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研究生:邱逢深
研究生(外文):Feng-Shen Chiu
論文名稱:以轉殖 aldehyde reductase 與 dehydrogenase 之重組大腸桿菌改善甘油利用率並生產聚羥基烷酯
論文名稱(外文):The improvement of glycerol utilization for poly- hydroxyalkanoates production from recombinant E. coli harboring aldehyde reductase and dehydrogenase genes
指導教授:藍祺偉
指導教授(外文):Chi-Wei Lan
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:93
中文關鍵詞:PHAs;粗甘油;雙質體表現系統;乙醇生產
外文關鍵詞:PHAscrude glyceroldual-plasmidethanol production
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隨著科學技術與工業經濟不斷進步,人們對於各項物質的需求提高,同時也帶來無法彌補的環境傷害,包括石化合成的塑膠的大量生產,因性質穩定萬年不化,造成嚴重的環境汙染。因此,替代性塑料因而被開發,其中最受矚目的是聚羥基烷酯。聚羥基烷酯 ( Polyhydroxyalkanoates, PHAs ) 是微生物中在營養不帄衡的條件下,將多餘的碳源轉換成脂質累積於胞內作為能量儲存或冺用之聚合物,因其物理性質近似石化塑膠,且具有生物可分解性與相容性;若將之量產化,以醱酵培養操作則是最有效且快速方法,但碳源成本佔醱酵原料成本中最重要的部分。目前,世界各國極力發展生質柴油,使得其產量大幅上升,但製造過程之轉酯化步驟,會產生粗甘油此副產物,其價格低廉且甘油比例佔絕大多數。因此,本研究希望藉由Escherichia coli 原生菌株之優勢,藉由基因重組方式提高粗甘油冺用率並轉化為PHA,不僅減少粗甘油對於環境汙染更能提升生質柴油的附加價值。
本研究使用雙質體重組菌株,帶有Aldehyde reductase與Aldehyde dehydrogenase二粗甘油冺用酵素與PHA合成酵素PhaCAB,使得E. coli 原生菌株增加對粗甘油冺用效率以及合成PHB。培養結果顯示雙質體重組菌株之粗甘油代謝率可達90 % 以上,相較於野生型E. coli ( WT ) 菌株之粗甘油冺用率,增加近2倍之代謝率,且具有29 % ( w/w ) 以上之PHB累積量。於耗氧與厭氧培養之比較,雙質體表現菌株培養於耗氧環境中,其粗甘油冺用效率與PHB累積含量明顯優於厭氧環境;但於厭氧環境中,其粗甘油冺用效率與PHB累積含量一樣優異於野生型E. coli BL21菌株之表現。而醱酵後的培養液經HPLC分析,發現有未知波峰出現,再藉由GC-MS定性比對,二結果皆證明醱酵後培養液含有乙酸、乙醇以及其他醇類產生,且重組E. coli ( dual-plasmid )菌株之乙醇產量約有0.8 g L-1,進而推斷重組E. coli ( dual-plasmid ) 菌株之乙醇產生的可能路徑。

Polyhydroxyalkanoates ( PHAs ) can be synthesized from numbers of bacteria and is characterized as biodegradable polymer with similar property to those petrochemical plastics. PHAs contain a variety of polymer properties based on the carbon chain length of the combination of functional groups. It is formed under the specific conditions including excess of carbon supply or limitation of nutrient sources such as nitrogen, phosphorus, sulfur or oxygen. The development of PHAs production has drawn much attention on fermentation strategies, strain development as well as reduction of production cost by substitution of low cost of nutrients ( ex. crude glycerol、whey and beet ). In which, crude glycerol is by-product from biodiesel transesterification process and can be applied as carbon source to produce chemicals in a fermentation process. The glycerol can be used as carbon supply for PHAs production E. coli but low utilization and conversion of glycerol became a key to success of such manner. In this study, the recombinant E. coli BL-21 harvested pARD33 and pBHB2 had developed. By using a dual-plasmid expression system not only provide an alternative pathway of glycerol metabolism but also improved synthesis of PHAs production under two-stage genetic regulation. This strategy can accelerate the efficiency of glycerol utilization as well as increasing PHAs formation. In addition, it was found the ethanol was produced in a concentration of 0.8 g L-1 by inducible Alrd and AldH in recombinant E. coli.

