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研究生:黃祐緯
研究生(外文):Huang, Yuwei
論文名稱:具有分解纖維素與木聚醣能力之環境菌(Bacillus sp.) MGM7 特性分析
論文名稱(外文):Characterization of a Cellulose and xylan Degrading Bacterial Strain Bacillus sp. MGM7
指導教授:柳源德
指導教授(外文):Liou, Yuande
口試委員:黎耀基孫芳君
口試委員(外文):Lai, YiukaySun, Fangchun
口試日期:2012-07-31
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物資源學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:51
中文關鍵詞:纖維素酶菌落檢測法生質酒精
外文關鍵詞:Biological energyBioethandCelluloseZymogram
相關次數:
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在能源危機的二十一世紀,替代能源的尋找已經是一個不可避免的問題。而以生質柴油與生質酒精最為世界所期待。生質酒精主要是利用微生物分解澱粉或纖維素產生葡萄糖,再將其發酵產生酒精得到。澱粉的分解主要是分解穀物澱粉而來,造成與人爭食以致糧食短缺、糧食價格飛漲的現象。而纖維素乃是地球上最大的碳資源,主要以葡萄糖之結晶組合而成,雖然有極大的碳量卻不容易分解。為此,纖維素的利用乃是亟待突破的重要課題。
本研究目的即在於自採集之土壤中分離出分解纖維素之Bacillus菌種,利用其孢子耐高溫特性,以菌落檢測法分離出具有纖維素酶活性之菌株,經由Zymogram分析活性蛋白,再將由Sau3A1限制酶隨機切割後的細菌染色體DNA與經Bam H1限制酶切割後的載體(pBCKS+)黏合,並將基因選殖出來至大腸桿菌中表現與分析。
在實驗後可以得知,此MGM7菌種分析後纖維素酶蛋白質大小為100~110 KDa,並經由限制酶Sau3 A1水解染色體DNA後,與載體(pBCKS +)接合且轉型至DH5α,再使用菌落檢測法作為篩選。

In 21th centry, energy sources can be absent due to overusase of oil. Thus, Biological replacement should be developed. In all biological energy, biodiesel and bioethanol are two most expected bio-energy sources. Bioethanol is genterated by microbial-fermented starch or cellulose and given glucose for ethand production.
The degradation of starch for bioethanol production mainly utilizes crops that can be used as food, therefore these processes will cause food shortening. In contrast, cellulose is the most aboundant organic carbon source in the world; it is composted of polyglucose that linked by -glycan bond and stored as crystal form. The structure of cellulose is extremely stable and is difficult to digest by normal animal; however, it can be degraded by microbes.
In this thesis, Bacillus sp. MGM7 was screened from soil samples by cellulolytic activity. Zymogram analysis showed that the cellulose degrading activity protein was a 100~110 kda protein.
Sau3AI partial digestion of genomic DNA was proceeded after genomic DNA extraction and the fragments were ligated into phagemid vector PBCKS+. The ligates were then franeformed into E. coli DH5α, and positive clones were screened by cellulolytic activities.

封面內頁
簽名頁
中文摘要 iii
Abstract iv
誌謝 v
目錄 vi
圖目錄 x
表目錄 xi

1. 文獻回顧 1
1.1 前言 1
1.2 纖維素(Carboxymethylcellulose) 2
1.2.1植物細胞壁的最主要組成分 2
1.2.2纖維素的分佈與構造 2
1.2.3纖維素酶作用機制 3
1.3 木聚醣 (Xylan) 5
1.3.1 半纖維素的主要組成分 5
1.3.2 木聚醣的分佈與構造 5
1.3.3木聚醣酶水解木聚醣之機制 6
1.4 枯草桿菌屬 (Bacillus sp.) 7
2. 實驗材料與設備 8
2.1 實驗材料 8
2.2 實驗設備 9
2.3 培養基與藥品配置 10
2.3.1 培養基配置 10
2.3.2 緩衝液與染劑配置 11
2.3.3 細菌染色體DNA純化溶液配置 12
2.3.4 SDS-PAGE 配置 13
3. 實驗方法 14
3.1 篩選、分離菌株 15
3.2 鑑定分離的菌株 15
3.2.1 利用PCR 方法鑑定菌株 16S rDNA 15
3.2.2 DNA 洋菜電泳 16
3.2.3 DNA 純化 17
3.2.4 DNA定序和比對 18
3.3 多醣類水解酶活性測定 18
3.3.1 選殖株菌落檢測 18
3.3.2 SDS聚丙烯醯胺膠體電泳(SDS-PAGE) 19
3.3.3 Zymogram 20
3.4 纖維素酶基因轉殖 21
3.4.1 細菌染色體DNA純化 22
3.4.2 限制酶隨機切割細菌染色體與染色體純化 23
3.4.3 限制酶切割載體、去磷酸化與載體純化 23
3.4.4 DNA黏合 25
3.4.5 勝任細胞製備 25
3.4.6電穿孔法與轉殖 26
3.4.7 轉殖效率計算 27
3.4.8複製盤和活性測試 28
3.4.9基因圖譜、定序和比對 29
4. 結果與討論 30
4.1 16S rDNA鑑定分離的菌株 30
4.2 酵素活性篩選與推估蛋白質大小 31
4.2.1 纖維素指示盤篩選菌種 31
4.2.2 SDS-PAGE電泳與Zymogram推估蛋白質大小 33
4.3 纖維素酶基因轉殖 34
4.4 未來研究 37
4.4.1 轉殖後纖維素酶基因表達 37
4.4.2引子設計和PCR程式 37
4.4.3限制酶切割轉殖基因片段和表達載體的純化 38
4.4.4表達寄主選殖和製備 38
4.4.5轉殖基因片段黏合、轉殖和酵素活性測試 39
4.4.6轉殖後纖維素酶的純化 39
4.5 結論 40
參考文獻 41
附錄一:配置 10 % running gels SDS-PAGE 44
附錄二:配置 5 % stacking gels SDS-PAGE 45
附錄三:PCR反應成分配方 46
附錄四:PCR反應條件 47
附錄五:染色體以限制酶水解配方 48
附錄六:pBCKS+以限制酶(Bam H1)切割配製方法 49
附錄七:pBCKS+去磷酸化 50
附錄八:黏合配方 51

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