臺灣博碩士論文加值系統

本研究利用剛果紅試驗自澎湖沿岸分離出108株產纖維素酶之海洋細菌，自8株具較高纖維素酶活性中篩選出二株產鹼性纖維素酶之菌株，經革蘭氏染色及16S rDNA序列比對結果鑑定Bacillus pumilus（C4）及Vibrio屬（C7）。C4生產之CMCase、Avicelase及β-glucosidase最適作用pH分別為pH9、pH8和pH9，最適作用溫度則分別為50℃、30℃和50℃；而C7生產之CMCase、Avicelase及β-glucosidase最適作用pH分別為pH8、pH8和pH6，最適作用溫度則分別為30℃、50℃和50℃。C4生產之Avicelase具有較廣之pH穩定性，在pH6-9範圍內，12小時後仍可保有70%以上之活性；而C7則是以CMCase具有較廣之pH穩定性，在pH3-10範圍內，12小時後仍可保有70%以上之活性；C4和C7生產之纖維素酶於10-40℃之間，5小時內，其活性可維持在80%以上。C4培養於纖維素酶發酵培養基72小時後，生產之CMCase及β-glucosidase活性，最高可達0.360 IU/ml和0.102 IU/ml，而C7則為0.812 IU/ml和0.102 IU/ml，培養96小時後，C4之Avicelase活性最高可達1.267 IU/ml，而C7則為1.262 IU/ml。C7之粗酵素液經凍乾濃縮後，利用DEAE FF陰離子交換管柱層析分離CMCase和Avicelase，經SDS-PAGE分析後發現蛋白質分子量介於97-116 kDa之間。

108 cellulase producing marine bacteria were isolated from Penghu coast by Congo red test and 8 isolates with higher cellulolytic activity were screened for the production of alkaline cellulase. C4 and C7 alkaline cellulase producing isolate were identified as Bacillus pumilus and Vibrio sp. by Gram stain and 16S rDNA sequence analysis. The optimal pH for CMCase, Avicelase and β-glucosidase activity of C4 isolate was 9, 8 and 9, respectively. The optimal temperature for CMCase, Avicelase and β-glucosidase activity of C4 isolate was 50, 30 and 50℃, respectively. The optimal pH for CMCase, Avicelase and β-glucosidase activity of C7 isolate was 8, 8 and 6, respectively. The optimal temperature for CMCase, Avicelase and β-glucosidase activity of C7 isolate was 30, 50 and 50℃. For C4 isolate, the Avicelase has broad pH stability ranging from pH 6 to pH9 and residual activity was above 70% of original activity after 12h incubation. For C7 isolate, the CMCase has broad pH stability ranging from pH 4 to pH10 and residual activity was above 70% of original activity after 12h incubation. More than 80％ of original cellulose activity was maintained at relative low temperature ranging from 10 to 40℃ after 5h incubation for C4 and C7 isolate. The highest activities of CMCase (0.360 IU/mL) and β-glucosidase (0.102 IU/mL) of C4 were observed in the cellulose fermentation medium for 72h. The highest activities of CMCase (0.812 IU/mL) and β-glucosidase (0.120 IU/mL) of C4 were observed in the cellulose fermentation medium for 72h.