摘要 I
Abstract II
致謝 III
目錄 IV
表目錄 VIII
圖目錄 X
第一章 緒論 1
1.1前言 1
1.2生物可分解性塑膠種類 3
1.2.1天然生物可降解塑料 3
1.2.2化學合成生物可降解塑料 3
1.2.3化學合成添加可降解塑料 4
1.2.4微生物合成可降解塑料 4
1.2.5可降解塑料之比較 5
1.3聚羥基烷酯介紹 6
1.3.1聚羥基烷酯 ( Polyhydroxyalkanoates, PHAs ) 6
1.3.2聚羥基烷酯性質 7
1.3.3聚羥基烷酯的代謝途徑 9
1.3.3.1 PHA代謝路徑I 9
1.3.3.2 PHA代謝路徑II 9
1.3.3.3 PHA代謝路徑III 10
1.3.4聚羥基烷酯的應用 11
1.4粗甘油 ( Crude glycerol ) 11
1.4.1 生質柴油介紹 11
1.4.2 粗甘油介紹 13
1.5研究動機與策略 14
1.5.1研究動機 14
1.5.2研究策略 15
第二章 雙質體表現系統構築與優勢菌株篩選 19
2.1前言 19
2.2 研究策略 19
2.3 Alrd與 AldH介紹 21
2.3.1 Aldehyde reductase 21
2.3.2 Aldehyde dehydrogenase 21
2.4表現載體 pET-23a、pBAD33與pBHB2介紹 21
2.4.1本研究表現載體pET-23a 21
2.4.2本研究表現載體pBAD33 22
2.4.3 PHB合成載體 pBHB2 24
2.5 質體構築流程 24
2.6 本章使用之菌株、藥品與儀器設備 27
2.6.1 菌株 ( strains ) 與質體 ( plasmids ) 27
2.6.2 培養基 28
2.6.3藥品與儀器設備 29
2.6.3.1藥品材料 29
2.6.3.2 儀器設備 31
2.7本章實驗方法與步驟 32
2.7.1菌株儲存 32
2.7.2菌株使用之前培養與主培養 32
2.7.3 R. eutropha H16 Genomic DNA 純化 32
2.7.4 Alrd與AldH引子設計 33
2.7.5 Alrd與AldH之酵素基因取得 33
2.7.6 目標基因藉洋菜膠體電泳分析與純化 35
2.7.7目標基因之膠體萃取與純化 36
2.7.8勝任細胞製備 36
2.7.9目標基因與TA vector 接合作用 37
2.9.10 TA產物轉型至勝任細胞 37
2.7.11目標基因定序 38
2.7.12 Plasmid DNA 純化方法 38
2.7.13限制酵素反應 39
2.7.14 mRNA純化方法 39
2.7.15目標蛋白基因之mRNA確認 40
2.7.16目標蛋白基因之酵素活性測試 41
2.7.16.1 總蛋白濃度測定 41
2.7.16.2 目標蛋白活性測定 42
2.7.17 SDS聚丙烯醯胺膠體電泳法 ( SDS-PAGE ) 43
2.7.18菌株培養 45
2.7.18.1 菌株前培養 45
2.7.18.2 主培養 45
2.7.18.3 厭氧培養 45
2.7.19 粗甘油定量分析 46
2.7.20 乙酸定量分析 47
2.8 結果與討論 48
2.8.1 重組菌株構築 48
2.8.1.1 alrd 與 aldH基因取得 48
2.8.1.2 alrd和aldH 二基因構築之質體確認 49
2.8.1.3 pARD23與pARD33重組質體之目標基因cDNA確認 49
2.8.1.4目標蛋白之SDS-PAGE確認 50
2.1.8.5目標蛋白酵素活性測試 51
2.1.8.6 E. coli ( pARD23 ) 與E. coli ( pARD33) 之粗甘油利用分析 52
2.8.2 雙質體構築 54
2.8.2.1 pARD33與pBHB2質體確認 54
2.8.2.1 E. coli ( dual-plasmid ) 之目標蛋白活性測試 54
2.9本章結論 55
第三章 甘油利用率和PHB合成比較 56
3.1前言 56
3.2樣品分析方法 57
3.2.1聚羥基丁酯定量分析 57
3.2.2乙醇定量分析方法 59
3.3使用之藥品與儀器設備 60
3.3.1使用藥品整理 60
3.3.2 儀器設備 61
3.4 結果與討論 62
3.4.1 Alrd與AldH於粗甘油利用效率 62
3.4.3 誘導與未誘導探討 64
3.4.4 誘導時間與培養溫度探討 67
3.4.5 厭氧培養探討 70
3.4.6 醱酵槽培養 72
3.4.7 其他產物分析 74
3.4.7.1 高效能液相層析儀分析 74
3.4.7.2 氣相層析質譜分析儀分析 76
3.5 本章結論 82
第四章 結論與未來工作規劃 83
4.1 結論 83
4.2 未來工作規畫 83
附件一 Aldehyde reductase基因定序結果 84
附件二 Aldehyde reductase 蛋白定序結果 86
附件三 Aldehyde dehydrogenase基因定序結果 87
附件四 Aldehyde dehydrogenase蛋白定序結果 90
參考文獻 91


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