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研究生:潘彥齊
研究生(外文):Yen-Chi Pan
論文名稱:Pseudomonas vesicularis MA103 所產 amylase Pv-AM-I 純化與澱粉酶基因 Amy-00047轉殖至大腸桿菌及其特性分析
論文名稱(外文):Purification and Characterization of Pseudomonas vesicularis MA103 Amylase Pv-AM-I as well as Cloning of Amylase Gene Amy-00047 in Escherichia coli
指導教授:潘崇良
指導教授(外文):Chrong-Liang Pan
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:120
中文關鍵詞:澱粉酶純化基因轉殖
外文關鍵詞:amylasepurificationclone
相關次數:
  • 被引用被引用:5
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  • 下載下載:18
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本研究目的在於探討 P. vesicularis MA103 經可溶性澱粉誘導後所產之澱粉誘導粗酵素液 (SI-Enzyme solution),分離純化後分別探討其生化特性和水解產物組成;另外嘗試以無限制酶剪切 (RF cloning) 方式將 MA103 澱粉酶基因 (Amy-00047) 轉殖至大腸桿菌中表現。MA103 澱粉誘導酵素液經過離心、超過濾和膠體過濾層析法無法得到單一純化後蛋白質,因而使用澱粉親和力純化法暨膠體過濾層析法,成功得到單一純化後酵素 Pv-AM-I,純化倍率為 7.94,其分子質量約為 37 kDa。MA103 澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I 之最適反應溫度及 pH 皆為 50oC, pH 7.0,而且 Ca2+ 皆可以使二者之相對活性提昇 (124%, 103%),但 Sn2+ 會使澱粉誘導粗酵素液及澱粉酶 Pv-AM-I 完全失去澱粉酶活性。在基質特異性部分Pv-AM-I 及澱粉誘導粗酵素液對龍鬚菜多醣與海菜多醣都具有降解能力,而 Pv-AM-I 亦具有降解馬尾藻多醣之能力。水解澱粉產物分析方面,MA103 澱粉誘導酵素液與純化後澱粉酶 Pv-AM-I 水解 0.2% 之可溶性澱粉產物,經高效能液相層析法 (HPLC) 以及薄層層析法 (TLC) 分析比對標準品後,推測其主要產物為麥芽糖。另一方面探討澱粉酶基因轉殖技術部分,嘗試從 MA103 genomic DNA定序報告中挑選一個澱粉酶序列,命名為 Amy-00047 且長度為 1071 bp,依序列設計專一性引子,以 RF cloning 進行基因轉殖至大腸桿菌表現,實驗過程中,使用二種不同之 DNA 聚合酶 (Taq, Pfu polymerase) 經過三次聚合酶連鎖反應 (polymerase chain reaction, PCR) 成功擴增後,利用 DpnI 剪切處理後以熱休克之方式轉型至宿主細胞 E. coli DH5 以及 BL21 皆發現具有抗生素抗性之轉殖株生長,但使用不具基因校正能力之 Taq polymerase 組別經 PCR 擴增後,於轉殖株中並未發現有與 Amy-00047基因大小相似之產物出現;反觀使用具校正能力之 Pfu polymerase 之組別於轉殖株中可以成功擴增出與欲轉殖之基因大小相似約1000 bp 之產物產生,此結果仍待進一步轉殖株之重組質體基因定序確認。
The aim of this study is using soluble starch to induce Pseudomonas vesicularis MA103 for amylase production, after enzyme purification the biochemical properties and hydroltsate of starch induced enzyme solution (SI-Enzyme solution) and purified amylase Pv-AM-I were both investigated; MA103 amylase sequence Amy-00047 was cloned to E. coli. MA103 starch induced enzyme solution was centrifuged, ultrafiltrated and chromatogramed by gel permeation. The