The highest activity of Avicelase was 1.267 IU/mL and 1.262 IU/mL obtained from C4 and C7 isolate after 96h incubation, respectively. The crude cellulose of C7 isolate were concentrated by freeze drying and then purified by the anion-exchange chromatography with a DEAE column. The molecular weights of purified proteins were between 97-116 kDa by SDS-PAGE analysis

表次.............................................................................................................................. IV 圖次............................................................................................................................... V 中文摘要.................................................................................................................... VII 英文摘要................................................................................................................... VIII
第一章 文獻回顧 ........................................................................................ 1
1. 纖維素 ........................................................................................................................... 1
1.1. 纖維素之組成 ........................................................................................................ 1
1.2. 纖維素之來源 ........................................................................................................ 1
1.3. 纖維素之水解 ........................................................................................................ 5
2. 纖維素酶 ....................................................................................................................... 7
2.1. 內切纖維素酶 ........................................................................................................ 7
2.2. 外切纖維素酶 ........................................................................................................ 7
2.3. β-葡萄糖苷酶......................................................................................................... 8
2.4. 纖維素酶的作用機制 ............................................................................................ 8
2.5. 纖維素酶的回饋抑制 ............................................................................................ 9
2.6. 纖維素酶測定 ........................................................................................................ 9
2.7. 纖維素酶的應用 .................................................................................................... 9
3. 降解纖維素之微生物種類 ......................................................................................... 15
4. 海洋細菌 ..................................................................................................................... 18
5. 鹼性纖維素酶 ............................................................................................................. 19
6. 鑑定海洋菌株之方法-16S rDNA (ribosomal deoxyribonucleic acid, rDNA) ........... 21
7. 聚合酶鏈鎖反應（polymerase chain reaction ,PCR） .............................................. 27
8. 酵素純化 ..................................................................................................................... 24
8.1. 鹽析分劃法 .......................................................................................................... 24
8.2. 透析法 .................................................................................................................. 24
II
8.3. 管住層析 .............................................................................................................. 25
第二章 前言 ............................................................................................................... 27 第三章 材料方法 ....................................................................................................... 28
1. 實驗儀器與藥品 ......................................................................................................... 29
1.1. 儀器設備 .............................................................................................................. 29
1.2. 藥品及試劑 .......................................................................................................... 31
1.3. 商業測試套組 ...................................................................................................... 32
1.4. 培養基的組成 ...................................................................................................... 32
1.5. 溶液及試劑配製 .................................................................................................. 34
2. 實驗方法 ..................................................................................................................... 36
2.1. 菌株來源 .............................................................................................................. 36
2.2. 菌株篩選、鑑定及保存 ...................................................................................... 36
2.2.1. 菌株篩選 ..................................................................................................... 36
2.2.2. 革蘭氏染色 ................................................................................................. 37
2.2.3. 16S rDNA菌種鑑定 ................................................................................... 37
2.3. 粗酵素液之製備 .................................................................................................. 42
2.4. 纖維素酶活性分析 .............................................................................................. 42
2.4.1. 剛果紅試驗 ................................................................................................. 42
2.4.2. 內切纖維素酶活性之測定 ......................................................................... 43
2.4.3. 外切纖維素酶活性之測定 ......................................................................... 43
2.4.4. β-葡萄糖苷酶活性之測定 .......................................................................... 43
2.4.5. 二硝基水楊酸(DNS)還原糖測定法 ........................................................... 44
2.5. 粗纖維素酶之生化特性 ........................................................................................ 44
2.5.1. 最適作用pH的測定 .................................................................................. 44
2.5.2. 最適作用溫度的測定 ................................................................................. 45
2.5.3. pH值穩定性測定 ....................................................................................... 45
