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研究生:謝依芸
研究生(外文):Yi-Yun Hsieh
論文名稱:蔗渣堆肥巨基因庫之新穎纖維素分解酶與脂肪分解酶基因的選殖與特性分析
論文名稱(外文):Cloning of a novel cellulase and lipase gene from bagasse compost metagenome and characterizations of their gene products
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming-Chung Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2009
畢業學年度:97
語文別:中文
論文頁數:135
中文關鍵詞:纖維素分解酵素生質酒精脂肪分解酵素巨基因庫分泌性轉脂化生質柴油
外文關鍵詞:BioethanolBiodieselCelluloseLipaseMetagenomic LibrarySecretionTransesterification
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隨著環保意識的抬頭與全球性的石油危機,生質能源逐漸受到世界矚目,主要因其原料多樣,對環境之衝擊低,使得生質能源成為最具潛力的替代性能源。生質能源主要分為生質酒精 (bioethanol) 與生質柴油 (biodiesel) 兩種能源形式,本實驗室分別針對參與於兩種生質燃料產程之關鍵酵素來進行研究,希望能尋找到於基因序列及酵素反應特色上皆具新穎性之纖維素分解酵素 (cellulase) 與脂肪分解酵素 (lipase),進而對生質能源之產程有所貢獻。藉由存在蔗渣堆肥中具有纖維素與脂肪分解分解活性潛力之細菌群聚建立其巨基因庫 (metagenome),分別透過序列性的篩選以菌落雜交法 (colony hybridization) 自巨基因庫中篩選出一屬於 GHF 6 (glycosyl hydrolase family 6) 之新穎纖維素分解酵素基因 YYH-1,以及透過功能性的篩選法自巨基因庫中篩選出具活性之新穎脂肪分解酵素基因 Lip2。進而分析 YYH-1,其基因大小共 1221 bp 且可轉譯出 407 個胺基酸,蛋白質約為 44.77 kDa,胺基酸序列中具 GHF 6 保留性序列;YYH-1 之粗酵素萃取液在 pH 6.5 下,於 55℃ 對於 β-1,4-glucan 結構之醣類具有較佳分解能力、於 45℃ 對於 p-Nitrophenyl 醣類衍生物具有較弱的分解能力,推測 YYH-1 蛋白屬於內切性纖維素分解酵素 (endoglucanase)且其最佳酵素反應條件同於蔗渣堆肥環境,推測未來可將 YYH-1 應用於各類農業廢棄物堆肥;另在分泌性分析中則顯示,YYH-1 蛋白可被分泌到培養液當中,進而預測其訊息胜肽,預測在 YYH-1 蛋白之 N 端之第 28 與第 29 個胺基酸或第 31 與第 32 個胺基酸可能具有訊息胜肽酶 (signal peptidase) 作用的位置,推測 YYH-1 蛋白因此具有良好的外泌能力,故 YYH-1 在基因序列、酵素特性與外泌特色上皆具新穎性,推測 YYH-1 未來可作用於稻桿或玉米桿之堆肥中進行纖維素的分解。另分析 Lip2,其基因大小共 879 bp 且可轉譯出 293 個胺基酸,蛋白質約為 32.23 kDa,胺基酸序列中具 esterase-lipase 保留性序列及 GXSXG 保留性模體 (motif);將 Lip2 基因利用 pET 蛋白質表現系統進行表現與純化,進而針對其酵素特性進行分析,結果顯示 Lip2 蛋白在 pH 8.0、45~65℃ 下具最佳反應溫度及 pH 值,並且對於 C2、C4、C6、C8 及 C14 之 p-Nitrophenyl 酯質衍生物具有較佳分解能力,推測 Lip2 未來可作用於中長鏈油脂之生質柴油轉脂化反應 (transesterification)。期望此研究能對生質酒精與生質柴油之製程與農業廢棄物及各類油脂來源的再利用有所貢獻。
The global petroleum crisis awakes human to concern about an exhaustive problem that energy will soon be insufficient. Hence the development of substitute energy becomes a newly anxious and worldwide subject. In all kinds of substitute energy, to use biomass derivatives as source for energy production is more environmental friendly than others. The biomass mainly takes the crops and the agricultural residues as raw materials and exhibits lower environment impacts. Biofuel composes two major types: bioethanol and biodiesel. We focused on key enzymes involved in bioethanol and biodiesel production, such as cellulases and lipases. In this study, we established microbial metagenomic library from bagasse compost and isolated two clones containing a novel glycosyl hydrolase family 6 (GHF 6) homologous partial sequence using colony hybridization. Subclone this gene revealed that both two clones composed of the same 1.5 kb DNA fragment, in which encoded a protein with cellulase activity (named as YYH-1). Amino acid sequences alignment showed YYH-1 contained a GHF 6 conserved domain sharing 56% similarity to those of cellulases belonged to this family. The molecular weight of YYH-1 was about 44.7kDa and its optimal enzyme activities reached under pH 6.5 and 45-55°C which is similar to the condition in bagasse compost. In substrate specificities of YYH-1, a wide range of polysaccharides including β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides could be hydrolyzed into sugars. In addition, we found that YYH-1 can be secreted into cultured medium of E. coli and also exhibited its cellulase activity. In signal peptide prediction, there were two putative cleavage sites at 28th or 31th amino acid of YYH-1. To study the extracellularly secreted mechanism of YYH-1 was undertaken. As for another section, we also isolated a clone with lipase activity that contained a lipase gene (named as lip2). The molecular weight of Lip2 is 32.3 kDa and contained an esterase-lipase conserved domain and a GXSXG motif. Expression and purification of recombinant Lip2 in E. coli using pET expression vector was performed. We determined the Lip2 optimum activity occurred at pH 8.0 and 45-65°C. Additionally, Lip2 could hydrolyze preferentially carbonated p-nitrophenyl esters, especially the C2, C4, C6, C8 and C14 ones. For all, it is expected that YYH-1 and Lip2 could be utilized in the fiber digestion in compost of rice and corn stalks and transesterification of middle or long chain lipid. It is anticipated that the research would contribute to bioehtanol and biodiesel production and recycle of agriculture waste and various lipid source.
中文摘要.........................................I
Abstract.........................................III
誌謝.............................................V
目錄.............................................VII
圖表目錄.........................................XI
縮寫表...........................................XII

