請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/23844
標題: Bacillus licheniformis CFFD2分離株之內切型纖維素分解酶基因的選殖及異源表現
Cloning and heterologous expression of the endoglucanase gene of Bacillus licheniformis CFFD2
作者: 王芝翔
Wang, Chih-Hsiang
關鍵字: cellulose
Bacillus licheniformis CFFD2
Bacillus licheniformis CFFD2
Clostridium xylanolyticum Ter3
Clostridium acetobutylicum
內切型纖維素分解酶Clostridium xylanolyticum Ter3
Clostridium acetobutylicum
出版社: 生命科學系所
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摘要: 因應石化燃料所引發的能源危機及環境污染等全球性問題,各國學者無不傾注心力地開發乾淨、零污染的替代能源,其中生質能更是這十幾年來的研發重點,致使生質能技術在短短十年內就歷經四代的演變。僅管如此,以木質纖維素為原料的第二代生質能技術仍是現今的主力,其原因就在於木質纖維素含有豐富的碳水化合物,來源也相當多樣化。木質纖維素是目前被公認用來生產乙醇、氫氣等生質能最好的再生性原料,主要成分包含纖維素、半纖維素和木質素。由於木質纖維素整體結構緊實,分解時需要多種酵素共同作用,包括纖維素分解酶、半纖維素分解酶和木質素分解酶,這些酵素多半來自微生物,但是每種微生物所產生的分解酵素種類和活性強弱不一,若要將木質纖維素做有效的分解利用,常需將具有不同分解能力的菌種和生產菌進行共培養,如此會產生菌種間競爭養分和空間而影響生產效率的問題,最佳解決之道就是讓一株好的生產菌同時具有強的分解能力。本研究擬從實驗室前人分離到具有糖化纖維素能力的Bacillus licheniformis CFFD1、CFFD2及CFFD4挑選一株分解纖維素能力較強的菌種,將其纖維素分解酶基因選殖到分解半纖維素能力較好也兼具產乙醇和氫氣的Clostridium xylanolyticum Ter3或C. acetobutylicum表現,補強兩菌種所缺乏的纖維素分解能力。從纖維素分解酶活性的定性和定量試驗發現Bacillus licheniformis CFFD2具有較強的內切型纖維素分解酶( CMCase ) 活性。將選殖B. licheniformis CFFD2之內切型纖維素分解酶基因( bglC )所獲得的重組質體pBglC送入E. coli DH5a之後,發現不論是培養於含有CMC、濾紙還是天然木質纖維素(蔗渣、稻桿、牧草)的培養液,E. coli DH5a ( pBglC ) 所表現的CMCase活性都比B. licheniformis CFFD2來得強。然而,將pBglC送入C. xylanolyticum Ter3表現卻一直無法成功。探討其原因發現C. xylanolyticum Ter3可能含有未知的限制酶系統切割分解pBglC,導致此重組質體無法存在於C. xylanolyticum Ter3。因而改將pBglC送入C. acetobutylicum表現,增強此菌種分解纖維素的能力。但截至截稿為止尚未獲得帶有pBglC的C. acetobutylicum轉形株。
In response to the global issues of energy crisis and environmental pollution caused by overuse of fossil fuels, researchers in various countries have devoted all efforts to develop clean, renewable alternative energy, biomass energy has been on research focus during the past decade, even biomass-energy technology has gone through four generations of evolution, among them, however, the second generation of biomass energy technology which uses lignocellulose as raw material still attracts more attention today. Lignocellulose is particularly well-suited for biomass energy production because of its high carbohydrate content, large-scale availability, low cost, and environmentally friendly production. Lignocellulose is composed of cellulose, hemicellulose ( both are carbohydrate polymers ) and lignin, and its whole structure is very compact, so its complete degradation needs the cooperative action of cellulase, hemicellulase and ligninase. These degrading enzymes are almost produced by microorganisms, but the degrading enzymes produced by one microorganism always differ from others in function and activity, different decomposing and producing microorganisms must be cultivated together in order to utilize lignocellulose to produce biomass energy. This growing mode would poses problem of competition in nutrients and growing space among microorganisms, the best solution is genetically to make a good producing microorganism also a good decomposer. Clostridium xylanolyticum Ter3 is an early bacterial isolate of our lab, it has good xylanolytic ability and can produce hydrogen, ethanol and methanol, but it has only a little cellulolytic ability. In this study, an attempt was made to strengthen the cellulolytic ability of C. xylanolyticum Ter3. At first, three Bacillus licheniformis isolates, CFFD1, CFFD2 and CFFD4 were tested on their cellulolytic ability, their endoglucanase ( CMCase ) genes were also sequenced, then the complete CMCase gene ( bglC ) of CFFD2 which expressed higher CMCase activity was inserted into a shuttle vector of E. coli and Clostridium spp., pIMP1. When the constructed plasmid pBglC was transfered into E. coli DH5a by transformation and the transformant E. coli DH5a ( pBglC ) was grown in PY medium containing carboxylmethylcellulose (CMC), filter paper or natural lignocellulose (such as bagasse, rice straw and forage), it displayed stronger CMCase activity than B. licheniformis CFFD2 grown in the same media. However, the transformation of C. xylanolyticum Ter3 was unsuccessful, it seems that C. xylanolyticum Ter3 contained unknown restriction enzyme system to digest pBglC. Alternatively, pBglC is tried to transfer into C. acetobutylicum which has known restriction-methylation system, experimental work is proceeding now.
URI: http://hdl.handle.net/11455/23844
其他識別: U0005-2908201117074200


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