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研究生:廖家淇
論文名稱:利用定向演化改善麥芽寡糖?海藻糖生成?之活性
論文名稱(外文):Improving the activity of maltooligosyltrehalose synthase by directed evolution.
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:2008
畢業學年度:96
語文別:中文
論文頁數:91
中文關鍵詞:海藻糖麥芽寡糖?海藻糖生成?定向演化基因重組隨機突變
外文關鍵詞:trehaloseMTSasedirected evolutionDNA family shufflingDNase IStEP
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麥芽寡糖?海藻糖生成? (MTSase) 為部份嗜高溫古細菌所具有的糖?轉移?-水解?系統中之酵素, MTSase 可催化分子內的轉糖?反應,將麥芽寡糖還原端上的 α-1,4 糖?鍵結轉變為 α-1,1 糖?鍵結,以產生麥芽寡糖?海藻糖。由於其活性催化區的 (β/α)8 桶狀結構適合進行定向演化,因此本實驗選用兩種嗜高溫古細菌 Sulfolobus solfataricus ATCC 35092 與 Sulfolobus acidocaldarius ATCC 33909 ,兩者 MTSase 基因序列 treY 核酸序列相似度為 64.4% ,胺基酸序列相似度為 51.3% ,利用兩種定向演化方法 DNA family shuffling 與 Stagger Extension Process (StEP) 使兩者 treY 基因藉由同源性進行重組,隨後進行活性篩選,期望能篩選出活性較高的突變株 MTSase 。
在 DNA family shuffling 實驗中,DNase I 的最適酵素剪切條件為:模板基因在 Mn2+ 離子的存在下,加入 0.1U DNase I 在 15℃ 下反應 5 分鐘,隨後以含有 50 mM EDTA 與 30% glycerol 之溶液冰浴 15 分鐘終止反應。取100-300 bp 短片段基因純化回收後進行隨機重組,再以帶有限制?切位之引子擴增出重組基因,得到與原生型 treY 大小相近之重組產物,由於重組基因濃度不足且成功率低無法進行後續之轉形株篩選實驗。
StEP 實驗中,可能由於兩者 treY 的同源性太低,因此 PCR 反應中黏合與延伸條件以及使用的引子備受限制,目前尚未成功獲得活性較高的突變型 MTSase ,但是可將多次實驗結果作為經驗不斷地修正 PCR 反應的黏合與延伸條件,並持續發展出高效率及高準確度的篩選方法。
目錄

一、 前言 1
二、 文獻整理 3
1. 海藻糖 3
1.1 研究背景 3
1.2 海藻糖之結構與特性 3
1.3 生產方式 5
1.4 應用與發展 7
2. 麥芽寡糖?海藻糖生成? 7
2.1 簡介 7
2.2 轉糖基反應機制 9
2.3 結構之探討 9
3. 定向演化 10
3.1 定向演化的概念 10
3.2 DNA shuffling 11
3.2.1 DNA shuffling 簡介 11
3.2.2 DNA family shuffling 之原理 13
3.2.3 DNA family shuffling 之方法 13
3.2.4 DNA family shuffling 之應用 14
3.3 Stagger Extension Process (StEP) 17
三、 實驗設計與流程 17
1. 實驗設計 17
2. 實驗流程 18
四、 實驗材料 19
1. 菌株與質體 19
1.1 treY 基因來源菌株 19
1.2 表現 treY 基因之質體 19
1.3 保存質體之菌株 19
1.4 蛋白質表現系統之生產菌株 19
2. 抗生素 19
3. 標準品 19
4. 培養基 20
5. 酵素 20
5.1 聚合? 20
5.2 限制? 20
5.3 接合? 20
5.4 去氧核糖核酸水解? 20
6. 化學藥品 21
7. 市售快速套組 22
8. 實驗設備 22
9. 軟體 24
五、 實驗方法 25
1. 模板基因之序列同源性分析 25
2. 製備欲進行定向演化之基因序列 25
2.1 製備小量質體 DNA 25
2.2 製備大量質體 DNA 26
2.3 DNA 濃度之定量 27
2.4 PCR反應 27
2.5 膠體電泳分析 28
2.6洋菜膠體回收純化 PCR 反應產物 28
2.6.1 選用 Micro-Elute DNA Clean/Extraction Kit 進行回收純化 28
