臺灣博碩士論文加值系統

本研究主要目的是利用Providencia rettgeri ATCC31052 的pac 基因在大腸桿菌（Escherichia coli）中進行異形表現來大量生產PAC酵素。我們將Providencia rettgeri ATCC31052的pac基因植株於一個多複製（multicopy）的質體上且pac基因的表現受到tac啟動子的調節控制。對於PAC酵素之大量生產中，我們可以發現到一些不可或缺的因子，包括：碳源的添加，培養溫度的不同，以及宿主細胞的影響。
對於PAC酵素生產的最適培養條件是改變不同的宿主/載體（host/vector）系統。在最適當的生產當中，我們可以發現到一個很重要的改善，不管是體積活性（volumetric PAC activity）或者是比活性（specific PAC activity），都比原始菌種Providencia rettgeri ATCC31052還要高出50倍左右。由此結果這樣我們則可以論證，在不同的培養溫度以及宿主/載體系統的影響下，PAC酵素之異形表現可能被限制在轉譯步驟（translation）或轉譯後修飾步驟（post-translational）上。
所以我們從這篇論文當中，可以知道Providencia rettgeri ATCC31052 PAC酵素形成之限制步驟以及Providencia rettgeri ATCC31052之PAC先驅物（precursor）的存在。也讓我們更能確定Providencia rettgeri 的PAC酵素的形成機制跟大腸桿菌是很類似的。

In this study, we used heterologous production of Providencia rettgeri ATCC31052 for production penicillin acylase in Escherichia coli .The pac gene from Providencia rettgeri ATCC31052 was cloned in a multicopy plasmid and expression of pac was regulated by tac promoter. For the enzyme production several factors were identified to be critical, including: carbon source, temperature, and host effect.
The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimum, both volumetric and specific PAC activity could be significantly improved at more than fifty folds compared to the native expression in Providencia rettgeri ATCC31052.With the result, we can find the heterologous production could be possibly limited by translation or post-translational steps,depending on the culture temperature and host/vector system.
In this study we can know the limiting step for the production of Providencia rettgeri ATCC31052 PAC and the existence of the Providencia rettgeri ATCC31052 PAC precursor. The observation supports a common speculation that the enzyme formation mechanism of Providencia rettgeri ATCC31052 PAC is similar to that of Providencia rettgeri ATCC31052 PAC.

目錄
中文摘要………………………………………………………………..Ⅰ
Abstract………………………………………………………………..Ⅱ
目錄……………………………………………………………………..Ⅲ
表目錄…………………………………………………………………..Ⅴ
圖目錄…………………………………………………………………..Ⅵ
第一章 緒論……………………………………………………………..1
1.1 前言………………………………………………………………...1
1.2 盤尼西林醯胺分解酵素的用途與介紹…………………………...1
1.3 PAC的來源與種類………………………………………………....2
1.4 大腸桿菌E.coli中PAC酵素的成熟與合成……………………....3
1.5 E.coli ATCC11105與Providencia rettgeri ATCC31052之比較.5
1.6 研究方向…………………………………………………………...6
第二章 實驗方法………………………………………………………....8
2.1 菌種的處理………………………………………………………...8
2.1.1 本研究所使用菌種種類……………………………………...8
2.1.2 培養基的製備………………………………………………...8
2.1.3 菌種的儲存與活化…………………………………………...8
2.1.4 菌種的培養…………………………………………………...8
2.1.5 細胞濃度……………………………………………………...10
2.2 處理DNA技術…………………………………………………......10
2.2.1 本研究所使用質體DNA種類……………………………......10
2.2.2 質體(Plasmid)DNA的萃取……………………………….....10
2.2.3 質體的建構…………………………………………………...12
2.3 轉形(Transformation)…………………………………………….17
2.3.1 可轉形細胞（competent cell）的製備…………………….17
2.3.2 轉形(Transformation)……………………………………….17
2.4 隨機突變 (Miller, 1992)………………………………………..17
2.5 最小抑制濃度的測試………………………………………….....18
2.6 以微生物篩選法測試PAC酵素產生菌………………………......18
2.7 批次菌種培養………………………………………………….....19
2.8 PAC酵素定量分析………………………………………………....19
2.8.1 酵素分析樣品的製備………………………………………...19
2.8.2 酵素反應……………………………………………………...19
2.8.3呈色反應……………………………………………………....20
2.9 蛋白質SDS-PAGE分析法…………………………………….......20
2.10實驗藥品……………………………………………………….....22
2.11實驗設備……………………………………………………….....24
第三章 結果與討論……………………………………………………....25
3.1培養環境的影響…………………………………………………....25
3.1.1 IPTG的誘導對於pac基因的表現測試……………………....25
3.1.2不同碳源對於PAC酵素產量的影響……………………….....29
3.1.3不同溫度對於PAC酵素產量的影響……………………….....37
3.2宿主細胞的影響…………………………………………………....41
3.2.1宿主細胞MDDP7之篩選…………………………………….....41
3.2.2宿主細胞的影響……………………………………………....42
3.3 PAC酵素製造的限制條件(limiting step)…………………….…48
3.4 PAC酵素製造的最適化………………………………………......51
第四章 結論與展望……………………………………………………....52
參考文獻……………………………………………………………….....54
表目錄
表 一.本研究使用菌種列表……………………………………………....9
表 二.本研究使用培養基的組成…………………………………….....10
表 三.本研究使用質體DNA列表……………………………………......11
表 四.蛋白質SDS-PAGE凝膠的製備……………………………….......21
表 五.不同的宿主細胞對於溫度的改變以及甘油加入與否的影響.....44
圖目錄
圖 一.酵素法生產6-APA……………………………………………….....2
圖 二.大腸桿菌中PAC蛋白成熟的途徑……………………………….....4
圖 三.質體pUTKnPAC2601的基因限制結構圖…………………….......13
圖 四.質體pUTKnPAC2611的基因限制結構圖…………………….......14
圖 五.質體pTrcKnPAC2601的基因限制結構圖……………………......15
圖 六.質體pTrcKnPAC2611的基因限制結構圖……………………......16
圖 七. IPTG對JM109（pUTKnPAC2601）與JM109（pTrcKnPAC2601）之PAC酵素活性定量分析…………………………………….................27
圖 八. IPTG對JM109（pUTKnPAC2611）與JM109（pTrcKnPAC2611）之PAC酵素活性定量分析…………………………………….................28
圖 九. PAA對PAC酵素活性之定量分析……………………………......31
圖 十.葡萄糖對PAC酵素生成的影響………………………………......32
圖 十一.果糖對PAC酵素生成的影響………………………………......33
圖 十二.山梨糖醇對PAC酵素生成的影響…………………………......34
圖 十三.蔗糖對PAC酵素生成的影響………………………………......35
圖 十四.甘油對PAC酵素生成的影響………………………………......36
圖 十五.溫度對PAC酵素生成的影響………………………………......38
圖 十六.溫度對菌種之蛋白質SDS-PAGE分析圖………………….......39
圖 十七.不同碳源對JM109（pUTKnPAC2601）的影響……………......40
圖 十八.溫度對PAC酵素生成的影響；甘油濃度5g/L………………....45
圖 十九.溫度對PAC酵素生成的影響；不添加碳源…………………....46
圖 二十.蛋白質 SDS-PAGE 分析圖；樣品為各菌種之可溶的部分.....47
圖 二十一.蛋白質 SDS-PAGE 分析圖；樣品為各菌種之不可溶的部分…………………………………………………………...............48

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