臺灣博碩士論文加值系統

摘要
嗜鹽性甲烷古生菌Methanohalophilus portucalensis是一株可生長在高鹽環境的太古生物，會利用在胞內大量累積甜菜鹼 (glycine betaine)作為相容質 (compatible solutes)來對抗外在的高滲透壓環境。NMR的分析及in vivo和in vitro的相容質甜菜鹼生合成實驗，已證實嗜鹽性甲烷古生菌以S-adenosyl-L- methionine (SAM)為甲基提供者，利用甲基化glycine的方式進行相容質甜菜鹼的生合成。將嗜鹽性甲烷古生菌M. portucalensis strain FDF1的細胞粗萃取液以DEAE-Sephacel陰離子交換樹脂進行純化後，得到一具有能將glycine甲基化成sarcosine活性的glycine N-methyltransferase (GNMT, EC 2.1.1.20)。進一步測試嗜鹽性甲烷古生菌的GNMT的受質專一性時發現，亦具有將sarcosine和dimethylglycine分別甲基化生成dimethylglycine和 glycine betaine的活性，較高等生物及高鹽細菌的GNMT具有較廣的受質使用性。此GNMT會受到鈉和鉀離子濃度不同影響其甲基轉移的能力，生成不同的甲基化產物。在高鉀離子濃度 (800 mM)時傾向生成glycine betaine，低鉀離子濃度 (400 mM)時則傾向生成sarcosine，而鈉離子在高低濃度時則有相反結果。利用2-D電泳及PBE 94 chromatofocusing column可將此嗜鹽性甲烷古生菌的GNMT分為四個分子量相似 (52 kDa)，但pI值分別為4.4、5.0、5.2和4.8的胜肽，分別命名為α、β、γ和δ。其中α具有較低的glycine sarcosine dimethylglycine N-methyltransferase (GSDMT)活性，β則具有較高的活性，而γ則僅具有sarcosine dimethylglycine N-methyltransferase (SDMT)的活性。同時α蛋白的GSDMT酵素活性亦受到鉀離子濃度的調控，在低鉀濃度 (≤ 0.4 M)具有較高活性，而γ蛋白的SDMT酵素活性卻只在高鉀濃度時具有較高活性。

Abstract
Methanohalophilus portucalensis FDF1 can de novo synthesize and accumulate glycine betaine through threefold methylation of glycine as compatible solutes to overcome the osmotic stress. The activity of glycine N-methyltransferase (GNMT) which formed sarcosine by transferring the methyl group from S-adenosyl-L- methionine (SAM) to glycine was detected by radiometric methods in extracts of M. portucalensis FDF1. The cell extract with GNMT activity was further purified by DEAE-Sephacel ion chromatograph with a 0.1~0.5 M potassium (step or linear) gradient. In addition to the transfer of methyl group from SAM to glycine, experimental results also showed that the GNMT of strain FDF1 was able to transfer the methyl group to sarcosine and N, N-dimethylglycine. GNMT methyltransferring activities on different substrate were effectted by different concentrations of sodium and potassium ions. By 2-D gel and PBE 94 chromatofocusing column, GNMT was further separated into four non-identical subunits (α, β, γ and δ). They have the same molecular weights (52 kDa) and different pI values of 4.4, 5.0, 5.2 and 4.8, respectively. Both the α and β subunits posses the glycine sarcosine N, N-dimethylglycine N-methyltransferase (GSDMT) activity, while the γ subunits only demonstrated the sarcosine N, N-dimethylglycine N-methyltransferase (SNMT) activity. The activities of methyltransferases were regulated by potassium concentrations； the optimal concentration of α, β subunits were around and below 0.4 M, whereas γ subunit was around 0.8 M.

