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論文名稱(中文) 固定化纖維素分解酵素水解微藻細胞壁應用於葉綠素萃取之研究
論文名稱(英文) Immobilized Cellulase for Chlorophyll Extraction from Microalgae
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 李文敏
研究生(英文) Wen-Min Lee
學號 n36981472
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 指導教授-吳文騰
口試委員-許梅娟
口試委員-張德生
中文關鍵字 纖維素分解酵素  氧化鐵磁性奈米粒子(Fe3O4)  葉綠素  微藻 
英文關鍵字 Cellulase  Magnetic nanoparticle(Fe3O4)  Chlorophyll  Microalgae 
學科別分類
中文摘要 由聚丙烯腈包覆之氧化鐵(Fe3O4)磁性奈米粒子,可透過amidination reaction活化粒子表面之聚丙烯腈的C≡N官能基,與纖維素分解酵素上之胺基形成共價鍵結。於實驗條件為活化時間2.5分鐘,固定化酵素濃度為0.5wt%,固定化時間60分鐘,固定化溫度50℃,pH值為7.8的條件下,可製得較高蛋白質固定量之固定化纖維素分解酵素。本實驗進而將固定化酵素應用於水解微藻細胞壁,並藉由產生還原醣之濃度,決定最佳反應條件。經實驗證實在適當的水解時間下,固定化纖維素分解酵素可水解微藻之細胞壁,使藻體細胞壁破裂,但破裂程度不致使葉綠素釋出胞外,造成損失,並可提高後續以丙酮萃取葉綠素之效益。經固定化纖維素分解酵素水解後之微藻,利用丙酮於常溫下進行萃取反應,100 mg之藻體可萃得葉綠素1.5mg;相較未經過水解反應之藻體,則只可萃得0.4mg之葉綠素。重複進行批次反應10次以後,萃取率仍維持初始萃取率之88%。由於氧化鐵磁性奈米粒子具備磁性易回收,因此實驗所製得之固定化纖維素分解酵素,證明不僅可多次重複使用,並具有回收容易之優點。
英文摘要 The enzyme, Trichoderma reesei cellulase was immobilized on PAN (polyacrylonitrile) coated magnetic nanoparticles(Fe3O4) by amidination reaction for long term operation. In addition, covalent bond formation between enzyme molecule and the PAN coated magnetic nanoparticles was confirmed via FT-IR measurement. The optimal operation conditions of cellulase immobilization for the highest protein loading were under the enzyme concentration of 0.5 wt%, immobilization time of 60 min, temperature at 50°C and pH value at 7.8. The optimal conditions of hydrolysis reaction were determined by measuring the concentration of producing reducing sugar. The immobilized cellulase was further employed for hydrolyzing the cell walls of microalgae. The experimental results indicate that under the specific reaction time, cell walls of microalgae were broken without releasing of chlorophyll. After breaking the cell walls of microalgae by hydrolysis reaction with immobilized cellulase, 1.5 mg chlorophyll was extracted with acetone, on the other hand, only 0.4 mg chlorophyll was obtained from the microalgae without hydrolysis before extraction. In reusability experiments, the efficiency retained 88% of its initial value after 10 time uses of the immobilized cellulase. Since magnetic nanoparticles are super-paramagnetic materials with strong magnetic properties, the immobilized cellulase on PAN coated magnetic particles can be easily recovered.
論文目次 摘要........................................................................................................Ⅰ
Abstract...................................................................................................Ⅱ
致謝.........................................................................................................Ⅲ
目錄.........................................................................................................Ⅴ
表目錄.....................................................................................................Ⅸ
圖目錄.....................................................................................................Ⅹ
第一章 緒論............................................................................................1
1.1前言..............................................................................................1
1.2研究動機與目的..........................................................................2
第二章 文獻回顧....................................................................................4
2.1葉綠素..........................................................................................4
2.1.1葉綠素的來源與選擇.........................................................6
2.1.2葉綠素的萃取.....................................................................8
2.2 纖維素........................................................................................11
2.2.1纖維素的水解....................................................................12
2.3纖維素分解酵素.........................................................................14
2.3.1纖維素分解酵素之應用....................................................14
2.3.2纖維素分解酵素之種類....................................................16
2.4酵素固定化.................................................................................18
2.4.1酵素固定化之簡介............................................................18
2.4.酵素固定化方法之介紹.......................................................19
2.5 磁性奈米粒子............................................................................23
2.5.1磁學理論............................................................................24
2.5.2氧化鐵磁性奈米粒子之簡介............................................25
第三章 實驗材料與方法.......................................................................28
3.1實驗藥品與設備.........................................................................28
3.1.1實驗藥品............................................................................28
3.1.2實驗設備............................................................................30
3.2實驗方法.....................................................................................31
3.2.1磁性奈米粒子之製備........................................................31
3.2.2以聚丙烯腈包覆磁性奈米粒子之製備............................31
3.2.3活化以聚丙烯腈包覆之磁性奈米粒子............................32
3.2.4纖維素分解酵素之固定化................................................33
3.2.5固定化纖維素分解酵素水解微藻細胞壁.........................35
3.2.6葉綠素之萃取.....................................................................35
3.3分析方法...................................................................................36
3.3.1 TEM粒徑分析.................................................................36
3.3.2磁性分析..........................................................................36
3.3.3傅立葉紅外光吸收光譜分析..........................................37
3.3.4蛋白質定量分析..............................................................37
3.3.5還原醣定量分析..............................................................38
3.3.6葉綠素定量分析..............................................................40
第四章 結果與討論.............................................................................41
4.1固定化前後以聚丙烯腈包覆之磁性奈米粒子物性分析.......41
4.1.1 TEM粒徑分析.................................................................41
4.1.2磁性分析..........................................................................43
4.1.3傅立葉紅外光吸收光譜分析..........................................44
4.2 酵素固定化之探討..................................................................46
4.2.1活化時間對酵素固定化的影響......................................46
4.2.2固定化溫度對酵素固定化的影響..................................47
4.2.3固定化pH值對酵素固定化的影響...............................48
4.2.4固定化時間對酵素固定化的影響..................................49
4.2.5酵素濃度對酵素固定化的影響......................................50
4.3水解反應之最適化條件探討.....................................................53
4.3.1反應溫度對固定化酵素水解微藻細胞壁的影響..........53
4.3.2反應pH值對固定化酵素水解微藻細胞壁的影響.......54
4.3.3藻液濃度對固定化酵素水解微藻細胞壁的影響..........56
4.4以固定化酵素水解後之微藻中萃取葉綠素.............................57
4.4.1萃取溶劑之選擇..............................................................58
4.4.2固定化酵素與微藻之最適水解時間..............................59
4.4.3丙酮體積對於葉綠素萃取效益的影響..........................60
4.4.4丙酮萃取時間對於葉綠素萃取效益的影響..................61
4.5固定化酵素重複使用性之探討.................................................62
第五章 結論與未來展望.....................................................................64
5.1結論.............................................................................................64
5.2未來展望.....................................................................................66
參考文獻...............................................................................................67
自述.......................................................................................................71
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