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研究生:張永盛
研究生(外文):Weng-Seng Cheong
論文名稱:Thermoanaerobacterium saccharolyticum NTOU1 肝醣磷酸水解酶在大腸桿菌之表現及特性探討
論文名稱(外文):Expression and characterization of glycogen phosphorylase from Thermoanaerobacterium saccharolyticum NTOU1 expressed in Escherichia coli
指導教授:方翠筠
指導教授(外文):Tsuei-Yun Fang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:73
中文關鍵詞:肝醣磷酸水解酶
外文關鍵詞:Thermoanaerobacterium saccharolyticum NTOU1glycogen phosphorylase
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肝醣磷酸水解酶 (glycogen phosphorylase, GP; EC 2.4.1.1) 為一磷酸化酵素,可磷酸化水解肝醣成葡萄糖 1-磷酸 (glucose 1-phosphate, G1P),來自 Thermoanaerobacterium saccharolyticum NTOU1 之 GP 具熱穩定性,熱穩定酵素在工業上有很大的應用價值。將含有 T. saccharolyticum NTOU1之 GP 基因的表現載體 pET-21b-NTOU2-gp 於宿主 Escherichia coli BL21(DE3)-CodonPlus-RIL 表現 GP。經鎳親和性管柱純化後的重組型 GP之比活性為 1.42 U/mg,活性回收率為 76.2%,純化倍率為 5.1 倍,單體分子量為約 63 kDa,主要以二聚體的形式存在。重組型 GP最適作用溫度為 50℃,最適作用 pH 為 6.5~7.0,溫度範圍於 40~55℃ 與 pH 值於 4.0~8.5 之間具有良好的穩定性,另外,5 mM 銅離子則會完全抑制重組型 GP 的酵素活性,而鈷、錳、鎳及鋅離子對重組型 GP 的酵素活性影響較小,鈉、鉀、鎂離子及則對重組型 GP 的酵素活性沒有明顯影響。重組型 GP 對磷酸及肝醣的 kcat 分別為0.224 與 0.29 s-1,Km 分別為 0.195 mM 與 2.26%,,kcat/Km 為 1.14 與 0.13 s-1.mM-1。GDP-glucose 和 ADP-glucose 為對磷酸與肝醣的競爭性抑制劑,GDP-glucose 對磷酸與肝醣之 Ki 分別為 0.44 mM 及 0.40 mM,而 ADP-glucose 對磷酸與肝醣之 Ki 分別為 0.79 mM 及 0.68 mM。
Glycogen phosphorylase (GP; EC 2.4.1.1) catalyzes the phosphorolytic degradation of glycogen and forms G1P. The expression vector pET-21b-NTOU2-gp carrying GP gene from Thermoanaerobacterium saccharolyticum NTOU1 was transformed into the host Escherichia coli BL21 (DE3)-CodonPlus-RIL to express the recombinant GP. The recombinant GP was purified by Ni2+-affinity column chromatography. The specific activity and purification fold of recombinant GP were 1.42 U/mg and 5.1 fold, respectively and with a yield of 76.2%. The molecular weight of one subunit of the recombinant GP is around 63 kDa and the enzyme is mainly a homo-dimer. The optimum temperature and pH of recombinant GP were observed at 50℃ and in the pH range of 6.5~7.0, respectively. The enzyme was quite stable at 40~55℃ and in the pH range of 4.0~8.5. The activity of recombinant GP would be completely inhibited by 5 mM Cu2+ ions, to a lesser extent by Co2+, Mn2+, Ni2+ and Zn2+ ions, and was unaffected by Na+, K+, Mg2+ and EDTA. The kcat values of recombinant GP were determined for phosphate and glycogen were 0.224 and 0.29 s-1, respectively, and the Km values were 0.195 mM and 2.26%, respectively. The kcat/Km values were 1.14 and 0.13 s-1.mM-1, respectively. GDP-glucose and ADP-glucose were competitive inhibitors toward phosphate and glycogen, The Ki values of GDP-glucose for phosphate and glycogen were 0.44 and 0.40 mM, respectively. The Ki values of ADP-glucose for phosphate and glycogen were 0.79 and 0.68 mM, respectively.
目錄
表目錄
圖目錄
附錄目錄
壹、前言
貳、文獻整理
1. 高溫酵素與嗜高溫菌
2. 肝醣磷酸水解酶
2.1肝醣磷酸水解酶生理活性
2.2肝醣磷酸水解酶的特性
2.3肝醣磷酸水解酶結構
2.3.1 結構
2.3.2 基質結合部位
2.4肝醣磷酸水解酶應用
参、實驗流程設計
肆、材料與方法
一、實驗材料
1. 菌株與載體
1.1 基因來源
1.2 菌株來源
1.3 質體來源
2. 標準品
2.1 DNA 標準品 (Marker)
2.2 蛋白質標準品 (Marker)
3. 酵素
3.1 聚合酶
3.2 限制酶
3.3 偶合酵素
4. 市售套組
5. 培養基材料
6. 化學藥品
7. 儀器設備
二、實驗方法
1. 含 Thermoanaerobacterium saccharolyticum NTOU1 的GP 基
之表現載體的製備與轉形至表現宿主
1.1 質體 DNA 之製備
1.2 製備電穿孔勝任細胞 E. coli BL21(DE3)-CodonPlus-RIL
1.3 DNA 濃度之定量
1.4 LB 培養基之製備
1.5 電穿孔轉形作用 (electroporation)
1.6 膠體電泳分析
1.7 序列分析
1.8 菌種保存
2. 重組型 GP 之蛋白質表現與純化
2.1 重組型 GP 於大腸桿菌中之表現
2.2 重組型 GP 的純化
2.3 蛋白質定量
2.3.1 定量蛋白質之相關試劑的製備
2.4 蛋白質電泳分析
2.4.1 膠體成份
2.4.2 膠體製備
2.5 膠電泳之操作方法
2.5.1 膠片的染色與脫色
2.5.2 染色液的製備
2.6.3 脫色液的製備
3. 重組型 GP 分子量之鑑定
4. 重組型 GP 的特性探討
4.1 肝醣磷酸水解酶的活性測定
4.2 最適作用溫度
4.3 最適作用pH探討
4.4 熱穩定性探討
4.5 pH穩定性探討
4.6 金屬離子對活性的影響
4.7 磷酸濃度之影響
4.8 不同效應子對重組型 GP 之影響
5. 酵素動力學之分析
5.1 重組型 GP 之酵素動力學分析
5.2 核苷糖類抑制作用之動力學分析
伍、結果與討論
1. 重組型 GP之蛋白質表現與純化
1.1 重組型 GP 之蛋白質純化的熱處理條件
1.2 重組型 GP之純化
2. 重組型 GP 分子量之鑑定
3. 重組型 GP 之特性探討
3.1 最適作用溫度
3.2 最適作用 pH
3.3 熱穩定性
3.4 pH 穩定性
3.5 金屬離子之影響
3.6 磷酸濃度之影響
3.7 不同效應子對重組型 GP 之影響
4. 酵素動力學分析
4.1 重組型 GP 之酵素動力學分析
4.2 核苷糖類抑制作用之動力學分析
4.3 與其他細菌來源之 GP 各項酵素特性之比較
陸、結論
柒、參考文獻


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