臺灣博碩士論文加值系統

因應全球石油危機，全世界都在尋找其他能源。而其將纖維素(cellulose)醣化後再發酵成酒精，可以產生生質能源。且纖維素廣泛的存在自然界的植物細胞壁中，其含量龐大、價格便宜、含糖量豐富、且非糧食作物，是再生能源最佳的原料之一。但缺點是自然界中的纖維素被層層包覆在木質纖維素(lignocellulose)複雜的結構中，使得纖維素不易裸露在外，而不易被纖維素酵素(cellulase)水解。此外還需要木聚醣酵素(xylanase)不等的各種酵素合作，先分解纖維素原料外層複雜的結構，使得纖維素結構能露出，而能被纖維素酵素水解，藉此提升分解複雜木質纖維素原料的效率。而要再把纖維素完整醣化成葡萄糖也需要endo- glucosidase和exo-glucosidase等纖維素酵素，大量酵素的使用和繁複的步驟，使得花費相當龐大而不符合成本效益。先前研究發現利用來自微生物Clostridium thermocellum的纖維素水解酵素複合體(cellulosome)的結構，可以同時聯合不同纖維素酵素在一個支架蛋白上，使酵素間的距離接近而產生的鄰近加成作用，使木質纖維素水解效率提升。纖維素水解酵素複合體分為支架蛋白 (scaffolding protein)和各式木質纖維素水解酵素兩個部分；所以本實驗選擇纖維素水解酵素複合體中的六個木質纖維素水解酵素相關基因以及兩個支架蛋白相關基因，透過OGAB(Ordered Gene Assembly in Bacillus subtilis)方法，分別構築並表現在Bacillus subtilis中，探討支架蛋白和水解酶之間的關係以及其酵素活性分析，期望利用纖維素水解酵素複合體分解並糖化木質纖維素，以提升木質纖維素之糖化水解效果。

The structure complexity of lignocellulose makes it recalcitrant to be hydrolysised. To enhance the efficiency of cellulose degradation is a crucial issue for futher application. It has been discovered that cellulosome could enhance the efficiency of cellulose degradation through cellulases assembling on scaffolding protein and their synersm. Though Clostridium thermocellum could produce cellulosome, its anaerobic culturing condition is difficult to handle for application. According to a proteome-wide analysis of Clostridium thermocellum ATCC27405, eight cellulosomal genes including two exo-glucosidase genes (celK and celS), two endo-glucosidase genes (celA and celR), two xylanase genes (xynC and xynZ), one scaffolding protein gene (cipA) and one anchoring protein gene (sdbA) were cloned and co-expressed in Bacillus subtilis WB800 by a method named ordered gene assembly in Bacillus subtilis (OGAB) to produce designer cellulosome in order to exploit the potential for enzyme industry. In addition, to investigate cellulose-scaffolding protein interaction, scaffolding protein gene and anchoring protein gene were constructed together in one strain, and each cellulase gene mentioned above was constructed in each B. subtillis strains. Those strains were cultured and the secreted proteins were mixed in different ratio for enhancing cellulose hydrolysis activity.

第一章、前言 1
第ㄧ節、生質能源 2
第二節、木質纖維素 2
第三節、木質纖維素分解酵素 3
第四節、纖維素酵素複合體(cellulosome) 7
第五節、Clostridium thermocellum 8
第六節、Bacillus subtilis 9
第七節、合成生物學 9
第二章、研究目的、策略及架構 13
第一節、研究目的 14
第二節、研究策略 14
一、基因重組宿主的選擇策略─Bacillus subtilis WB800 14
二、木質纖維素水解酵素生產基因群之選擇策略 15
第三節、研究架構 16
第三章、研究材料及方法 17
第一節、研究材料 17
ㄧ、菌種與質體 17
二、藥品及酵素 17
第二節、研究方法 17
一、培養基配製 17
二、試劑與緩衝溶液 19
三、DNA之製備 21
四、質體之構築與選殖（cloning） 24
五、Ordered Gene Assembly in Bacillus subtilis (OGAB) method 26
六、轉殖基因結果確認 30
七、表現蛋白確認 32
八、纖維素分解酵素複合體活性分析 37
第四章、研究結果與討論 39
第一節、製備所需Cellulosome相關基因片段 39
一、cipA基因的製備與鑑定： 39
二、sdbA基因的製備與鑑定： 41
第二節、分開構築基因並以B. subtillis WB800為宿主表現 42
一、表現支架蛋白及錨蛋白基因 42
二、表現單一纖維素水解酵素之相對應基因 43
第三節、表現蛋白的產出及功能性確認 44
一、單一纖維素水解酵素及支架蛋白的產出。 44
二、單一纖維素水解酵素及支架蛋白的功能性確認。 47
第四節、收集蛋白並分析比較各別酵素活性 49
一、表現單一纖維素水解酵素之菌株的胞內及胞外酵素活性分析 49
二、培養不同時間對酵素活性的影響 51
三、cellulosome之支架蛋白及纖維素分解酵素的交互作用分析 54
第五章、結論與未來展望 61
第六章、參考文獻 62
附錄一 67
1. 支架蛋白基因cipA以blast與原始菌株基因序列比對結果 67
2. 支架蛋白基因cipA以blastx與原始菌株胺基酸序列比對結果 72
3. 錨蛋白基因sdbA以blast與原始菌株基因序列比對結果 73
4. 製備錨蛋白基因sdbA所使用引子 75
5. 確認pGETS118質體所使用引子 75
附錄二 76
1. 5th International Conference on Industrial Bioprocesses與會海報 76
2. The ASM 115th General Meeting: Fermentation and Biotechnology與會海報 79

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