purification methods above were not able to acquire single protein band. On the other hand, using starch affinity method and gel permeation chromatography successfully obtained single protein band by SDS-PAGE, named as Pv-AM-I. The purification fold was 7.94 and the molecular weight was approximately 37 kDa. The optimal reaction pH and temperature of SI-Enzyme solution and purified amylase Pv-AM-I were pH 7.0 and 50oC. Ca2+ was able to raise the relative activity of both SI-Enzyme solution and Pv-AM-I (103-124%), on the other side, Sn2+ was with the ability to completely inhibit the enzyme activity of both enzymes. On substrate specificity , SI-Enzyme solution and Pv-AM-I can both hydrolyze Garcilaria sp. and Monostroma sp. polysaccharide, in addition, purified amylase Pv-AM-I was capable to hydrolyze Sargassum sp. polysaccharide. Analyzing the hydrolysate of SI-Enzyme solution and Pv-AM-I by HPLC and TLC, compared to the standards, discovered that the main hydrolysate were both assumed to be maltose. Further, research on amylase cloning. Amy-00047, 1071 base pairs, was one of the amylase gene sequence from the sequencing report of MA103 genomic DNA. According to Amy-00047 gene sequence a pair of primer was designed, and the primer pair was used in 3 times of PCR (RF cloning) to acquire recombinant plasmid DNA. During the 3 times PCR process, two different kinds of DNA polymerase (Taq, Pfu) were used. After 3 times of PCR, the Amy-00047 inserted plasmid DNA was transformed into E. coli DH5 and BL21 by heat shock. The plasmid accepted E. coli colonies were capable to grow on ampicilin added LA plate, but there were no colony PCR products with the colonies which plasmids were amplified by Taq polymerase. In contrast, the colonies with Pfu polymerase amplified plasmids was discovered with approximately 1000 bp product. The result still needs further recombinant plasmid DNA sequencing of to confirm the inserted gene sequence.
目錄 i
表目錄 v
圖目錄 vi
附錄目錄 ix
中文摘要 x
英文摘要 (Abstract) xi
壹、前言 01
貳、文獻整理 03
一、澱粉的組成與結構 03
二、澱粉酶 (amylase) 03
II-1. -澱粉酶 (-amylase) 04
II-2. -澱粉酶 (-amylase) 05
