臺灣博碩士論文加值系統

第二代生質酒精的生產是希望利用農業或廚房廢棄物中的木質纖維素提供糖做為發酵生產酒精的原料，然而，現有的纖維素水解酶，分解纖維素的效率不高是造成第二代生質酒精的價格比石油更貴的原因之一。因此，找到具有高催化活性的纖維素水解酶是必要的。證據顯示，牛的瘤胃真菌所含的纖維素水解酶具有相當高的催化活性，是高效率纖維素水解酶的良好來源。本研究的目的是分析一株選殖自瘤胃真菌的纖維素水解酶，CelC6，評估其催化活性及用於生質酒精生產的可能性。經由胺基酸序列比對預測，CelC6可能是一株內切型葡萄醣苷酶（endoglucanase, EC 3.2.1.4），屬於醣類水解家族family 9。將其選殖到pET21a載體並轉殖至大腸桿菌BL21(DE3)，發現無法使CelC6大量表達，所以再將CelC6轉殖到其他四種大腸桿菌菌株表達，分別是BL21-pLys(DE3)、Rosetta2、Rosetta2-pLys、Rosetta2 gami-pLys，但是，依然無明顯表達。我們嘗試表達CelC6的truncated forms，分別截斷CelC6 N端的transmembrane helix及C端的CBM，稱為CelC6(-1CBM)、CelC6(-3CBM)、CelC6(NT)、CelC6(NT-1CBM)、CelC6(NT-2CBM)及CelC6(NT-3CBM)，但是，依然無法在大腸桿菌中大量表達，所以，再將CelC6選殖到pMAL-c5X載體，稱為MBP- CelC6並轉殖至五種大腸桿菌表達，在上清液及沉澱物中均可發現蛋白質明顯的表達。經由amylose管柱純化出MBP- CelC6，其最佳反應條件為45 ℃，pH 7.0。耐熱性於50 ℃仍保有60 %之活性；酸鹼耐受範圍為pH 5~10。對受質β-glucan的比活性為12.62 U，與一般同類型的酵素相比，CelC6的催化活性並不高。

Production of the second generation bioethanol is to use lignocellulose from agricultural or kitchen waste as the source of fermentable sugars. However, a key problem for this strategy is the inefficiency of cellulose hydrolysis by currently available cellulases, which is one of the major reasons causing bioethanol more expensive than the petroleum. Thus, it is necessary to explore cellulases with high catalytic activities over lignocellulose. Evidence indicated that rumen fungi contain cellulase with high catalytic activities. Thus, the objective of this study is to analyze a celluase gene CelC6 cloned from a rumen fungus Orpinomyces sp.Y102. The deduced amino acid sequence of CelC6 predicted an endoglucanase belonging to glycoside hydrolases family 9. The predicted open reading frame of CelC6 was subcloned into pET21a and expressed in Escherichia coli BL21(DE3). However, there was no obvious expression of CelC6 protein in this strain. Therefore, the expression of CelC6 was tested in another four E.coli strains, i.e. BL21-pLys(DE3), Rosetta2, Rosetta2-pLys and Rosetta2 gami-pLys. Consequently, there was still no obvious expression in these strains. Thus, the expression of truncated forms of CelC6 were tried., However, there was still no obvious expression in these strains. Thus, CelC6 was subcloned into pMAL-c5X and expressed in five E.coli strains, designated as MBP-CelC6. Consequently, the expression of CelC6 was obvious. Thus, MBP-CelC6 was purified and characterized for substrate specificity, thermal stability and pH tolerance. The optimal reaction conditions of CelC6 were 45℃ and pH 7.0. The thermal stability for CelC6 was at 50 ℃, and 60 % activity was remained. The pH stability from pH 5 to pH 10. The specific activity for β-glucan was 12.62 U. The activity of CelC6 was found lower than other the same enzymes.

