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標題: 嗜鹽性甲烷古生菌相容質glycine betaine生合成調控與其生合成酵素 glycine N-methyltransferase的純化
The biosynthesis of glycine betaine in halophilic methanogenic archaea Methanohalophilus portucalensis FDF1 and the purification of glycine N-methyltransferase
作者: 莊旻真
Chuang, Ming-Jen
關鍵字: 相容質
halophilic methanogenic archaea
glycine betaine
Methanohalophilus portucalensis FDF1
glycine N-methyltransferase
出版社: 植物學系
摘要: 生長於高鹽環境中的嗜鹽性甲烷古生菌Methanohalophilus portucalensis FDF1,會於細胞內累積glycine betaine, (-glutamine ,N(-acetyl-(-lysine及鉀離子為相容質來平衡細胞內外的滲透壓差。在 已知相容質中glycine betaine具有最高的滲透壓保護係數,且大部份生 物都能利用glycine betaine做為相容質。為了探討glycine betaine生合 成途徑及其調控因子,我們利用Mh. portucalensis FDF1細胞萃取液分析 glycine betaine的生合成,發現必須有鉀離子存在才有glycine betaine 生成,而鋰、鈉等單價陽離子可完全或部份取代鉀離子在反應中的功能。 利用(methyl14C(-S-adenosyl-L-methionine(SAM)為甲基供應者,以 glycine,sarcosine,N,N-dimethylglycine及glycine betaine為基質的 實驗證實glycine betaine的生合成可由SAM提供甲基,經三次甲基化作用 由glycine?sarcosine?N, N-dimethylglycine合成glycine betaine。 而鉀離子濃度可調節產物的累積與生合成,高濃度的鉀離子會促使 sarcosine轉化成glycine betaine,但N, N-dimethylglycine會迅速轉化 成glycine betaine,而不能在胞內累積。 於純化glycine betaine生合 成酵素-glycine N-methyltransferase(GNMT)的過程中,將Mh. portucalensis FDF1厭氧細胞萃取液通過DEAE-Sephacel陰離子交換樹脂 ,並以0.1~1.0 M的鉀離子濃度梯度沖洗管柱後,在1.0 M鉀離子濃度的沖 洗液中發現具有GNMT酵素活性的蛋白。此純化的GNMT以glycine為基質生 合成sarcosine的比活性為6.12 nmole/(,約佔細胞總蛋白量的0.12 ﹪,分子量約為240 kD。於SDS-PAGE電泳上發現GNMT可分成100 kD,55 kD,40 kD三個polypeptides。
Halophilic methanogen Methanohalophilus portucalensis FDF1 is able to accumulate glycine betaine, (-glutamine, N(-acetyl-(- lysine and potassium ion as the compatible solutes to survive in the hypersaline environment. Among all known compatible solutes, glycine betaine has the highest osmoprotective efficiency and most halotolerant and halophilic organisms can accumulate it as the compatible solutes. The anaerobic crude extract of glycine betaine synthesizing Mh. portucalensis FDF1 were used in accompany with (14C(-glycine or (methyl14C(-S-adenosyl-L- methionine (SAM) to study the glycine betaine biosynthetic pathway and its regulatory factors. Results showed that potassium is required for the formation of glycine betaine. Other monovalent cations, lithium or sodium, could partly or completely substitute potassium function. Results also indicated that SAM can be the methyl donor in the process of glycine b etaine formation. It also confirmed that the de novo biosynthesis of glycine betaine proceed from glycine to sarcosine and N, N-dimethylglycine. High potassium level enhanced the formation of glycine betaine while lower potassium level favored the sarcosine accumulation. The glycine betaine formation enzyme ?glycine N-methyltransferase (GNMT) was purified by using the DEAE-Sephacel anion exchange column with the linear gradient of potassium chloride (0.1~1.0 M) with the crude extract of Mh. portucalensis FDF1. Elutant with 1.0 M potassium showed the GNMT activity and later demonstrated as purified protein by native gel electrophoresis. The estimated molecular weight of the GNMT was about 240 kD and can be separated into three subunits as 100 kD, 55 kD and 40 kD in sodium dod ecyl sulfate electrophoresis. GNMT composes 0.12﹪of the total cell protein of Mh. portucalensis FDF1 and the specific activity of this enzyme with glycine as substrate is 6.12 nmole/(
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