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研究生:江衍毅
研究生(外文):Yen-I Chiang
論文名稱:Bacillussp.YJ6聚木醣?之純化與特性分析
論文名稱(外文):Purification and Characterization of Xylanase from Bacillus sp. YJ6
指導教授:江善宗殷儷容殷儷容引用關係
指導教授(外文):Shann-Tzong JiangLi-Jung Yin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:112
中文關鍵詞:Bacillus sp. YJ6聚木醣?純化生化特性
外文關鍵詞:Bacillus sp. YJ6xylanasepurificationbio-properties
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本研究目的為純化 Bacillus sp. YJ6 聚木醣? (xylanase) 並探討其生化特性。菌株接種於培養基後,置於 25oC、150 rpm 振盪培養,第四天可以得到最高聚木醣?活性為 2.26 U/mL。收集胞外酵素液,經硫酸銨分劃、透析後,以 CM-Sepharose Fast Flow 管柱層析及 sephacryl S-100 HR 管柱層析可得到具有活性之聚木醣?,其比活性為 1435.98 U/mg,回收率為 3.46%,純化倍率為 678.1 倍。經 SDS-PAGE 電泳分析呈現單一色帶,其分子量為 19 kDa。純化之聚木醣?最適反應 pH 值為 pH 6.0,在 pH 5.0-9.0 間有較佳安定性,最適反應溫度為 50oC,在 40oC 以下安定性較佳,其活性會受到金屬離子 Hg2+、Cu2+、Fe2+ 及抑制劑 PMSF、TPCK、NEM、Leupeptin 抑制,而金屬離子 K+、Na+、Co2+、Mg2+ 及還原劑 b-mercaptoethanol、glutathione 則會提高其活性。此聚木醣?僅對櫸木、樺木及燕麥聚木醣有活性,推論此酵素為一內切型聚木醣?;N 端定序前八個胺基酸序列為 ASTDYWQN,設計引子經聚合?連鎖反應得到基因全長,此序列具有642 個核甘酸,與其他菌株之聚木醣?基因序列比較其相似度為 95% 左右,顯示該菌為一新穎的菌株。
The cells of Bacillus sp. YJ6 were removed by passing through a 0.45 μm membrane after 4 days incubation at 25oC, which had the highest xylanase activity (2.26 U/mL). The xylanase was purified to electrophorectical homogeneity by precipitating at 40-60% saturation of ammonium sulphate, passing through CM-Sepharose FF and Sephacryl S-100 HR chromatographs. About 3.5% of xylanase was recovered and 678.1 purification fold were obtained. The purified xylanase was with a molecular mass of 19 kDa and specific activity of 1435.98 U/mg. It had an optimal pH and temperature at 6.0 and 50oC, respectively, and was stable at pH 5.0-9.0 and below 40oC. This xylanase was inhibited by Cu2+, Fe3+, Hg2+, PMSF, TPCK, NEM and Leupeptin, but activated by K+, Na+, Co2+, Mg2+, b-mercaptoethanol and glutathione. According to substrate specificity, the purified xylanase had high specificity to beechwood, birchwood and oat spelts xylans. The N-terminal sequence of xylanase was ASTDYWQN and used for desiging the primers to amply nucleotide sequence by PCR. This xylanase DNA consisted of 642 nucleotides, encoded 213 amino acid residues and exhibited upward 95% homology compared with 7 strains of Bacillus by BLAST system in NCBI database.
