臺灣博碩士論文加值系統

致謝…………………………………………………………………………… i
摘要…………………………………………………………………………… ii
Abstract……………………………………………………………………...… iv
目錄………………………………………………………………………….... vi
表目錄…………………………………………………………………........ x
圖目錄………………………………………………………………………… xi
附表目錄……………………………………………………………………… xii
附圖目錄……………………………………………………………………… xiii
第壹章 前 言………………………………………………………………… 1
一、 聚羥基烷酸酯……………………………………..………………… 1
二、 PHA之特性………………………………………………………..... 1
1. 化學結構…………………………………………………………….. 1
2. 物理性質…..………………………………………………………… 2
3. 生物降解性與再生性….……………………………………………. 2
三、 PHA之應用………………………………………………………..... 3
四、 PHA之生合成……………………………………………………… 3
五、 生合成PHA之相關基因……………………………………………. 4
六、 微生物種類與生合成PHA之關係…………………………………. 6
七、 碳源種類與生合成PHA之關係……………………………………. 7
八、 PHA合成酶的蛋白質工程…………………………………………. 7
九、 研究起源與目的…………………………………………………….. 8
第貳章 材料與方法………………………………………………………….. 9
一、 化學藥品…………………………………………….......................... 9
二、 實驗器材及儀器…………………………………………………… 9
三、 PhaCH16點變異株菌E. coli / pBCspAB-H16CX之製備…………… 9
1. 熱休克勝任細胞之製備...................................................................... 10
2. 轉形作用……………………………..……………………………… 10
四、 胺基酸位置D233，A466以及A493飽和突變株質體之建構…… 10
1. 飽和突變模板股之建立………………………………….................. 10
2. 飽和突變之聚合酶連鎖反應……………………………………….. 10
2-1 飽和突變B片段之增幅………………..…………………………… 12
2-2 飽和突變megaprimer之增幅……………….……………………… 12
2-3 飽和突變A片段之增幅…………………………………………….. 12
2-4 飽和突變A片段及B片段之重組………………..………………… 13
2-5 增幅重組之飽和突變A片段及B片段…………………..………… 13
3. 接合作用……………………………………………..……………… 13
4. 轉形作用…………………………………………………………….. 14
5. 質體DNA之萃取…………………………………………………… 14
6. 核酸定序……………………………………………..……………… 14
五、 定點突變株之製備…………………………………..……………… 15
1. 定點突變之聚合酶連鎖反應……………………………………….. 15
2. 限制酶截切………………………………………………………….. 15
3. 轉形作用…………………………………………………………….. 15
六、 PHA產量分析………………………………………………………. 15
1. PHA之累積……………………………………………………….. 15
2. PHA甲基化分解………………...………………………………….. 16
3. PHA定量分析……………………...………………………….......... 16
七、 重組蛋白質PHA synthase之表現………………………………….. 16
1. 表現質體之建構…………………………………………………….. 16
2. 重組蛋白質PHA synthase之表現………...………………………... 16
八、 重組蛋白質PHA synthase之純化………………………………...... 17
1. 樣品前處理………………………………………………………….. 17
2. 硫酸胺沉澱………………………………………………………….. 17
3. 疏水性管柱層析…………………………………………………….. 17
九、 十二烷基硫酸鈉-聚丙烯醯胺膠體電泳…………………………… 18
1. 電泳膠體之配製………………………………………...................... 18
2. 樣品處理與蛋白質電泳…………………………………………...... 18
3. 蛋白質膠片的染色與退染………………………………………….. 18
十、 蛋白質濃度之測定………………………………………………….. 19
十一、 重組PHA synthase之生化特性分析…………………………... 19
1. 最適反應溫度……………………………………………………….. 19
2. 熱失活半衰期……………………………………………………….. 19
十二、 PHA合成酶基質專一性分析………………………………...... 20
1. 質體pBHR-H16Cx之建構………...………………………………... 20
2. 電穿孔勝任細胞之製備…………………………………………….. 20
3. 基質專一性分析菌株P. putida GPp104 PHA-/pBHR-H16CX之PHA累積………………………….………………………………… 20
十三、 蛋白質動態結構分析………………………………………….. 21
第參章 結 果………………………………………………………………… 22
一、 提升聚羥基烷酸酯合成酶PhaCH16熱穩定性與酵素比活性的關鍵胺基酸位置………..……………………………………………... 22
二、 PhaCH16飽和突變實驗對PHB累積之影響………………………... 23
1. PHA合成酶胺基酸位置D233飽和突變變異株PHB累積結果….. 23
2. 飽和突變變異點於PHA合成酶胺基酸位置A466的PHB累積….. 23
3. PHA合成酶胺基酸位置A493飽和突變變異株PHB累積試驗….. 24
三、 多個胺基酸變異點對PhaCH16酵素於PHB累積之影響…………... 25
四、 PHA合成酶變異株之蛋白質表現與純化…………………………. 25
五、 PHA合成酶變異株酵素比活性與熱穩定性分析…………………. 26
1. 最適反應溫度……………………………………………………….. 26
2. 熱失活半衰期試驗………………………………………………….. 26
六、 PHA合成酶變異株之酵素基質專一性分析……………………..... 27
七、 蛋白質螢光消光 (fluorescence quenching) 試驗…………………. 27
第肆章 討 論………………………………………………………………… 29
第伍章 結 論………………………………………………………………… 32
參 考 文 獻………………………………………………………………….. 34