目錄
中文摘要 Ⅰ
英文摘要 Ⅱ
表目錄 Ⅵ圖目錄 Ⅶ
壹、前言 1
貳、前人研究 3
一、相容質 3
1.相容質的種類和特性 4
2.滲透壓調節 5
3.相容質累積 8
二、嗜鹽性甲烷古生菌相容質 9
三、相容質glycine betaine 11
1. Glycine betaine的功能 11
2. Glycine betaine的生合成途徑 12
3. Glycine betaine的生合成調控因子 14
4. Glycine betaine自體生合成酵素和GNMT 15
四、SAH對N-methyltransferase的抑制作用 19
五、嗜鹽性蛋白的結構與特性 20
參、材料與方法
一、菌種 22
二、配置含12 % NaCl的無氧培養液H-P medium 22
三、厭氧接菌與菌體生長 24
四、厭氧細胞萃取液的製備 25
五、蛋白質定量 26
六、管柱層析分離GSDMT 26
七、濃縮蛋白質溶液 30
八、酵素蛋白的保存 31
九、蛋白質電泳 31
十、二維電泳分析 (2-D Electrophoresis) 34
十一、銀染色法 (Silver staining) 35
十二、N-methyltransferase酵素活性分析 36
十三、核酸分析技術 40
十四、聚合酶連鎖反應(Polymerase chain reaction, PCR) 43
十五、GSDMT基因的選殖 45
十六、蛋白質轉印（Protein blotting） 48
肆、結果與討論
一、純化相容質甜菜鹼生合成酵素 51
1.陰離子交換樹脂DEAE-Sephacel純化GNMT (P5) 51
2.以膠體層析Superose 12 column再純化P5蛋白 53
3.利用Native gel和SDS-PAGE電泳分析P5蛋白 54
4.二維電泳 (2-D gel)分析P5 55
5. Chromatofocusing樹脂PBE 94分析 56
6. Chymotrypsin處理與N端胺基酸定序 57
7.蛋白純化效率 58
8.甜菜鹼生合成酵素蛋白結構探討 59
9.酵素穩定性 59
二、受質專一性測試 60
1. M. portucalensis FDF1 P5的受質專一性 60
2. M. portucalensis FDF1 α、β和γ的受質專一性 62
三、P5受質濃度測試 63
四、鉀離子對甜菜鹼生合成酵素活性的影響 64
1.鉀離子濃度對M. portucalensis FDF1 P5的影響 65
2.鉀離子濃度對M. portucalensis FDF1α、β和γ的影響 65
五、鈉離子對甜菜鹼生合成酵素活性的影響 66
六、反應時間對甜菜鹼生合成酵素活性的影響 67
七、GSDMT的基因分析 68
伍、結論 69
陸、表與圖 71
柒、參考文獻 94
表目錄
Table 1. Methyltransfer activities of P5 from M. portucalensis assayed with the
TLC phenol-water system and acid-washed charcoal methods. 72
Table 2. The yield and activity of GNMT purified from M. portucalensis strain
FDF1. 73
圖目錄
Figure 1. The fate of compatible solutes in the halophilic methanogens. 74
Figure 2. N-methylamine separation in betaine formation assays under a
TLC phenol-water system. 75
Figure 3. Purification of P5 from M. portucalensis strain FDF1 by DEAE-
Sephacel chromatography with KCl step gradient. 76
Figure 4. Gel electrophoresis of P5 from the crude extract of M. portucalensis after being separated by DEAE-Sephacel chromatography. (A)12 % native Poly-
acrylamide gel and (B)12.5 % SDS-PAGE. 77
Figure 5. Heat treatment P5 from M. portucalensis strain FDF1 showed the mono-
mer 52 kDa protein in 12.5 % SDS-PAGE. 78
Figure 6. Native polyacryamide gel electrophoresis (9 %, 11 %, 13 % and 15 %)
of P5 from M. portucalensis strain FDF1 showed the different migration pattern. 79
Figure 7. Determination of native P5 molecular weights based on Bollag’s equation. 80
.
Figure 8. 2-D gel electrophoresis of purified P5 (pI 3~10). 81
Figure 9. 2-D gel electrophoresis of purified P5 (pI 4~7). 82
Figure 10. P5 of M. portucalensis were separated by PBE 94 chromatofocusing
column with pH gradient of 7.4. 83
Figure 11. Chymotrypsin digestion and N-terminal sequencing results
of P5. 84
Figure 12. A. Standard methylamines were separated by a TLC phenol-water
system. B. P5 methyltransfering activities assays were under a TLC phenol-water
system. 85
Figure 13. Substrates and potassium effects on the activity of P5. 86
Figure 14. Substrate and potassium effects on the α, β and γ. 87, 88, 89
Figure 15. Substrates and sodium effects on the activity of P5. 90
Figure 16. The effect of substrate sarcosine concentrations on SNMT activity. 91
Figure 17. The effect of substrate N, N-dimethylglycine concentrations on DNMT
activity. 92
Figure 18. The effect of reaction time periods at GNMT activity of P5. 93

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