第一部分、
蔗渣堆肥巨基因庫之新穎纖維素分解酶基因的選殖與特性分析..................................................1
第一章、緒論........................................2
  一、世界能源發展現況.............................2
  二、生質酒精 (Bioethanol)........................3
  三、木質纖維素 (Lignocellulose)..................4
  四、生質酒精的產程...............................5
  五、纖維素分解酵素 (Cellulase)...................6
  六、具有纖維素分解酵素活性的生物.................8
  七、纖維素分解酵素於醣苷水解酵素 (Glycosyl hydrolase)
中的分類.....................................8
八、巨基因庫 (Metagenomic library 或稱 metagenome)9
九、大腸桿菌的分泌系統...........................10
十、實驗目標 (Aim).............................. 12

第二章、材料與方法..................................13
一、巨基因庫的建立...............................13
1-1. 所使用的菌株、載體與培養基................13
1-2. 剛果紅染色試驗 (Congo Red test)...........18
1-3. 嵌入DNA (Insert) 的備製...................18
1-4. 載體 (Vector) 的備製......................22
1-5. 巨基因庫 (metagenomic library) 的建立與完整度
評估...........................................23
二、纖維素分解酵素的篩選與次選殖.................27
2-1. 菌落雜交法 (Colony hybridization).........27
2-2. 次選殖 (Subcloning).......................29
三、纖維素分解酵素的表現與特性分析...............32
3-1. 纖維素分解酵素的備製......................32
3-2. 蛋白質濃度定量............................35
3-3. Zymogram..................................36
3-4. 纖維素分解酵素活性分析....................39
3-5. SDS-PAGE 蛋白質電泳分析...................45
3-6. 西方墨點法 (Western blotting).............47

