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系統識別號 U0002-3006202014392000
中文論文名稱 枯草桿菌 TKU049 所生產果膠酶的純化與定性
英文論文名稱 Purification and characterization of a pectinase from Bacillus subtilis TKU049
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 108
學期 2
出版年 109
研究生中文姓名 謝孝婷
研究生英文姓名 Hsiao-Ting Hsieh
學號 607160248
學位類別 碩士
語文別 中文
口試日期 2020-06-05
論文頁數 42頁
口試委員 指導教授-王三郎
共同指導教授-阮文邦
委員-呂誌翼
委員-王全祿
中文關鍵字 枯草桿菌  果膠  果膠酶 
英文關鍵字 Bacillus subtilis  pectin  pectinase 
學科別分類 學科別自然科學化學
中文摘要 由於果膠酶的重要生物技術用途,在食品和飲料行業具有廣闊的工業前景。本研究使用的菌株Bacillus subtilis TKU049係從淡江大學土壤中篩選出來,探討香蕉皮、橙皮、柚子皮、麥麩,米糠以及咖啡渣等六種農業副產品作為B. subtilis TKU049生產果膠酶之碳和氮的之可行性。 結果顯示於含有1.5%果膠的培養基中培養三天,B. subtilis TKU049所產生果膠酶活性最高(1.19 U / mL)。 SDS-PAGE分析純化所得TKU049果膠酶之分子量為60.5 kDa。 TKU049果膠酶之最適反應溫度及酸鹼值分別為60°C和pH 9,酸鹼穩定性則是pH6至pH10。
此外,於無機鹽對TKU049果膠酶酵素活性影響方面,Mn2+、Mg2+、Ca2+以及Fe2+金屬離子具促進酵素活性之作用。 而界面活性劑EDTA、Triton X-100、Tween 40以及Tween 20則對酵素活性影響不大。
英文摘要 Pectinases have tremendous industrial prospects to be used in the fields of food and beverage. In this study, pectin and six types of agricultural by-products (banana peel, orange peel, pomelo peel, wheat bran, rice bran, and coffee ground) were used as the sole carbon and nitrogen sources for pectinase production by Bacillus subtilis TKU049 which was isolated from the soil of Tamkang University. B. subtilis TKU049 produced highest pectinase activity of 1.19 U/mL when cultured for three days on a medium containing 1.5% of pectin. The molecular mass of TKU049 pectinase was determined to be 60.5 kDa by SDS-PAGE. TKU049 pectinase displayed optimal activity at 60 °C and pH 9, with a pH range of stability from 6 to 10. The enzyme activity of TKU049 pectinase was stimulated with Mn2+, Mg2+, Ca2+, and Fe2+ metal ions. Additionally, the TKU049 pectinase was stable in the presence of EDTA, Triton X-100, Tween 40, and Tween 20.
論文目次 誌謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 VII
表目錄 VIII
第一章 緒論 1
第二章 文獻回顧 2
2.1枯草桿菌之簡介 2
2.2果膠 2
2.3果膠酶 4
2.4果膠酶的應用 4
第三章 材料與方法 7
3.1實驗菌株 7
3.2藥品與試劑 7
3.3實驗儀器 7
3.4生產菌株之篩選與分離 8
3.5革蘭氏染色 8
3.6菌種之鑑定 9
3.6.1 rDNA 序列分析 9
3.6.2 API 菌種之鑑定 9
3.7果膠酶活性測定 10
3.8果膠酶生產條件探討 10
3.8.1碳源種類之影響 10
3.8.2碳源濃度之影響 11
3.8.3培養溫度之影響 11
3.8.4培養基酸鹼值之影響 11
3.9果膠酶之分離與純化 12
3.9.1粗酵素液的備製 12
3.9.2離子交換樹脂層析法 12
3.9.3蛋白質電泳分析 12
3.10 酵素之特性分析 13
3.10.1酵素最適反應溫度 13
3.10.2酵素熱安定性 13
3.10.3酵素最適反應pH 13
3.10.4酵素之pH安定性 14
3.10.5金屬離子與抑制劑對酵素活性之影響 14
3.10.6界面活性劑對酵素活性之影響 15
第四章 結果與討論 16
4.1 果膠酶生產之菌種鑑定 16
4.2 果膠酶生產條件之探討 18
4.2.1碳源種類之影響 18
4.2.2碳源濃度之影響 19
4.2.3培養溫度之影響 20
4.2.4培養基酸鹼值之影響 21
4.2.5較適培養條件結果與比較 22
4.3果膠酶純化與分離 23
4.3.1粗酵素液製備 24
4.3.2離子交換層析 24
4.3.3純化與分離之綜合結果 26
4.3.4蛋白質電泳分析 27
4.4果膠酶之特性分析 29
4.4.1酵素最適反應溫度及熱安定性 29
4.4.2酵素最適反應pH及pH安定性 31
4.4.3金屬離子與抑制劑對酵素活性之影響 34
4.4.4界面活性劑對果膠酶之影響 35
第五章 結論 36
參考文獻 37

圖目錄
圖2. 1 低(a)和高(b)甲基化果膠物質的結構(主鏈)以及參與降解酶之作用點 3
圖4. 1 Bacillus subtilis TKU049之顯微鏡照片 16
圖4. 2不同碳源對B. subtilis TKU049生產果膠酶之影響 18
圖4. 3果膠濃度對B. subtilis TKU049生產果膠酶之影響 19
圖4. 4培養溫度對B. subtilis TKU049生產果膠酶之影響 20
圖4. 5培養pH值對B. subtilis TKU049生產果膠酶之影響 21
圖4. 6 B. subtilis TKU049 果膠酶之分離純化流程圖 23
圖4. 7果膠酶之Macro-Prep High Q層析圖譜 25
圖4. 8果膠酶之純化SDS-PAGE分析圖 28
圖4. 9果膠酶之最適反應溫度及熱安定性 30
圖4. 10果膠酶之最適反應pH及pH安定性 32

表目錄
表2. 1果膠酶生產菌種類 5
表2. 2果膠酶的應用 6
表3. 1緩衝溶液種類 14
表4. 1TKU049之16S rDNA 部分基因序列 17
表4. 2API鑑定結果 17
表4. 3B. subtilis TKU049 生產果膠酶之較適培養條件 22
表4. 4B. subtilis TKU049果膠酶之純化總表 26
表4. 5芽孢桿菌菌株產生的果膠酶之特性比較 33
表4. 6金屬離子對果膠酶活性之影響 34
表4. 7界面活性劑對果膠酶活性之影響 35

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