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研究生：	陳靜儀 Chen, Jing-Yi
論文名稱：	固定化纖維素分解酵素於微藻細胞壁水解之研究 Immobilized Cellulase for Microalgal Cellulose Hydrolysis
指導教授：	吳文騰 Wu, Wen-Teng
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	83
中文關鍵詞：	固定化纖維素分解酵素、電紡織奈米纖維膜、微藻
外文關鍵詞：	microalgae, electrospun nanofiber, immobilized-cellulase
相關次數：	點閱：90 下載：3
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本研究將纖維素分解酵素固定於高比表面積之電紡織聚丙烯腈奈米纖維膜上，透過amidination reaction活化聚丙烯腈奈米纖維膜上的C≡N官能基，與纖維素分解酵素上的胺基官能基形成共價鍵結，並藉由FT-IR的分析，證明纖維素分解酵素以共價鍵結方式固定在奈米纖維膜上。在奈米纖維膜活化時間為7.5分鐘，固定化酵素濃度為2 wt%、固定化時間為30分鐘、固定化溫度為40oC及固定化pH值為4.6的條件下，製備出較高蛋白質固定量及高比活性之固定化纖維素分解酵素，其蛋白質固定量與比活性分別為28 mg-protein /g-material及3.68U/mg-protein。將本研究所製備之纖維素分解酵素應用於水解微藻細胞壁，用以生產還原醣，於最適化水解反應條件下之水解產率及葡萄糖產率分別為62%及50%，且重複進行批次反應5次後，其水解產率仍可維持起始水解產率的40%，證明本研究所製備之固定化纖維素分解酵素，具有重複使用的優點。由於微藻中含有油脂可作為其他工業應用，透過固定化纖維素水解酵素水解製程，亦有助於增加藻體之油脂萃取率，大幅提升酵素製程於工業應用上之競爭力。

Polyacrylonitrile (PAN) nanofibrous membrane providing huge surface area could be manufactured by electrospinning as support for enzyme immobilization. The enzyme, Aspergillus niger cellulase was immobilized on electrospun PAN nanofibrous membranes by amidination reaction for long term operation. In addition, covalent bond formation between enzyme molecule and the nanofiber was confirmed from FT-IR measurement. The optimal operation conditions of cellulase immobilization for the highest specific activity as the enzyme concentration of 2 wt%, immobilization time of 30 min, temperature at 40°C and pH value at 4.6. The immobilized cellulase onto PAN nanofibrous membranes was further employed to investigate the hydrolysis reaction of microalgal cellulose. The highest yields of reducing sugars and glucose were 62% and 50% respectively under the optimal conditions. Since microalgal compositions were not only cellulose but also lipid, the lipid can be extracted for other applications. After the enzymatic process, the yield of extraction was 1.75 times higher than that of unpretreated by cellulase. The observed results indicate that the enzymatic hydrolysis of microalgal cellulose has the potential industrial applications for producing reducing sugars.

目錄
摘要....................................................................................................................I
Abstract ............................................................................................................ II
誌謝................................................................................................................. III
目錄.................................................................................................................IV
表目錄.......................................................................................................... VIII
圖目錄.............................................................................................................IX
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 纖維素................................................................................................. 4
2.1.1 纖維素之水解........................................................................... 5
2.1.2 纖維素的來源與選擇............................................................... 6
2.2 纖維素分解酵素............................................................................... 11
2.2.1 纖維素分解酵素之應用......................................................... 11
2.2.2 纖維素分解酵素之種類......................................................... 13
2.2.2.1 內切型纖維素分解酵素(endo-β-1,4-D-glucanase) ..... 13
2.2.2.2 外切型纖維素分解酵素(exo-β-1,4-D-glucanase) ....... 14
2.2.2.3 纖維雙醣分解酵素(cellobiase) .................................... 14
2.3 酵素固定化....................................................................................... 15
2.3.1 固定化之定義與性質............................................................. 15
2.3.2 固定化之方法......................................................................... 17
2.4 奈米纖維膜....................................................................................... 21
2.4.1 奈米纖維膜之製備程序......................................................... 21
2.4.2 電紡織技術簡介..................................................................... 23
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 聚丙烯腈奈米纖維膜之製備................................................. 31
3.2.2 酵素固定化之方法................................................................. 32
3.2.3 總醣定量之方法..................................................................... 34
3.2.4 還原醣定量之方法................................................................. 35
3.2.5 葡萄糖定量之方法.................................................................. 37
3.2.6 蛋白質定量分析..................................................................... 38
3.2.7 酵素活性之分析..................................................................... 39
3.2.8 藻類油脂定量分析.................................................................. 40
3.2.9 固定化纖維素分解酵素水解微藻細胞壁............................. 42
3.2.10 微藻溶液經酵素反應後之油脂萃取可行性分析................ 44
第四章 結果與討論....................................................................................... 45
4.1 固定化前後奈米纖維膜之比較....................................................... 45
4.1.1 奈米纖維膜之表面分析.......................................................... 45
4.1.2 奈米纖維膜之FT-IR 分析...................................................... 50
4.2 酵素固定化參數之探討................................................................... 52
4.2.1 活化時間對酵素固定化的影響.............................................. 52
4.2.2 酵素濃度對酵素固定化的影響............................................. 53
4.2.3 固定化時間對酵素固定化的影響......................................... 55
4.2.4 固定化溫度對酵素固定化的影響......................................... 56
4.2.5 固定化pH 值對酵素固定化的影響.................................... 58
4.3 固定化酵素水解微藻細胞壁生產還原醣........................................ 60
4.3.1 固定化纖維素分解酵素之水解時間曲線............................. 60
4.3.2 不同溶液系統對於藻類細胞壁水解產率之影響.................. 61
4.3.3 微藻油脂含量對於藻類細胞壁水解產率之影響................. 62
4.3.3 水解反應之最適化條件探討................................................. 65
4.3.3.1 反應溫度對於藻類細胞壁水解產率之影響............... 65
4.3.3.2 反應pH 值對於藻類細胞壁水解產率之影響............ 68
4.3.3.3 藻液濃度對於藻類細胞壁水解產率之影響............... 70
4.3.4 固定化酵素重複使用性之探討............................................. 72
4.4 酵素反應後藻類萃取油脂之可行性分析........................................ 74
第五章 結論與未來展望............................................................................... 75
5.1 結論................................................................................................... 75
5.2 未來展望........................................................................................... 77
參考文獻......................................................................................................... 78
自述................................................................................................................. 83

