Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99562
標題: 固定化枯草桿菌Bacillus subtilis WB800N 生產之纖維素體於再生纖維膜上之探討
Study on immobilization of cellulosome produced by Bacillus subtilis WB800N on the regenerated cellulose membrane
作者: 楊量宇
Liang-Yu Yang
關鍵字: 纖維素水解酵素複合體
固定化酵素
環氧氯丙烷(EPI)
枯草桿菌
Cellulosome
Bacillus subtilis
Immobilization enzyme
RC membrane
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摘要: 鑒於石油枯竭的危機,尋找可再生的替代生質能源為當務之急的議題。生質酒精(bioethanol)是目前具有高開發價值的生質能源之一。主要是利用纖維素(cellulose)醣化後,再經由發酵生成酒精。纖維素廣泛分布在自然界植物的細胞壁中,其含量最龐大、價格便宜、含糖量豐富、且非糧食作物。但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,導致不易被纖維素酵素水解(cellulase)。微生物Clostridium thermocellum 所產出的纖維素水解酵素複合體(cellulosome)被發現能提升木質纖維素的水解效率。本研究使用的纖維酵素複合體是利用重組枯草桿菌(recombinant B.subtilis WB800N),其帶有8個C.thermocellum 纖維素基因片段,包含一個支架蛋白(scaffolding protein)、一個錨蛋白(anchoring protein)、兩個內切葡聚醣酶(endoglucanase)、兩個外切葡聚醣酶(exoglucanase)以及兩個木聚醣酶(xylanase)。 本研究以再生纖維素薄膜(RC membrane)首先利用Epichlorohydrin(EPI)來改質官能基,再以1,4-Diaminobutane(DA)及Glutaric dialdehyde(GA)完成改質,最後再接上酵素。第一部份是針對不同濃度的NaOH、GA、時間、溫度對固定化酵素的影響,以及SEM之照攝。第二部份則是針對固定化酵素對基質水解的探討,其中包含了pH、溫度以及純化分析。最後是針對固定後纖維素水解酵素複合體之動力學探討。結果顯示,固定化酵素之Vmax = 2.376 U/ml,Km= 12.54 g/L。
According to the possibility of fossil fuels sources exhaustion, it is an imperative issue to explore renewable alternatives energy. Lately, various renewable energy sources have been offered as alternatives to petroleum. The production of bioethanol was conducted via the saccharification and fermentation of cellulose, and then fermentation to ethanol. Cellulose is widely disturbed in the cell wall of natural plants. It is abundant, cheap, and does not belong to food crops. In natural, cellulose is wrapped by the complex structure of lignocellulose. It is difficult for the plant cell wall with cellulose to subject to enzymatic hydrolysis by cellulase. Hence, enhancing the efficiency of cellulose degradation is a matter of great importance. Cellulosomes are multi-subunit protein complexes having great potential in degradation of cellulose. The recombianat Bacillus subtilis WB800N harboring pGETS 118 cellulosome genes could produce the artificial cellulosome including six cellulose hydrolysis enzyme subunits. In this study, regenerated cellulose membrane (RC) was used as a solid matrix for cellulosome immobilization. The modification of RC was conducted by coupling EPI as a spacer, followed by binding the active reagents(ie., DA,GA), to immobilize cellulosome on the membrane. This study included three parts. The first part discuss the effect of different concentration of NaOH, GA, time, temperature for enzyme immobilization. The SEM was used check the membrane morphology. The second part discuss the optimal pH and temperature for membrane regeneration. Finally, the kinetics studies of immobilized cellulosome on RC membrane and the free enzyme. The results showed that Vmax and Km for immobilized cellulosome on RC membrane showed better performance than that of free enzyme.
URI: http://hdl.handle.net/11455/99562
文章公開時間: 2020-07-31
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