2.6.2 選用 WizardR SV Gel and PCR Clean-Up System 進行回收
純化 29
2.6.3 以膠回收機器與酒精沉澱進行回收純化 30
2.7 PCR 反應產物回收 30
2.7.1 選用 Micro-Elute DNA Clean/Extraction Kit 進行回收 30
2.7.2 選用 WizardR SV Gel and PCR Clean-Up System 進行回收 31
3. DNA family shuffling 實驗 31
3.1 DNase I 隨機剪切與膠回收純化 DNA 31
3.2 第一階段 PCR 反應 (fragment reassembly) 32
3.3 設計 Nhe I 與 Hind III 限制?切位之引子 33
3.4第二階段 PCR 反應 (PCR amplification of reassembled products) 33
4. Stagger Extension Process (StEP) 實驗 34
5. 將重組基因構築至表現載體中進行轉形作用 35
5.1 限制?剪切 35
5.2 接合反應 36
5.3 RF cloning 36
5.4電穿孔轉形作用 (electroporation) 37
5.4.1 製備電穿孔勝任細胞 (E. coli Rosetta (DE3)) 37
5.4.2 電穿孔轉形作用 (electroporation) 38
6. 小量培養轉形株並進行熱穩定性酵素之初步篩選 38
6.1 colony PCR 38
6.2 小量培養轉形株並萃取粗酵素液 39
6.2.1 小量培養方法(一) 39
6.2.2 小量培養方法(二) 40
7. 以 DNS 法初步篩選具麥芽寡糖?海藻糖生成?活性之突變株 40
7.1 製備 MTSase 所需粗基質 (0.6% maltodextrin D10) 40
7.2 活性定義 41
7.3 製備 DNS 試劑 (DNS-reagent) 41
7.4 以 DNS 法測麥芽寡糖?海藻糖生成?之轉糖?活性 41
7.4.1 酵素活性測定法(一)- 5.2.1 小量培養方法(一)使用 41
7.4.2 酵素活性測定法(二)- 5.2.2 小量培養方法(二)使用 42
8. 序列分析 43
9. 核?酸序列比對 43
六、 結果與討論 44
1. 利用 DNA family shuffling 方法進行基因重組 44
2. Stagger Extension Process (StEP) 48
2.1 利用質體 DNA 作為模板股 48
2.2 利用質體 PCR 反應擴增之 SStreY 及 SAtreY作為模板股 50
2.3 以帶有限制?切位之引子進行反應 52
未來展望 54
七、 結論 55
八、 參考文獻 56

圖表目錄
圖一、麥芽寡糖?海藻糖生成?之 3D 立體結構 62
圖二、(β/α)8 桶狀構形 63
圖三、定向分子演化的過程 64
圖四、DNA shuffling 的重組過程 65
圖五、Stagger Extension Process (StEP) 的重組過程 66
圖六、利用洋菜瓊脂膠電泳分析模板股之質體 DNA 與基因 67
圖七、利用洋菜膠電泳分析兩種不同方式終止反應的效果 68
圖八、利用洋菜瓊脂膠電泳分析 DNase I 以二次去離子水稀釋後之剪切
效果 69
圖九、利用洋菜瓊脂膠電泳分析 DNase I以 1 X reaction buffer 稀釋後
之剪切效果 70
圖十、利用洋菜瓊脂膠電泳分析 DNase I 隨機剪切之產物 71
圖十一、利用洋菜瓊脂膠電泳分析DNA family shuffling 之產物 72
圖十二、利用洋菜瓊脂膠電泳分析DNA family shuffling 實驗中失敗的
情形 73
圖十三、利用洋菜瓊脂膠電泳分析 StEP 之重組產物 74
圖十四、利用洋菜瓊脂膠電泳分析 RF cloning 之產物 75
圖十五、突變型 treY 與原生型 SStreY 序列比對後之重組情形 76
圖十六、突變型 treY 與原生型 SAtreY 序列比對後之重組情形 77
圖十七、突變型 treY 與原生型 SStreY 序列比對後之重組情形 78
圖十八、突變型 treY 與原生型 SAtreY 序列比對後之重組情形 79
圖十九、突變型 treY 與原生型 SStreY 序列比對後之重組情形 80
圖二十、突變型 treY 與原生型 SAtreY 序列比對後之重組情形 81
表一、 StEP 實驗中帶有載體 (pET-15b)基因序列之特異性引子序列 82
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