2.5.4. 溫度穩定性測定 ......................................................................................... 45
III
2.6. 酵素純化 ................................................................................................................ 46
2.6.1. 培養時間對菌株生產纖維素酶之影響 ..................................................... 46
2.6.2. 硫酸銨沉澱 ................................................................................................. 46
2.6.3. 丙酮沉澱 ..................................................................................................... 47
2.6.4. 透析 ............................................................................................................. 47
2.6.5. 陰離子交換樹脂 ......................................................................................... 47
2.7. 蛋白質總量測定 .................................................................................................... 48
2.8. SDS-PAGE電泳分析 ............................................................................................ 49
第四章 結果與討論 ................................................................................................... 51
1. 菌株分離 ..................................................................................................................... 51
2. 產纖維素黴菌株之篩選 ............................................................................................. 51
3. 菌落型態 ..................................................................................................................... 52
4. 革蘭氏染色 ................................................................................................................. 52
5. 菌種16S rDNA序列之定序分析 .............................................................................. 52
6. 分離株粗纖維素酶之特性分析 ................................................................................. 54
6.1. 粗纖維素酶之最適作用pH ................................................................................ 54
6.2. 粗纖維素酶之最適作用溫度 .............................................................................. 57
6.3. 粗纖維素酶之穩定性 .......................................................................................... 58
6.3.1. pH穩定性 ................................................................................................... 58
6.3.1. 溫度穩定性 ................................................................................................. 59
7. 纖維素酶之純化 ......................................................................................................... 60
7.1. 培養時間對菌株生產纖維素酶之影響 .............................................................. 60
7.2. 粗酵素之回收 ...................................................................................................... 61
7.3. DEAE FF陰離子交換管柱層析 ......................................................................... 61
7.4. SDS-PAGE分析 .................................................................................................. 62
第五章 結論 ............................................................................................................... 63 第六章 參考文獻 ....................................................................................................... 88
IV
表次 表1. 纖維素酶的組成分對纖維素之作用方式........................................................ 10 表2. 測定纖維素酶活性的基質及分析方法............................................................ 12 表3. 纖維素酶在工業上之應用................................................................................ 13 表4. 產纖維素酶之微生物........................................................................................ 17 表5. 利用剛果紅測試法所得菌株纖維素酶之活性................................................ 64 表6. 分離菌株之特徵及鑑定結果............................................................................ 69
V
圖次 圖1. 纖維素的結構...................................................................................................... 3 圖2. 植物細胞壁中纖維素的結構.............................................................................. 4 圖3. 纖維素酶之協同機制 . ................................................................................... 11 圖4. 聚合酶連鎖反應................................................................................................ 23 圖5. 纖維素酶生產菌形成之酵素圈........................................................................ 67 圖6. 分離株在TSBN培養基培養於25℃ 24小時後之型態 ................................ 68 圖7. 菌株生長在TSBN下顯微鏡之觀察 ............................................................... 70 圖8. pH對各分離株之內切纖維素酶反應活性之影響 .......................................... 71 圖9. pH對各分離株之外切纖維素酶反應活性之影響 .......................................... 72 圖10. pH對各分離株之β-葡萄糖苷酶反應活性之影響 ......................................... 73 圖11. 溫度對各分離株之內切纖維素酶反應活性之影響...................................... 74 圖12. 溫度對各分離株之外切纖維素酶反應活性之影響...................................... 75 圖13. 溫度對各分離株之β-葡萄糖苷酶反應活性之影響 ....................................... 76 圖14. pH對C4產之內切纖維素酶穩定性之影響 ................................................. 77 圖15. pH對C4產之外切纖維素酶穩定性之影響 ................................................. 78 圖16. pH對C4產之β-葡萄糖苷酶穩定性之影響 ................................................... 79 圖17. pH對C7產之內切纖維素酶穩定性之影響 ................................................. 80 圖18. pH對C7產之外切纖維素酶穩定性之影響 ................................................. 81 圖19. pH對C7產之β-葡萄糖苷酶穩定性之影響 ................................................... 82 圖20. 溫度對C4產之內切纖維素酶穩定性之影響 ............................................... 83 圖21. 溫度對C4產之外切纖維素酶穩定性之影響 ............................................... 84 圖22. 溫度對C4產之β-葡萄糖苷酶穩定性之影響 ................................................ 85 圖23. 溫度對C7產之內切纖維素酶穩定性之影響 ............................................... 86 圖24. 溫度對C7產之外切纖維素酶穩定性之影響 ............................................... 87 圖25. 溫度對C7產之β-葡萄糖苷酶穩定性之影響 ................................................ 88
圖26. 培養時間對分離株C4產之纖維素酶活性的影響 ....................................... 89
VI
圖27. 培養時間對分離株C7產之纖維素酶活性的影響 ....................................... 90 圖28. 陰離子交換管柱層析...................................................................................... 91 圖29. 纖維素酶之蛋白質電永圖 ................................................................................. 92

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