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研究生:游靜茹
研究生(外文):Ching-Ju Yu
論文名稱:嗜高溫古細菌之重組肝醣支切酶的純化及其特性之探討
論文名稱(外文):Purification and Characterization of Recombinant Glycogen Debranching Enzyme from the Hyperthermophilic Archaeum, Sulfolobus solfataricus ATCC 35092
指導教授:方翠筠
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:2004
畢業學年度:92
語文別:中文
論文頁數:93
中文關鍵詞:嗜高溫古細菌重組肝醣支切酶
外文關鍵詞:Hyperthermophilic ArchaeumGlycogen Debranching EnzymeSulfolobus solfataricus
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已建構於不同表現載體中的Sulfolobus solfataricus ATCC 35092 treX基因再轉形於Escherichia coli宿主細胞後,唯含有T7 起動子 (promoter)之pET-15b-treX具有較佳的肝醣支切酶(glycogen debranching enzyme, GDE) 的表現。利用SDS-聚丙烯醯胺電泳分析,結果發現重組GDE次體分子量落在約83 KDa的位置,與由其核酸序列所預測之蛋白質次體分子量一致。且為了簡化酵素純化步驟,我們選用的pET-15b表現載體,在所表現的目標蛋白質的胺基端,含有一段由六個組胺酸 (histidine) 連續的序列 (His-tag),因此可利用鎳螯合親和性膠體管柱來純化重組GDE。以80℃熱處理與鎳螫合親和性層析膠體 (His‧Bind Resin) 純化的重組GDE,其最適作用溫度為75℃、最適作用酸鹼值 (pH) 為5。另外並將已刪除的pET-15b-treX質體上treX基因前端His-tag序列,產生的pET15b-△H-treX表現載體,轉形於E. coli BL21(DE3)-CodonPlus-RIL 宿主細胞中,表現的GDE為原生型GDE (wild-type GDE),利用膠體 (Sephacryl S-200) 過濾純化原生型GDE,其最適作用溫度為75℃、最適作用酸鹼值為pH 5。原生型GDE在85℃保溫兩個小時後相對殘留活性降為82%,在90℃下僅殘留16%的酵素活性。重組GDE在85℃保溫兩個小時後相對殘留活性降為59%,在90℃下則完全喪失活性;顯示出重組GDE熱穩定性比原生型GDE差。重組GDE的在pH 8.0時有最高的酵素殘留活性,在pH 7, pH 9酵素殘留活性分別為31%, 68%, 在pH 3.0 ~ 6.0中僅具有13% 以下的殘留活性。原生型GDE在pH 8.0時有最高的酵素殘留活性,在pH 7, pH 9酵素殘留活性分別為36%, 70%,與重組GDE相似。GDE酵素在pH 6以下緩衝溶液中明顯喪失其酵素活性,表示GDE酵素不適合保存於pH 6以下的緩衝液中。鉀、鈉、鈣、鎂、鋅、銅、鎳離子等金屬離子,皆會促進GDE酵素活性,尤其以銅離子 (5 mM) 提高GDE酵素活性最高,其提高原生型GDE相對酵素活性達225%,提高重組GDE相對酵素活性達185%。
The cloned treX gene from Sulfolobus solfataricus ATCC 35092 was transformed to Escherichia coli. The recombinant treX gene was expressed well under the T7 expression system compared to those of T5, Tac, and ara expression systems. The subunit molecular weight of GDE estimated by SDS-PAGE was about 83 kDa. Since this gene was fused in frame with the His-tag coding sequence on pET-15b-treX expression vector, we purified recombinant GDE by metal chelating chromatography after heat treatment at 80oC. In order to express wild-type GDE, we transformed pET-15b-DH-treX, which containing no His-tag coding sequence, to E. coli BL21 (DE3)-CodonPlus-RIL. The wild-type GDE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The recombinant his-tagged GDE was purified by using heat treatment, and metal-affinity chromatography. The obtained recombinant GDEs in both forms showed similar enzymatic properties. They all had an apparent optimal pH of 5 and an optimal temperature at 75oC. The enzymes were quite stable at the temperature up to 80oC for 2-h incubation. These GDEs were activated by K+、Na+、Ca2+、Mg2+、Zn2+、Cu2+、Ni2+ ions. The Cu2+ ion increased the activities of wild-type and recombinant GDEs up to 225% and 185%, respectively.
