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研究生:魏萍雀
研究生(外文):Wei, Pingchiue
論文名稱:Burkholderia cepacia complex咖啡因去甲基酵素之基因選殖及表現
論文名稱(外文):Molecular Cloning and Expression of Caffeine Demethylase from Burkholderia Cepacia Complex
指導教授:鍾雲琴
指導教授(外文):Chung, Yunchin
口試委員:陳俊宏吳如雯
口試委員(外文):Chen, GenhungWu, Ruwen
口試日期:2013-12-06
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2013
畢業學年度:102
語文別:中文
論文頁數:108
中文關鍵詞:Burkholderia cepacia complex咖啡因去甲基酵素基因重組包含體
外文關鍵詞:Burkholderia cepacia complexcaffeine demethylasegenetic recombinationinclusion body
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咖啡因(1,3,7-trimethylxanthine)廣泛存在於多種日常食品中,過量攝取對人體健康具不良影響,如誘發心血管疾病、造成胎兒畸形和降低生育率等,因此去除食品中的咖啡因成為一項重要的加工技術。本研究旨於自種植咖啡樹之土壤中篩選具分解咖啡因能力之菌株,而後選殖其咖啡因去甲基酵素(Caffeine demethylase,CDase)基因,並於Escherichia coli細胞中大量表現,期望此基因重組之咖啡因去甲基酵素可應用於食品加工業。首先,自咖啡豆培植土中篩選200株具降解咖啡因能力之菌株,利用High-performance liquid chromatography (HPLC)評估咖啡因降解活性後,挑選活性最佳之菌株進行菌種鑑定,鑑定結果顯示其為Burkholderia cepacia complex中的一員。以polymerase chain reaction選殖B. cepacia之咖啡因去甲基酵素基因,並將其重組於表現載體pTrcHis A,之後於E. coli TOP10中大量表現。結果顯示,B. cepacia之咖啡因降解活性為0.056 g/L/h,經基因定序後得知其咖啡因去甲基酵素基因含有973 bp,且其DNA定序與Takeuchi, K.和Koide, Y.在1996年所發表Pseudomonas putida之咖啡因去甲基酵素相似度為99 %。由SDS-PAGE得知基因重組之咖啡因去甲基酵素之蛋白質分子量約為41.58 kDa。Western blot和咖啡因降解試驗結果顯示,重組之咖啡因去甲基酵素為包含體(inclusion body),不具酵素活性;但利用0.5 % SDS使重組之咖啡因去甲基酵素蛋白質變性,再以2 mM reduced glutathione和0.2 mM oxidized glutathione使之復性後,此重組酵素降解咖啡因的能力提升為0.67 mg/L/min。
Caffeine (1,3,7-trimethylxanthine) is widely present in many foods, excessive intake may have adverse effects on human health. For example, cardiovascular disease, fetus malformation and reduction of fertility rate may cause by the consumption of high caffeine-containing foods. Hence, de-caffeine is an important food processing technique for improving consumers’ health. The aims of this study were to isolate caffeine degrading strains from the soil of coffee plantation area, to clone the caffeine demethylase (CDase) gene from the strains and over-express in Escherichia coli. The recombinant CDase could be used in the food industry for de-caffeine process. In this study, 200 strains were isolated from the soil and the caffeine degradation activities of the strains were assessed by High-performance liquid chromatography (HPLC). The strain with the highest ability to degrade caffeine was identified as Burkholderia cepacia complex. The caffeine demethylase gene from B. cepacia was cloned by polymerase chain reaction, recombined in expression vector (pTrcHis A) and over-expressed in Escherichia coli TOP10. The results showed that the caffeine degradation activity of B. cepacia was 0.056 g/L/h. DNA sequencing indicated that the CDase gene had 973 bp and showed the homology of 99 % with the CDase reported from Pseudomonas putida by Takeuchi, K. and Koide, Y. in 1996. Molecular weight of the recombinant CDase was about 41.58 kDa based on the measurement of SDS-PAGE. Western blot and caffeine degradation test results showed that the recombinant CDase was an inclusion body and showed no caffeine degradation activity. However, after the denaturation of the recombinant CDase with 0.5% SDS and the renaturation using 2 mM reduced glutathione and 0.2 mM oxidized glutathione, the refolded recombinant CDase showed caffeine degradation activity of 0.67 mg/L/min.
