臺灣博碩士論文加值系統

為解決能源危機，各國都在努力開發各種替代能源，其中，生質能源特別有其開發價值，因為全球利用光合作用所生成的生物質量(Biomass)就有1,730億噸，且利用率僅不到10%。以台灣為例，一般可用於再生能源生產的生物質量多為稻草、雜草、廢木料等等，這些農業廢棄物每年都會產生，但目前的處理方式多為燃燒，浪費了這些生物質量，還會造成環境汙染，若能轉換為生質能源，將不啻為一解決問題的方法。天然的纖維素被包覆在結構複雜的木質纖維素（lignocellulose）之中，若僅透過纖維素分解酵素（cellulase）將其分解，須花費較長的時間，因此還得與木聚醣酵素（xylanase）等各種不同功能的酵素合作才能減少分解時間。其機制為：先分解外層的木質纖維素，使內部纖維素裸露出來，再藉endo-glucosidase和exo-glucosidase等纖維素分解酵素的水解，將纖維素醣化為葡萄糖。在先前的研究中指出，來自Clostridium thermocellum的纖維素水解酵素複合體（cellulosome），可以將纖維素水解酵素結合在一個支架蛋白上，使酵素的距離拉近而產生加乘作用，讓水解效率提升。該複合體的結構分為兩部分，其一為支架蛋白（scaffolding protein），其二為各種水解酵素。而本實驗從中選擇三個水解酵素及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上作為研究，探討支架蛋白與水解酵素的關係及不同組合與作用時間對效率的影響，期望透過纖維素水解酵素複合體分解木質纖維素，以提升生物質量的利用進而減緩能源危機的發生。

本研究由蛋白活性測試發現，木質纖維素水解酵素若單獨使用時，cipA對xynC的結合效率高於celA與celK，所以xynC在有cipA時效果較佳；另外也發現，celA、celK與xynC似乎彼此有交互作用，若同時用於木質纖維素水解，比單一酵素的效率高出三至六倍。

The possibility of exhaustion of fossil fuels has drawn global attention to the issue of exploring renewable alternatives to fossil fuel. Lately, various renewable energy sources have been offered as alternatives to petroleum, such as bioethanol. It is a biofuel produced from grasses, wood, algae, or other plants. Lignocellulosic biomass is the non-edible portion of the plant, which is underutilized, but could be used for biofuel production. The structure complexity of lignocellulose makes it recalcitrant to be hydrolysised. How 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 interact with cellulose. The cellulose hydrolysis efficiency could be increased by proximity synergy of cellulases. Though Clostridium thermocellum could produce cellulosome, but the culturing condition should be anaerobic and that it’s difficult to handle for application. According to a proteome-wide analysis of Clostridium thermocellum ATCC27405, five cellulosomal genes including one exo-glucosidase gene (celK), one endo-glucosidase gene (celA), one xylanase gene (xynC), 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. To instigate cellulose interaction, cellulosome were mixed in different way and different time 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 4
第四節、纖維素酵素複合體(cellulosome) 7
第五節、Clostridium thermocellum 9
第六節、Bacillus subtilis 9
第七節、合成生物學 10
第二章、研究目的、策略及架構 14
第一節、研究目的 15
第二節、研究策略 15
一、基因重組宿主的選擇策略─Bacillus subtilis WB800 15
二、木質纖維素水解酵素生產基因群之選擇策略 16
第三節、研究架構 18
第三章、研究材料及方法 17
第一節、研究材料 18
ㄧ、菌種與質體 18
二、藥品及酵素 18
第二節、研究方法 18
一、培養基配製 18
二、試劑與緩衝溶液 21
三、DNA之製備 22
四、質體之構築與選殖（cloning） 25
五、Ordered Gene Assembly in Bacillus subtilis (OGAB) method 27
六、表現蛋白確認 31
七、纖維素分解酵素複合體活性分析 33
第四章、研究結果與討論 35
第一節、Cellulosome相關基因片段與使用之基質 35
第二節、收集蛋白並分析比較各別酵素活性 36
一、表現單一纖維素水解酵素與菌液之酵素活性比較 37
二、表現混和纖維素水解酵素之菌株的酵素活性比較 39
三、酵素混和液與單一酵素之效率比較 41
四、酵素混和液與支架蛋白的交互作用分析 43
五、單一酵素與混和酵素之效率分析 45
六、以稻稈為基質探討木質纖維素水解酵素之效率 47
第五章、初步結果與未來展望 53
第六章、參考文獻 54

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