目錄
中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 X
第1章 前言 1
第2章 文獻回顧 2
2.1 化石燃料概況及替代性能源 2
2.2 生質能源與第二代生質酒精 2
2.2.1生質能源 2
2.2.2第二代生質酒精 2
2.3纖維素的結構 3
2.4 纖維素水解酶的分類及作用機制 5
2.4.1 醣類水解酶家族(glycoside hydrolase family) 5
2.4.2 纖維素水解酶 6
2.4.3 酵素的協同作用 7
2.5 醣類結合模組(CBM) 8
2.5.1醣類結合模組(CBM)之分類 8
2.5.2 CBM的功能 9
2.6纖維素水解酶的選殖與研究 10
2.6.1纖維素水解酶的來源 10
2.6.2 分析纖維素水解酶活性的方法 11
2.6.3纖維素水解酶活性分析所使用之各類受質 (substrates) 11
2.6.3.1 可溶性受質(soluble substrates) 12
2.6.3.2 不可溶性受質(insoluble substrates) 13
2.7 纖維素水解酶之應用 14
2.8研究目的與架構 15
第3章 材料與方法 17
3.1 實驗材料 17
3.1.1 微生物菌株 17
3.1.2 載體 17
3.1.3 聚合酶連鎖反應之引子(委託生工有限公司合成) 18
3.1.4 大腸桿菌培養基 18
3.1.5 試劑及緩衝溶液 19
3.1.5.1 5X TBE (用於DNA電泳) 19
3.1.5.2 5X gel loading dye (用於DNA電泳) 19
3.1.5.3 DNS試劑 (用於纖維素水解酶活性測試) 19
3.1.5.4蛋白質分析用試劑 20
3.1.6 分子量標記 (molecular weight markers) 22
3.1.7 蛋白質電泳分析膠體 22
3.1.7.1 3 %焦集膠 (stacking gel) 22
3.1.7.2 10 %分離膠(separating gel) 22
3.1.8 抗體 23
3.1.9 其他化學藥品 23
3.2 實驗方法 24
3.2.1 CelC6之次選殖(subcloning) 24
3.2.1.1 pTrip1EX2/ CelC6質體之小量純化 24
3.2.1.2 pTrip1EX2/ CelC6質體之洋菜膠電泳分析 24
3.2.1.3 CelC6 基因之擴增與純化 25
3.2.2 表現載體(pET21a及pMAL-c5X)之製備 25
3.2.2.1 質體(pET21a及pMAL-c5X)之小量純化 25
3.2.2.2 限制酶反應與純化 26
3.2.3 CelC6基因與表現載體之接合反應(ligation) 26
3.2.4 CelC6基因轉型至E.coli DH5α 26
3.2.5CelC6基因轉型至E.coli各種菌株 26
3.2.6 CelC6(truncated forms)之次選殖 27
3.2.7 CelC6以IPTG誘導表現之最適時間點測試 27
3.2.8 蛋白質定量 28
3.2.9 以聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electerophoresis, SDS-PAGE)分析CelC6之表達情形 28
3.2.10 西方墨點法(Western blot) 28
3.2.11 CelC6誘導表現之最適溫度測試 29
3.2.12 CelC6之大量表達 30
3.2.13以DNS比色法分析酵素活性 30
3.2.14以離子交換樹脂管柱(DEAE-sepharose)純化CelC6 31
3.2.15 以親和性管柱(amylose)純化CelC6 31
3.2.16 CelC6最適反應溫度測試 32
3.2.17 CelC6最適反應pH值測試 32
3.2.18 CelC6之熱穩定性測試 32
3.2.19 CelC6之酸鹼穩定性測試 32
3.2.20 CelC6之受質專一性分析 33
第4章 結果 34
4.1 CelC6的結構分析與表現 34
4.1.1 CelC6的結構分析 34
4.1.2 CelC6-6xHis在E.coli之表達情形 35
4.2CelC6 truncated forms 在E.coli之表達情形 38
4.3 MBP-CelC6在E.coli之表達情形 43
4.3.1 MBP-CelC6初步表達情形 43
4.3.2 MBP-CelC6 的最佳誘導條件 46
4.4 MBP-CelC6的純化 48
4.5 CelC6生化特性分析 51
4.5.1 CelC6最適反應溫度 51
4.5.2 CelC6最適反應pH 52
4.5.3 CelC6熱穩定性測試 53
4.5.4 CelC6酸鹼耐受性測試 54
4.5.5 酵素受質專一性分析 55
第五章 討論 56
第六章 結論 61
參考文獻 62
作者簡介 71

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