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標題: 嗜鹽性甲烷菌之相容質-glycinebetaine的合成相關酵素及其影響因子之研究
The regulatory factors of glycinebetaine synthetic associated enzymes in Methanohalophilus strain FDF1
作者: 楊道任
Yang, Daw-Renn
關鍵字: Methanohalophilus
compatible solute
出版社: 植物學系
摘要: 嗜鹽性甲烷菌 Methanohalophilus strain FDF1 是株自高鹽生態環境下 純化出來的絕對厭氧古生菌。於高鹽環境,其胞內可累積高濃度的 glycinebetaine、beta-glutamine和N-acetyl-beta-lysine作為相容質, 以平衡調節胞體內外滲透壓差。Glycinebetaine是滲透保護系數最高的有 機相容質。本研究將探討glycinebetaine的生合成途徑和探討glycine- betaine的合成相關酵素及其影響因子。於strain FDF1的細胞萃取液添 加 [methyl14C]S-adenosyl-L-methionine(SAM) 作為甲基提供者時,可 形成 14C標定的 sarcosine、N,N-dimethylglycine 和 glycinebetaine 。証實 glycinebetaine 生化合成路徑和 13CNMR 分析結果相符合,乃由 glycine經甲基化而形成 glycinebetaine。高濃度鉀離子( 800mM KCl ),會促進 sarcosine 和 glycinebetaine 的形成;反之,隨著鉀離子濃 度減少, sarcosine 和 glycinebetaine 的生成累積逐漸減少至無。顯 示鉀離子濃度的高低,會影響 glycinebetaine 生化合成的正逆向反應。 以 14C- glycine 探討 glycinebetaine 合成的第一個產物sarcosine的 形成酵素 glycine N-methyltransferase,結果顯示添加 20ug蛋白的細 胞萃取液,在四小時的反應時間下,能產生最多量的 sarcosine。於定量 細胞萃取液下,sarcosine的生成量會隨著glycine或SAM濃度的增加而增 加,到了2mM的glycine或4.6mM的SAM後,酵素反應逐漸達到飽和程度。此 外,氧氣的存在,並不會抑制由 glycine 生成 sarcosine 的酵素反應。 在含20ug蛋白的細胞萃取液中,添加 800mM 的 KCl、2mM 的 glycine 和 4.6mM 的 SAM,於 37℃、四小時的反應條件下, glycine N- methyltransferase的比活性是 290 nmol/。
Methanohalophilus strain FDF1 was an anaerobic archaea isolated from the hypersaline environment. Cells accumulated glycine- betaine, beta-glutamine and N-acetyl-beta-lysine as the major compatible solutes to survive in the high salt environment. Among the organic solutes, glycinebetaine has been demonstrated with the highest osmoprotective efficiency. This investigate analyzed the glycinebetaine synthetic pathway and studied the regulatory factors which involved in the glycinebetaine synthetic enzymes. With the addition of [methyl14C]S-adenosyl-L- methionine(SAM) into the crude extract of Methanohalophilus strain FDF1 as methyl donor, 14C labeled sarcosine, N,N- dimethylglycine and glycine- betaine were formed. This result demonstrated that the pathway of glycinebetaine synthesis proceeded from the methylation of glycine as indicated by 13CNMR analysis. The addition of potassium chloride (up to 800mM) increased the formation of sarcosine and glycinebetaine. In contrast, low and none potassium addition enhanced the reversed process. The concentration of potassium may play an important role in the glycinebetaine synthesis. The assay conditions for the sarcosine forming enzyme, glycine N- methyltransferase were set up. With the addition of 14C- glycine, the highest amount of sarcosine formed under the 20 ug protein of the crude extract of strain FDF1 in 4 hours reaction time. This enzyme was not sensitive to the oxygen. The optimal enzyme assay condition of glycine N-methyltransferase reached a specific activity as 290 nmol/ after 4 hr reaction with the addition of 800 mM KCl, 2 mM glycine and 4.6 mM SAM into 20 ug protein of strain FDF1 crude extract at 37℃.
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