II-3. -澱粉酶 (-amylase) 05
三、澱粉酶對澱粉水解的作用機制 05
四、澱粉酶之生化特性 06
IV-1. pH 值與溫度 06
IV-2. 各種離子對 -澱粉酶之影響 07
IV-2-A. 陽離子 07
IV-2-B. 陰離子 08
IV-3. 分子質量 08
五、澱粉酶之應用 08
六、澱粉酶之不同純化方式 10
七、澱粉酶的選殖 11
八、Restriction-free cloning 11
九、海藻產製生質酒精的優勢與開發現況 12
十、澱粉酶水解澱粉後產物分析 13
X-1. 薄層層析法 (Thin-layer chromatography, TLC) 13
X-2. 高效能液相層析法 (High-performance liquid chromatography, HPLC) 13
參、實驗設計 14
肆、實驗材料與方法 15
一、實驗材料 15
I-1. 實驗菌株 15
I-2. 試驗藥品 15
     I-2-A. 藥品 15
     I-2-B. 載體 17
     I-2-C. 引子 17
     I-2-D. 聚合酶鏈鎖反應試劑 17
     I-2-E. 限制酶 18
     I-2-F. 蛋白質及DNA 翠取純化套裝試劑 18
     I-2-G. 電泳標準品 18
     I-2-H. 培養基組成 19
     I-2-I. 澱粉酶反應基質 20
     I-2-J. DNS 溶液 20
I-2-K. 純化過程緩衝液 21
     I-2-L. 電泳膠片配置 21
I-2-M. 電泳溶液 22
I-2-N. 無菌過濾 22
     1-2-O. 離心式超過濾濃縮 22
I-4. 儀器設備 22
二、實驗方法 24
    II-1. 菌株保存與活化 24
     II-1-A. 菌株保存 24
     II-1-B. 菌株活化 24
    II-2. 澱粉誘導酵素液的純化分離 25
     II-2-A. 澱粉誘導酵素液之生產 25
     II-2-B. 酵素液之濃縮純化 25
     II-2-C. 澱粉酶的活性測定 25
II-2-D. 蛋白質的定量 26
     II-2-E. 分子量的鑑定 26
     II-2-F. 澱粉酶 N 端胺基酸定序 26
    II-3. my 基因選殖 27
     II-3-A. MA 103 染色體 DNA的萃取純化 27
     II-3-B. 引子設計 27
     II-3-C. 無限制酶剪切位基因選殖 (RF cloning) 及 DpnI
限制酶剪切 27     
II-3-D. 勝任細胞 (competent cell) 之製作 30
II-3-E. 熱休克 (heat shock) 轉形 31
II-3-F. 利用 PCR 確認插入之基因片段大小 31
    II-4. MA103 澱粉誘導粗酵素液的純化分離 32
     II-4-A. 酵素液之濃縮純化 32
     II-4-B. 澱粉酶酵素液親和力純化法進行純化 32
     II-4-C. Sephacryl® S-200 HR 膠體過濾層析 33
II-5. MA103 澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I 之生化性質測試 33
II-5-A. 基質特異性 33
II-5-B. 最適作用pH 值 33
II-5-C. pH 值的安定性 33
II-5-D. 最適作用溫度 34
II-5-E. 熱安定性 34
II-5-F. 金屬離子的影響 34
II-6. MA103 澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I之水解產物分析 34
II-7-A. TLC 鑑定 34
II-7-B. HPLC鑑定 35
伍、結果與討論 36
一、P. vesicularis MA103不同誘導物酵素液之澱粉酶活性 36
二、Amylase Pv-AM-I 之純化分離 36
II-1. MA103澱粉誘導粗酵素液以超過濾及膠體過濾層析進行純化 36
II-2. MA103 澱粉誘導酵素液以澱粉親和力純化法及膠體過濾層析進行純化 37
三、P. vesicularis MA103 澱粉誘導粗酵素液及純化後澱粉酶 Pv-AM-I 生化性質試驗 38
III-1. 最適作用溫度及溫度安定性 38
III-2. 最適作用 pH 值及 pH 安定性 39
III-3. 不同金屬離子之影響 41
III-4. 基質特異性 41
III-5. 水解產物分析 42
四、Amy-00047 基因選殖之結果 44
IV-1. MA103 澱粉酶基因的篩選 44
IV-2. 引子之設計 45
IV-3 以 RF cloning 進行基因轉殖 45
陸、結論 48
柒、參考文獻 50
捌、附錄 95