表目錄
表2-1 強酸水解與酵素水解特點之比較...................................................... 6
表2-2 小球藻之營養組成比例.................................................................... 10
表2-3 小球藻之礦物質或維生素含量........................................................ 10
表2-4 纖維素分解酵素在工業上的應用.................................................... 12
表2-5 纖維素分解酵素之分類與作用機制................................................. 13
表2-6 各種固定化方法之優缺點比較表.................................................... 20
表2-7 奈米纖維製備程序比較一覽表........................................................ 22
表2-8 奈米纖維製備程序之優缺點比較表................................................ 22
表2-9 1994 年起至2002 年電紡織相關之期刊文章數統計資料.............. 24
表2-10 美國電紡織技術之應用與專利統計圖.......................................... 24
表2-11 電紡織參數對奈米纖維形態之影響.............................................. 27
表3-1 DNS 試劑成分表................................................................................ 36
表4-1 水解反應前後藻體之油脂分析........................................................ 64
表4-2 萃取時間與萃取率之關係................................................................ 74

圖目錄
圖2-1 纖維素之化學結構.............................................................................. 4
圖2-2 植物纖維結構....................................................................................... 7
圖2-3 顯微鏡下所觀察之小球藻 (放大400 倍).......................................... 9
圖2-4 酵素熱展開失活之概念圖................................................................ 16
圖2-5 固定化方法之一般分類法................................................................ 19
圖2-6 奈米纖維之應用領域與用途............................................................ 25
圖2-7 電紡織過程示意圖............................................................................ 27
圖3-1 電紡織設備示意圖............................................................................ 32
圖3-2 酵素固定化反應示意圖.................................................................... 33
圖3-3 總醣測定法之檢量線........................................................................ 35
圖3-4 DNS 反應示意圖................................................................................ 36
圖3-5 還原醣測定法之檢量線.................................................................... 37
圖3-6 葡萄糖水劑測定法之檢量線............................................................ 38
圖3-7 蛋白質定量分析檢量線.................................................................... 39
圖3-8 酵素固定化薄膜反應器示意圖........................................................ 43
圖4-1 聚丙烯腈奈米纖維膜之SEM 圖（放大5000 倍）........................ 47
圖4-2 聚丙烯腈奈米纖維膜之SEM 圖（放大50000 倍）...................... 47
圖4-3 活化後聚丙烯腈奈米纖維膜之SEM 圖（放大5000 倍）............ 48
圖4-4 活化後聚丙烯腈奈米纖維膜之SEM 圖（放大50000 倍）.......... 48
圖4-5 固定化纖維素分解酵素（放大5000 倍） ...................................... 49
圖4-6 固定化纖維素分解酵素（放大50000 倍） .................................... 49
圖4-7 FT-IR 圖譜........................................................................................... 51
圖4-8 活化時間對蛋白質固定量之影響.................................................... 53
圖4-9 酵素濃度對蛋白質固定量與比活性之影響.................................... 54
圖4-10 固定化時間對蛋白質固定量與比活性之影響.............................. 56
圖4-11 固定化溫度對蛋白質固定量與比活性之影響.............................. 57
圖4-12 固定化pH 值對蛋白質固定量與比活性之影響........................... 59
圖4-13 固定化纖維素分解酵素水解藻類細胞壁之時間曲線.................. 61
圖4-14 海水素存在對於固定化纖維素分解酵素水解微藻細胞壁之影響62
圖4-15 不同油脂含量之微藻對固定化纖維素分解酵素水解微藻細胞壁
之影響............................................................................................... 64
圖4-16 反應溫度對於水解產率之影響...................................................... 67
圖4-17 反應溫度對於葡萄糖產率之影響.................................................. 67
圖4-18 反應pH 值對水解產率的影響....................................................... 69
圖4-19 反應pH 值對葡萄糖產率的影響................................................... 69
圖4-20 藻液濃度對水解產率的影響.......................................................... 71
圖4-21 藻液濃度對於葡萄糖產率的影響.................................................. 71
圖4-22 固定化纖維素分解酵素之重複使用性測試................................ 73
                                    

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