目錄 1
圖目錄 5
表目錄 6
附錄目錄 7
中文摘要 8
英文摘要 9
壹、前言 10
貳、文獻整理 12
一、海藻糖之簡介 12
1. 海藻糖的研究歷史 12
2. 海藻糖之生物學特性 13
3. 海藻糖之功能 15
4. 海藻糖之應用潛力 16
5. 海藻糖之生產方法 18
5.1微生物代謝發酵法 18
5.2酵素生物轉換法 18
二、熱穩定海藻糖生成相關酵素之簡介 20
三、支切酶的簡介與應用 21
1. 異澱粉酶 (isoamylase) 21
2. 普魯南糖酶 (pullulanase) 22
四、熱穩定肝醣支切酶 23
參、實驗材料與方法 26
一、實驗材料 26
1. treX基因來源菌株 26
2. 實驗用表現treX基因的質體種類 26
3. 重組肝醣支切酶的生產菌株 26
4. 實驗藥品 27
5. 實驗儀器設備 29
二、實驗方法 31
(一)treX基因在大腸桿菌的蛋白質表現 31
1. 蛋白質表現載體和宿主細胞的選擇 31
2. 重組GDE的誘導表現方法 31
2.1 胞內蛋白質表現系統之誘導 31
2.2 胞外蛋白質表現系統之誘導 32
3. 重組GDE表現量的初步分析 32
4. 膠電泳分析 32
4.1 試劑製備 33
4.2 膠體之製備 34
4.3 膠電泳之操作方法 34
4.4 膠片染色與脫色 35
4.4.1染色液之製備 35
4.4.2脫色液之製備 35
4.4.3操作方法 36
(二)肝醣支切酶的酵素活性測定方法 36
1. 酵素活性測定方法 36
1.1試劑之製備 36
1.2酵素活性測定之操作方法 36
2. 酵素活性單位 37
3. 蛋白質定量方法 37
3.1試劑之製備 37
3.2 BSA標準品之製備 38
3.3操作方法 38
(三)重組蛋白質的生產與純化方法 38
1. 大腸桿菌的培養與重組GDE的表現 38
2. 蛋白質的抽取 39
3. 重組GDE的純化 39
3.1重組GDE之熱處理 39
3.2 親和性膠體管柱層析法 39
(四)原生型GDE的生產與純化方法 40
1. 大腸桿菌的培養與原生型GDE的表現 40
2. 原生型GDE的抽取 41
3. 原生型GDE的純化 41
3.1原生型GDE之熱處理 41
3.2超過濾膜濃縮與緩衝液置換 41
3.3 膠體管柱層析法 41
(五)肝醣支切酶之特性探討 42
1. 肝醣支切酶的最適作用溫度之探討 42
2. 肝醣支切酶的最適作用酸鹼值之探討 42
3. 肝醣支切酶的熱穩定性之探討 43
4. 肝醣支切酶的酸鹼值穩定性之探討 43
5. 金屬離子對肝醣支切酶酵素活性之影響 43
肆、結果與討論 45
一、蛋白質表現載體和宿主細胞的選擇 45
二、重組GDE的生產與純化 46
三、原生型GDE的生產與純化 47
四、肝醣支切酶特性之探討 48
1. 肝醣支切酶的最適作用溫度之探討 48
2. 肝醣支切酶的最適作用酸鹼值之探討 49
3. 肝醣支切酶的熱穩定性之探討 49
4. 肝醣支切酶的酸鹼值穩定性之探討 50
5. 金屬離子對肝醣支切酶酵素活性之影響 51
伍、結論 53
陸、參考文獻 54
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