 
表 目 錄
表一、 本研究使用之引子…………………………………………………… 39
表二、 本研究使用之飽和突變引子………………………………………… 40
表三、 本研究使用之定點突變引子………………………………………… 42
表四、 各個PhaCH16點變異株菌E. coli/pBCspAB-H16CX於37 oC及42 oC
之PHB累積分析……..……………………………………………… 44
表五、 胺基酸位置D233飽和突變變異株於37 oC及42 oC之PHB累積分析……………………...…………………………………………… 46
表六、 胺基酸位置A466飽和突變變異株於37 oC及42 oC之PHB累積分析………………………..…………………………………………. 47
表七、 胺基酸位置A493飽和突變變異株於37 oC及42 oC之PHB累積分析……………………………………………………….………….. 48
表八、 多點變異點組合變異株於30 oC、37 oC及42 oC之PHB累積分
析…………………………………………………………..………… 49
表九、 野生株與變異株PHA合成酶之最適反應溫度、最適反應比活性與45 oC溫度熱失活半衰期的分析………...…………………….... 50
表十、 重組P. putida GPp104 PHA－於30 oC累積PHA及單體組成分析….. 51

 
圖 目 錄
圖一、 R. eutropha H16 PHA合成酶於胺基酸位置D233飽和突變變異株在37 oC及42 oC的PHB累積分析…………..………………………. 52
圖二、 R. eutropha H16 PHA合成酶於胺基酸位置A466飽和突變變異株在37 oC及42 oC的PHB累積分析…………………………….…….. 53
圖三、 R. eutropha H16 PHA合成酶於胺基酸位置A493飽和突變變異株在37 oC及42 oC的PHB累積分析……………………….………….. 54
圖四、 重組PHA synthase之純化…………………………………………... 55
圖五、 野生株與突變株PHA合成酶最適反應溫度的分析……………...… 56
圖六、 各個PHA合成酶於45 oC之熱失活半衰期…………………………. 57
圖七、 野生株與變異株PHA合成酶的螢光消光實驗分析……………....... 58

 
附 表 目 錄
附表一、 PHA與聚丙烯的物理性質……..………………………………...... 59
附表二、 PHA synthase的分類……...……………………..………………… 60

 
附 圖 目 錄
附圖一、 PHA化學結構……..…………………………..………………...... 61
附圖二、 P(3HB) 的合成路徑……………………………………………… 61
附圖三、 PHA生合成之代謝路徑…………………………..……………… 62
附圖四、 中溫菌與嗜熱菌PHA合成酶之胺基酸序列比對………………... 63

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