Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22335
標題: 嗜鹽甲烷古生菌之相容質甜菜鹼生合成酵素Sarcosine Dimethylglycine N-Methyltransferase的純化及特性探討
Purification and Characterization of Osmolyte Betaine Synthesizing Enzyme-Sarcosine Dimethylglycine N-Methyltransferase from Methanohalophilus portucalensis strain FDF1
作者: 陳詩雅
Chen, Shih-Ya
關鍵字: compatible solutes;甲基轉移酶甜菜鹼;methyltransferase;glycine betaine
出版社: 生命科學系所
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摘要: 
Methanohalophilus portucalensis FDF1是一株可生長在高鹽環境下的嗜鹽甲烷太古生物。為了維持細胞的正常生長及代謝,FDF1可在胞內累積甜菜鹼(glycine betaine)作為相容質以對抗胞外的高滲透壓逆境。經由NMR分析及in vivo和in vitro甜菜鹼生合成實驗證實FDF1可利用S-adenosyl-L-methionine(SAM)作為甲基提供者,將受質glycine進行三次甲基化的方式以生合成甜菜鹼。本研究將strain FDF1的細胞萃取液經由DEAE-sephacel陰離子交換樹脂,Phenyl Sepharose 6 fast flow疏水性交換樹脂及Sephadex G-75膠體層析管柱純化甜菜鹼生合成酵素Sarcosine Dimethylglycine N-Methyltransferase (SDMT)。SDMT酵素具有將sarcosine甲基化成dimethylglycine的SMT活性,亦具有DMT活性能進一步將dimethylglycine甲基化成glycine betaine,其SMT和DMT活性分別為0.71 nmol/μg/min及3.06 nmol/μg/min。在一維及二維的蛋白質電泳及膠體層析上,SDMT的分子量約為33 KD,pI為5.03,可能是一monomer構形的蛋白。利用結構相似的化合物進行受質專一活性測試,結果SDMT蛋白僅專一性地辨識sarcosine及dimethylglycine。另外,在酵素動力特性上,SDMT在甲基化sarcosine以生成dimethylglycine方面(SMT),和sarcosine結合的Km值為2.29 mM,和SAM結合的Km值為0.21 mM,最大反應速率為0.83 nmol/μg/min,酵素轉換數Kcat值為0.44 (1/s);在甲基化dimethylglycine以生成glycine betaine方面 (DMT),和dimethylglycine結合的Km值為3.76 mM,和SAM結合的Km值為0.59 mM,最大反應速率為4.88 nmol/μg/min,Kcat值為2.68 (1/s)。以終產物甜菜鹼探討回饋抑制對SDMT活性的影響,在2.0 M 甜菜鹼的反應條件下,甲基化sarcosine以生成dimethylglycine的活性並不受影響;而甲基化dimethylglycine以生成甜菜鹼方面仍保有65 %的活性。另外,SDMT蛋白不論在200~1000 mM鉀離子或鈉離子濃度下,活性皆無顯著性的差異且沒有隨著鹽濃度的增加而大量降低活性,證實SDMT蛋白可維持穩定的活性並不受鉀離子及鈉離子的調控。

Methanohalophilus portucalensis strain FDF1 can de novo synthesize glycine betaine as compatible solute to overcome the osmotic stress. The 13C-NMR and radiometric studies in extracts of M. portucalensis FDF1 proved that glycine betaine can de novo synthesize by transferring threefold methyl groups from S-adenosyl-L-methionine (SAM) to glycine. In this report, sarcosine dimethylglycine N-methyltransferase (SDMT) was purified from crude extract of FDF1 through DEAE-Sephacel ion exchange chromatography、Phenyl Sepharose hydrophobic interaction chromatography and Gel filtration Sephadex G-75. The specific methyltransferase activities of purified SDMT were 0.71 nmol/μg/min on sarcosine (SMT) and 3.06 nmol/μg/min on dimethylglycine (DMT). Analysis of 1 & 2-D gel electrophoresis and gel filtration suggests that SDMT was a monomer protein with the molecular weight of 33 KD and pI at 5.03. SDMT showed narrow substrate specificity with sarcosine and dimethylglycine only. On SDMT kinetic properties, the Km value of sarcosine and SAM were 2.29 mM and 0.21 mM respectively, the Vmax value was 0.83 nmol/μg/min and Kcat value was 0.44 (1/s) for SMT;the Km value of dimethylglycine and SAM were 3.76 mM and 0.59 mM respectively, the Vmax value was 4.88 nmol/μg/min and Kcat value was 2.68 (1/s) for DMT. The end product glycine betaine (2.0 M) did not effect the SMT activity but slightly repressed the DMT activity (with 65 % remains). On potassium and sodium effect, the SMT and DMT activity remains fairly constant with the addition of 200~1000 mM KCl or NaCl suggest that both potassium and sodium do not effect the SDMT activity.
URI: http://hdl.handle.net/11455/22335
其他識別: U0005-1408200611491600
Appears in Collections:生命科學系所

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