目錄 I-XII
圖目錄 VI-VIII
表目錄 IX
中文摘要 X
英文摘要 XI-XII
第一章 前言 1
第二章 文獻回顧 4
一、咖啡因簡介 4
1. 來源 4
2. 屬性 5
3. 化學結構 6
4. 對人體健康的影響 7
5. 環境問題(含咖啡因的農業廢棄物) 8
二、去除咖啡因之方法 9
1. 水處理法(water decaffeination) 9
2. 有機溶劑萃取法(solvent decaffeination) 11
3. 超臨界二氧化碳萃取法(supercritical carbon dioxide extraction) 12
4. 微生物法(Microbial methods) 13
(1) 使用細菌降解咖啡因 14
(2) 使用真菌降解咖啡因 14
研究目的 18
第三章 材料與方法 19
第一節、實驗架構 19
第二節、實驗材料 20
1. 菌種與載體 20
2. 化學藥品 20
3. 儀器設備 21
第三節、實驗方法 22
一、咖啡因降解菌株之篩選 22
1. 篩菌 22
2. 革蘭氏染色 23
3. 咖啡因降解活性分析-1 23
(1) 咖啡因降解菌株之生長與誘導 23
(2) 咖啡因降解試驗(caffeine degradation experiments) 24
4. 菌種鑑定 25
二、咖啡因降解菌株CLM 83 (Burkholderia cepacia complex)之咖啡因去甲基酵素(caffeine demethylase)基因之選殖與表現 26
1. 咖啡因去甲基酵素(caffeine demethylase)基因之primers設計 26
2. Genome DNA的萃取與保存 26
3. 咖啡因去甲基酵素(caffeine demethylase,CDase)基因之選殖 27
(1) 聚合酶連鎖反應(Polymerase Chain Reaction,PCR) 27
(2) 純化PCR產物 28
(3) 接合作用(Ligation) (與pGEMT Easy Vector ligation) 29
(4) 製備通透性細胞(Competent Cell) 29
(5) 轉型(Transformation)---轉殖入宿主細胞(Cloning host) 30
(6) 製備小量質體(Mini-preparation of Plasmid DNA) 30
(7) DNA鑑定 31
A. 建立限制酶圖譜(Mapping) 31
B. 聚合酶連鎖反應(Polymerase Chain Reaction;PCR) 31
C. DNA定序 32
4. 咖啡因去甲基酵素(caffeine demethylase,CDase)基因轉殖於表現系統(Expression vector) 32
(1) 製備大量表現載體 32
(2) 表現載體pTrcHis A與pGEMT-CDase質體之限制酶剪切與膠上純化 33
(3) 接合作用(Ligation) (與pTrcHis A ligation) 33
(4) 製備通透性細胞(Competent Cell) 34
(5) 轉型(Transformation)---轉殖入表現宿主細胞(Expression host) 34
(6) 製備小量質體(Mini-preparation of Plasmid DNA) 34
(7) DNA鑑定 34
A. 建立限制酶圖譜(Mapping) 34
B. 聚合酶連鎖反應(Polymerase Chain Reaction;PCR) 34
C. DNA定序 35
三、大量表現 (Over expression)基因重組之咖啡因去甲基酵素 36
1. 咖啡因去甲基酵素之誘導與大量表現 36
2. SDS-PAGE 36
3. Western blot 40
4. 咖啡因降解活性分析-1 42
四、包含體(inclusion body)之變性、復性 43
1. 製備透析袋 43
2. 試劑配製 43
3. 實驗步驟 43
4. 咖啡因去甲基酵素之酵素活性分析-2 44
第四章 結果與討論 45
一、咖啡因降解菌株之篩選 45
1. 篩菌 45
2. 咖啡因降解活性 52
3. 菌種鑑定 53
二、咖啡因降解菌株CLM 83 (Burkholderia cepacia complex)之咖啡因去甲基酵素(caffeine demethylase,CDase)基因之選殖與表現 59
1. 咖啡因去甲基酵素(caffeine demethylase)基因之選殖 59
2. 咖啡因去甲基酵素(caffeine demethylase)基因之鑑定 65
3. 咖啡因去甲基酵素基因之表現與鑑定 65
三、咖啡因去甲基酵素基因之誘導與大量表現 79
1. pTrcHis A-CDase之IPTG最佳誘導條件 79
2. 將轉殖株(E. coli-pTrcHis A-CDase)、未轉殖株(E. coli TOP 10)及野生株(CLM 83)於最佳IPTG誘導條件下進行SDS-PAGE、Western blot和咖啡因降解試驗 83
四、包含體(inclusion body)之變性及復性 82
第五章 結論 104
參考文獻 105
附錄一

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