表目錄

表一、 Pseudomonas vesicularis MA103 澱粉誘導粗酵素液以澱粉親和力純化法及 S-200 膠體過濾層析純化法純化表 63
表二、 MA103澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I 之生化特性表 64
表三、 Pseudomonas vesicularis MA103 全基因體定序報告中 13 個可能之澱粉酶 65
表四、RF cloning 所使用之 primer 序列 66



















圖目錄

圖一、 P. vesicularis MA103 由不同誘導物所產之酵素液其澱粉酶活性 67
圖二、 由澱粉誘導之 P. vesicularis MA103生長曲線圖 68
圖三、 MA103澱粉誘導酵素液經超過濾部分之 S-200 膠體過濾層析圖 69
圖四、 MA103澱粉誘導酵素液經澱粉親和力純化法後部分純化酵素液之 S-200 膠體過濾層析圖 70
圖五、 MA103澱粉誘導粗酵素液純化過程之10% SDS-PAGE 電泳分析圖 71
圖六、 溫度對 MA103澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I 水解 0.2% 澱粉基質之影響 72
圖七、 溫度對 MA103澱粉誘導粗與純化後澱粉酶 Pv-AM-I澱粉酶活性安定性之影響 73
圖八、 pH 值對水解 0.2% 澱粉基質之影響 74
圖九、 pH 值對純化後澱粉酶 Pv-AM-I 水解 0.2% 澱粉基質之影響 75
圖十、 pH 值對MA103澱粉誘導粗酵素液澱粉酶活性安定性之影響 76
圖十一、 pH 值對純化後澱粉酶 Pv-AM-I 之澱粉酶活性安定性之影響 77
圖十二、 金屬離子對MA103澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I酵素活性之影響 78
圖十三、 P. vesicularis MA103 澱粉誘導粗酵素液、純化後澱粉酶 Pv-AM-I 與市售澱粉酶 (Sigma) 對不同藻類及澱粉基質之特異性之比較 79
圖十四、 MA103 澱粉誘導粗酵素液水解可溶性澱粉 0-24 hr 之薄層色層分析圖 80
圖十五、 純化後澱粉酶 Pv-AM-I 水解可溶性澱粉 0-24 hr 之薄層色層分析圖 81
圖十六、 MA103 澱粉誘導粗酵素液降解 0.2% 可溶性澱粉0-24 hr之高效能液相層析圖 82
圖十七、 純化後澱粉酶 Pv-AM-I 降解 0.2% 可溶性澱粉0-24 hr之高效能液相層析圖 83
圖十八、 MA103 澱粉誘導粗酵素液降解 0.2% 麥芽糖 24 hr之
高效能液相層析圖 84
圖十九、 純化後澱粉酶 Pv-AM-I 降解 0.2% 麥芽糖24 hr之
高效能液相層析圖 85
圖二十、 MA103 所產 amylase Amy-00047 之完整序列 86
圖二十一、MA103 Amy-00047 與alpha-amylase Aeromonas
salmonicida subsp. salmonicida A449 胺基酸序列
比對 87
圖二十二、以 Taq polymerase 擴增之第一次及第二次之PCR 產物其 1% 洋菜糖膠電泳分析 88
圖二十三、 以 Taq polymerase擴增之第三次之 PCR 產物其 1% 洋菜糖膠電泳分析 89
圖二十四、以Pfu polymerase 擴增之第二次PCR 產物經膠純化後1% 洋菜糖膠電泳分析 90
圖二十五、以 Pfu polymerase擴增之第三次PCR 產物1% 洋菜糖膠電泳分析 91
圖二十六、轉殖成功之單一菌落以 Amy-for 及 Amy-rev 作為引子利用 PCR 確認插入基因片段之 1% 洋菜糖膠電泳分析 92


附錄目錄

附錄一、 澱粉之基本結構 93
附錄二、 不同澱粉酶之作用位置 94
附錄三、 不同藻類來源澱粉含量 95
附錄四、 麥芽七糖、麥芽五糖麥芽糖與葡萄糖之高效能液相層析
圖譜 96
附錄五、 麥芽五糖、麥芽三糖與麥芽糖之高效能液相層析圖譜 97
附錄六、 麥芽七糖之高效能液相層析圖譜 98
附錄七、 麥芽五糖之高效能液相層析圖譜 99
附錄八、 麥芽三糖之高效能液相層析圖譜 100
附錄九、 麥芽糖之高效能液相層析圖譜 101
附錄十、 MA103 澱粉誘導粗酵素液之高效能液相層析圖譜 102
附錄十一、純化後澱粉酶 Pv-AM-I 之高效能液相層析圖譜 103
附錄十二、0.2% 可溶性澱粉之高效能液相層析圖譜 104
附錄十三、RF cloning 的實驗原理流程圖 105
附錄十四、pET-21a (+) 的限制酶切位及基因圖譜 106
附錄十五、pET-21a-d (+) 上基因片段插入位區域的 DNA 序列 107




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