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研究生:葉幸兒
研究生(外文):Xing-Erh Yeh
論文名稱:利用基因重組大腸桿菌使用澱粉生產聚羥基烷酯之探討
論文名稱(外文):Production of polyhydroxybutyrate (PHB) using starch as substrate by genetic engineered Escherichia coli
指導教授:簡志青
指導教授(外文):Cih-Ching Chien
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:60
中文關鍵詞:澱粉酶澱粉酶澱粉酶澱粉酶澱粉酶澱粉酶
外文關鍵詞:polyhydroxybutyratealpha-amylasegene recombinantcloningEscherchia coli gene expression
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聚羥基烷酯 polyhydroxyalkanoate (PHA) 為一種由微生物所合成之生物性高分子聚合物,因它自生長環境中攝取過量的碳源在胞內累積而成的生物性高分子聚合物,而這些生物高分子經由萃取純化後,亦可應用至我們的日常生活中。在工業上大量應用時,培養基碳源的選用將是微生物生產聚羥基烷酯的最大考量因素。目前雖然已知有上百種不同的微生物可以累積聚羥基烷酯,但是在培養過程中,微生物通常無法直接運用澱粉作為產生聚羥基烷酯的碳源,其中不乏包括利用基因重組之微生物。
因此本研究之目的乃是將澱粉酶基因建構於大腸桿菌內,挑選出具有澱粉酶之大腸桿菌後,同時將合成聚羥基丁酯(polyhydroxybutyrate; PHB)的基因 (phaCAB) 轉殖入此大腸桿菌中。本研究選擇以格蘭氏陰性菌之產氣單胞菌屬的澱粉酶基因與Ralstonia eutropha H16之phaCAB為基因來源,建構可分解澱粉之重組大腸桿菌,並進一步探討這些重組菌株利用澱粉及其他不同培養基的生長情形,以及合成聚羥基丁酯的能力。
研究結果顯示,經由基因重組後之大腸桿菌在含有澱粉的培養基中,將其於 37℃ 培養72小時後,以碘液染色觀察其周圍有一圈透明環出現,確定基因轉殖大腸桿菌具有分解澱粉的能力。再加入phaCAB基因,基因重組後亦可生產少許之聚羥基丁酯,然而若僅使用澱粉則合成PHB之結果仍不甚理想,須再進一步的進行菌種改良或是培養條件之最適化探討。


Polyhydroxyalkanoates (PHA) are biopolymers synthesized by microorganism when grown under unbalanced conditions when the carbon substrate is in excess to other nutrients such as nitrogen, sulfur, phosphorus or oxygen. These bio-polymers may be able to use as nonpetroleum-based biodegradable polyesters. However, the high cost associated with production of PHA in industry is a major consideration for the production polyhydroxyalkanoates. Although there are numerous microorganisms can accumulate PHA, only few can use starch as carbon source and synthesize PHA.
Therefore, the goal of this study is to construct the recombination Escherchia coli by introducing alpha amylase gene and PHB synthesis genes in the cells inorder to obtain the recombinant strains of E. coli that are able to synthesize PHB using starch as substrate. The results showed that we have successfully cloned alpha amylase gene of Aeroomonas sp. into E. coli. We also introduced phaCAB of Ralstonia eutropha H16 into the recombinant E. coli strain. Although the recombinant strain was able to hydrolyse starch, the production of PHB using strach as carbon source by the recombinant strain is not apparent. Further investigation will be needed for the strain’s improvement.


中文摘要 i
Abstract ii
誌 謝 iii
目錄 iv
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第二章 文獻回顧 3
2.1 PHAs簡介 3
2.2 PHAs的發展歷史 3
2.3 PHAs的結構 4
2.4 PHAs的生合成途徑 5
2.5 PHAs的應用 7
2.6 產氣單胞桿菌屬 (Aeromonas hydrophila) 之介紹 7
2.7 產氣單胞桿菌屬與 α-amylase 的分泌 8
2.8 產氣單胞桿菌屬之培養特性 8
2.9 starch and α-amylase之簡介 9
2.10 α-澱粉水解酵素 (α-amylase) 在工業上之應用 10
第三章 材料與方法 11
3.1 材料 11
3.1.1 實驗儀器 11
3.1.2 實驗藥品與廠牌 12
3.1.3 培養液與培養基 14
3.1.4 抗生素與酵素 14
3.1.5 菌株與質體 15
3.2 實驗架構與流程 16
3.2.1 PHB 分析方法 18
3.2.2 菌株和質體 18
3.2.3 培養基的配方 18
3.2.4 培養流程 18
3.2.5 菌種保存與前培養 19
3.2.6 實驗用引子 19
3.3 聚合酶鏈鎖反應與基因重組質體實驗流程 19
3.3.1 抽取KC007 genome DNA 19
3.3.2 聚合酶鏈鎖反應PCR 20
3.4 重組菌株建構 (以pGEM-T為載體) 20
3.4.1 製作含有α-amylase的基因重組質體 20
3.4.2 轉型作用 20
3.4.3 小量質體DNA抽取之步驟 21
3.4.4 質體DNA限制酵素切割反應 21
3.4.5 DNA 定序 21
3.4.6 α-amylase SDS-PAGE analysis 22
3.4.7 蛋白質電泳鑄膠流程 22
3.4.8 菌體粗萃蛋白質表現之樣品製作流程 23
3.5 澱粉培養盤碘液染色測試 25
3.6 酵素活性分析(DNS呈色法) 25
3.7 單一質體含phaCAB與amylase基因之建構(pBHB2::amy) 25
3.8 重組菌株之建構 26
3.9 培養基測試 26
3.10 以澱粉做為單一碳源培養大腸桿菌菌株生產PHB 27
3.10.1 Amylase活性測試 27
3.10.2 以可溶性澱粉做為單一碳源培養 27
3.10.3 添加yeast extract對PHAs產率的影響 27
3.10.4 轉型菌株之生長濃度測定 27
3.11 以氣相層析法分析PHB產量 28
3.11.1 菌體培養及乾菌回收 28
3.11.2 PHB 標準品分析 28
3.11.3 樣品之PHB 分析 29
3.12 穿透式電子顯微鏡(TEM)觀察 30
3.12.1 TEM 樣品製備步驟 30
第四章 結果與討論 33
4.1 抽取篩選菌株KC007之細菌染色體DNA 33
4.2 α-amylase之聚合酶鏈鎖反應PCR 34
4.3 以pGEM-T為主建構含α-amylase之質體建構 35
4.4 大腸桿菌轉殖菌株之質體抽取 36
4.5 轉型菌株質體DNA限制酵素切割反應 37
4.6 重組菌之基因組分析 38
4.7 重組大腸桿菌之α-amylase SDS-PAGE蛋白質表現量 39
4.8 澱粉培養盤碘液染色測試 41
4.9 酵素活性分析(DNS呈色法) 42
4.10 建構含phaCAB與amylase基因之質體 43
4.11 質體限制酶酵素反應測試 44
4.12 重組菌株之建構 45
4.13.1 轉殖菌株之生長曲線測試一 46
4.13.2 轉殖菌株之生長曲線測試二 47
4.13.3 轉殖菌株之生長曲線測試三 48
4.14 PHB標準品之氣相層析分析 49
4.15 大腸桿菌轉型菌株之PHB GC 分析 50
4.16 大腸桿菌轉型菌株之TEM觀察 52
第五章 結論與未來展望 53
第六章 參考文獻 54
第七章 附錄 58



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