鍵字: 甜菜鹼;betaine;滲透壓調控;glycine N-methyltransferase (GNMT);Methanohalophilus portucalensis strain FDF1;osmoregulation
出版社: 植物學系
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。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℃。

Methanohalophilus 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 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.
Appears in Collections:生命科學系所

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