中文摘要 1
Abstract 2
貳、文獻整理 5
1. 植物之纖維質成分 5
2. 半纖維素之種類 6
3. 植物之聚木醣 7
4. 聚木醣之降解與利用 9
4.1. 木糖 10
4.2. 木糖醇 (xylitol) 10
4.3. 寡木醣 11
5. 寡木醣之簡介 12
5.1. 寡木醣之化學結構 12
5.2. 寡木醣對腸道的作用 12
5.3. 寡木醣的其他特性 13
5.4. 寡木醣的其他生理功能 14
5.5. 寡木醣的市場需求及經濟效益 14
6. 聚木醣? 15
6.1. 聚木醣?的來源與生化特性 15
6.2. 聚木醣?的種類與反應機制 16
6.3. 聚木醣?的生產 20
6.4. 聚木醣?的多樣性 21
6.5. 聚木醣?的分類 21
6.6. 聚木醣?的應用 23
?、材料與方法 26
Ι. 實驗材料 26
1. 菌株 26
2. 菌株之製備與保存 26
3. 藥品與培養基 26
Π. 實驗方法 29
一、 聚木醣?菌株的培養 29
二、 聚木醣?的純化 30
三、 聚木醣?之生化特性試驗 34
肆、結果與討論 42
一、 Bacillus sp. YJ6 酵素活性、生長曲線及 pH 變化 42
二、 Bacillus sp. YJ6 聚木醣?之純化 42
1. 粗酵素液的製備 42
2. 硫酸銨劃分 42
3. CM-Sepharose Fast Flow 管柱層析 43
4. Sephacryl S-100 High Resolution 膠過濾層析 44
5. 純度鑑定 44
三、 Bacillus sp. YJ6 生產聚木醣?之生化特性 45
1. 最適反應 pH 值 45
2. pH 值安定性 46
3. 最適反應溫度 46
4. 溫度安定性 47
5. 金屬離子之影響 48
6. 抑制劑之影響 49
7. 還原劑之影響 50
8. 基質特異性 50
9. 酵素水解產物分析 51
10. 聚木醣?之 N 端序列與 DNA 序列分析 52
伍、結論 53
陸、參考文獻 55
表目錄
表一、不同微生物來源之聚木醣?及特性 74
表二、Bacillus sp. YJ6 純化的聚木醣?之硫酸銨分劃表 76
表三、 Bacillus sp. YJ6 聚木醣?純化表 77
表四、金屬離子對 Bacillus sp. YJ6 聚木醣?活性之影響 78
表五、抑制劑對 Bacillus sp. YJ6 純化的聚木醣?活性之影響 79
表六、還原劑對 Bacillus sp. YJ6 純化的聚木醣?活性之影響 80
表七、Bacillus sp. YJ6 聚木醣?基質特異性 81

圖目錄
圖一、植物細胞壁之結構 82
圖二、不同來源半纖維素之結構 83
圖三、不同來源聚木醣之結構 84
圖四、聚木醣結構及聚木醣酵素群之作用位置 85
圖五、聚木醣酵素的合成與代謝抑制作用 86
圖六、Bacillus sp. YJ6 活性、生長曲線及 pH 變化 87
圖七、Bacillus sp. YJ6 聚木醣?經 CM-sepharose fast flow 陽離子交換管柱層析圖 88
圖八、Bacillus sp. YJ6 聚木醣?經 Sephacryl S-100 HR 管柱層析圖 89
圖九、Bacillus sp. YJ6 聚木醣?之 SDS 電泳圖 90
圖十、Bacillus sp. YJ6 聚木醣?之最適反應 pH 值 91
圖十一、Bacillus sp. YJ6 聚木醣?之 pH 值安定性 92
圖十二、Bacillus sp. YJ6 聚木醣?之最適反應溫度 93
圖十三、Bacillus sp. YJ6 聚木醣?之溫度安定性 94
圖十四、部分純化和純化的聚木醣?水解樺木聚木醣 24 小時之還原糖量 95
圖十六、pGEMT-XynB 之 DNA 定序圖譜 (使用 T7 promoter primer) 97
圖十七、Bacillus sp. YJ6 聚木醣?之基因序列 98
圖十八、不同 Bacillus 來源聚木醣?之演化關係圖 99
圖十九、不同 Bacillus 來源聚木醣?序列之相似程度 100
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