第三章、實驗結果....................................49
一、巨基因庫的建立...............................49
1-1. 建立甘蔗渣堆肥微生物群之巨基因庫..........49
1-2. 基因庫完整度之評估........................49
二、自巨基因庫中篩選新穎之纖維素分解酵素.........50
2-1. 序列性篩選屬於 GHF 6 的纖維素分解酵素.....50
2-2. 1-12 轉殖株之序列分析.....................51
三、以次選殖分離出 1-12 轉殖株中具纖維素分解活性之DNA
片段.........................................52
3-1. Zymogram 偵測 1-12 之纖維素分解酵素活性並預測
其基因大小................................52
3-2. 1-12 之第一次次選殖.......................52
3-3. 1-12 之次選殖株胺基酸序列的預測與分析.....53
四、誘導 YYH-1 蛋白表現..........................53
4-1. YYH-1 以 pET21a 系統進行表現..............53
4-2. YYH-1 之序列分析與演化特性................54
4-3. YYH-1 的表現與純化........................55
五、YYH-1 之特性分析.............................55
5-1. 最佳反應溫度與 pH 值......................55
5-2. 受質特異性之分析..........................56
六、YYH-1 之外泌性分析...........................56
6-1. 外泌特性..................................57
6-2. 訊息胜肽之預測............................58

第四章、實驗討論....................................59
一、篩選新穎纖維素分解酵素.......................59
二、序列性篩選屬於 GHF 6 的纖維素分解酵..........59
三、YYH-1 之序列分析.............................59
四、YYH-1 的表現與純化...........................60
五、YYH-1 之外泌性分析...........................61
六、YYH-1 之活性、新穎性與應用展望...............61

第五章、實驗圖表....................................89

第二部分、蔗渣堆肥巨基因庫之新穎脂肪分解酶基因的選殖與特性分
析..................................................64

第一章、緒論........................................65
一、生質柴油 (biodiesel).........................65
二、生質柴油的產程...............................66
三、脂肪分解酵素 (lipase)........................67
1. 脂肪分解酵素的催化反應......................67
2. 脂肪分解酵素的催化特異性....................68
3. 脂肪分解酵素的結構特性......................69
4. 脂肪分解酵素在生質柴油上的使用..............71
四、實驗目標 (Aim)...............................73

第二章、材料與方法..................................74
一、大範圍篩選具有脂肪分解酵素活性的菌株.........74
1-1. 所使用的培養基..................................................74
二、脂肪分解酵素活性的測試與次選殖...............76
2-1. pH STAT 法................................76
2-2. 次選殖....................................77
三、脂肪分解酵素的表現與特性分析.................79
3-1. p-NP- 酯類衍生物比色法 (p-NP-derivatives
colorimetric assay).......................79

第三章、實驗結果....................................83
一、自巨基因庫中篩選新穎之脂肪分解酵素...........83
1-1. 功能性的篩選具有活性的的脂肪分解酵素......83
1-2. Lip2 轉殖株之序列分析.....................83
二、以次選殖分離出 Lip2 轉殖株中具脂肪分解活性之 DNA
片段.........................................84
2-1. Lip2 轉殖株之第一次次選殖.................84
2-2. Lip2 之次選殖株胺基酸序列的預測與分析.....85
三、誘導 Lip2 蛋白表現...........................85
3-1. Lip2 以 pET21a 系統進行表現...............85
3-2. Lip2 之序列分析...........................86
3-3. Lip2 的表現與純化.........................86
四、Lip2 之特性分析..............................87
4-1. 最佳反應溫度與 pH 值......................87
4-2. 不同鏈長之受質特異性分析..................87

第四章、實驗討論....................................88
一、功能性的篩選具有活性的的脂肪分解酵素.........88
二、Lip2 的表現與純化............................88

第五章、實驗圖表....................................89

參考文獻............................................112

附錄................................................118

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