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研究生:葉佐珊
研究生(外文):Cally Yap Joe San
論文名稱:利用枯草桿菌Bacillus subtilis WB800N strain Type I生產人工纖維素水解酵素複合體之生產條件與酵素特性之探討
論文名稱(外文):The study of production conditions and enzymatic property of artificial cellulosome by recombinant Bacillus subtilis WB800N strain Type I
指導教授:劉永銓
指導教授(外文):Yung-Chuan Liu
口試委員:易逸波黃介辰
口試委員(外文):Yet-Pole IChieh-Chen Huang
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:56
中文關鍵詞:木質纖維素纖維素水解酵素複合體纖維素水解酵素枯草桿菌
外文關鍵詞:CellulosomeBacillus subtiliscellulaseendoglucanaseexoglucanasexylanasekinetics
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鑑於石油枯竭的可能性,尋找可再生的替代能源市一項非常重要的課題。生質酒精(bioethanol)市其中一個非常具有開發價值的生質能源。其主要方法是將纖維素(cellulose)醣化後,再經由發酵生成酒精。纖維素是自然界中分佈最廣泛,含量最龐大的一種多醣,也是植物細胞壁的主要成份。木質纖維素(lignocellulose)之組成主要是纖維素,半纖維素(hemicellulose)和木質素(lignin)。由於纖維素被層層包覆在木質纖維素的複雜結構中,導致其無法被纖維素酵素(cellulase)水解。厭氧菌 Clostridium thermocellum 所產出之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。本實驗所使用的纖維素酵素複合體是利用重組枯草桿菌(recombinant B. subtilis WB800N)生產,其帶有8個C. thermocellum 纖維素酵素基因片段。纖維素水解酵素複合體包含一個支架蛋白(scaffolding protein),一個錨蛋白(anchoring protein),兩個內切葡聚糖酶(endoglucanase),兩個外切葡聚糖酶(exoglucanase)以及兩個木聚糖酶(xylanase)。

本實驗分為兩個部份進行研究。第一個部份是針對纖維素水解酵素複合體的生產條件作探討,在培養基中添加不同之碳源。另外,也在培養的過程中添加不同濃度之 IPTG,結果顯示使用LB培養基並添加1 Mm 的IPTG能提升纖維素水解酵素複合體的產量。除此之外,本實驗也針對pH和溫度對纖維素水解反應之影響進行探討。第二部分則是將分泌至發酵液中(SC)和掛載細胞膜上(AC)的纖維素水解酵素複合體收集後,分別用來探討其各別的酵素性質,並利用HPLC分析出酵素水解反應後的產物。 最後則是針對纖維素水解酵素複合體中各個功能的酵素,進行動力學的探討。結果顯示,SC中的內切酵素與木聚糖酶之Vmax值較AC高。SC中的內切酵素與木聚糖酶之Km值比AC低,這表示AC之內切酵素與木聚糖酶與基質有較佳的親和力。

The possibility of exhaustion of fossil fuels sources 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. Lignocellulosic biomass is the non-edible portion of the plant, which is underutilized, but could be used for biofuel production. However, the lignocellulose is recalcitrance to enzymatic hydrolysis due to its structure complexity. Hence, enhancing the efficiency of cellulose degradation is a matter of great importance. Cellulosomes are multi-subunit protein complexes which possess great potential in degradation of cellulose. The recombinant Bacillus subtilis WB800N harboring pGETS 118 cellulosome genes could produce the artificial cellulosome including six cellulose hydrolysis enzyme subunits.
This study includes two parts. The first part is the production conditions of artificial cellulosome. The cultivation of recombinant B. subtilis was conducted with addition of carbon source and different concentrations of IPTG into LB medium. LB medium with 1mM IPTG was found to be the best condition for cellulosome production. In addition, the effects of pH and temperature on cellulosome activity were also studied. The second part is the study of enzymatic property of cellulosome. Both of the supernatant cellulosome (SC) and the anchored cellulosome (AC) were obtained after centrifugation, their respective enzymatic properties were investigated. It was noted that the cellulosome activity can be found in both supernatant of the harvested broth and cell pellets. Later, the end products of enzymatic reaction were collected and identified via HPLC analysis. The main products of cellulosome hydrolysis were obtained as oligosaccharides and cellobiose. The kinetics studies of cellulosome showed that Vmax value for endoglucanase and xylanase in SC was higher than that in AC. However, the endoglucanase and xylanase in AC demonstrated lower Km values, indicating a higher substrate affinity than that of SC. This study provides a better understanding of artificial cellulosome, which might be useful in the study of renewable energy sources.

Chapter 1 Introduction 1
1.1 Overview 1
1.2 Objective 2
Chapter 2 Literature review 3
2.1 Lignocellulose 3
2.2 Cellulase 4
2.3 Hemicellulase 5
2.4 Cellulosome 6
2.5 Characteristics of Clostridium thermocellum & Bacillus subtilis 10
Chapter 3 Materials and Methods 12
3.1 Materials 12
3.2 Instruments 14
3.3 Expression host 16
3.4 Strain cultivation 17
3.5 Additive effects of carbon sources on cellulosome production 18
3.6 Effect of IPTG on cellulosome production 18
3.7 Effect of pH on hydrolysis activity 19
3.8 Effect of temperature on hydrolysis activity 19
3.9 Total cellulosome activity assay 20
3.10 Determination of respective enzyme activity of cellulosome 20
3.11 HPLC assays 23
3.12 Determination of kinetics parameters 23
Chapter 4 Results and discussion 25
4.1 Effect of carbon sources on cellulosome production 25
4.2 Effect of IPTG on cellulosome production 28
4.3 Effect of pH on the activity of cellulosome 30
4.4 Effect of temperature on cellulosome activity 33
4.5 Study on respective enzyme activity of cellulosome 36
4.6 Analysis of end products of enzymatic hydrolysis 37
4.7 Kinetics study of cellulosome 42
Chapter 5 Conclusions and future perspective 44
5.1 Conclusions 44
5.2 Future perspective 46
References 47
Appendices 54
Appendix 1 Plasmid vector construction for production of artificial cellulosome 54
Appendix 2 DNS assay 56

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