博碩士論文 100326004 詳細資訊




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姓名 王俐涵(Li-han Wang)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 以游離及固定化酵素促進氯酚化合物偶合反應之研究
(Oxidative coupling of chlorophenol catalyzed by isolated and immobilized enzymes)
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摘要(中) 氯酚類化合物常見之處理方式有物理、化學、生物等,其中生物處理方法係利用微生物酵素去除氯酚被認為是對環境較為友善。本研究跳脫生物處理必需將污染物予以降解或被吸收移除的傳統思維,主要藉助微生物酵素催化氯酚產生氧化偶合反應以合成巨大、環境穩定且不具生物利用性之聚合物。為克服游離酵素之不穩定性和易受環境條件影響,選取廉價、易獲得之海藻酸鈉及幾丁聚醣製備固定化酵素並探討其活性影響條件及應用於催化氯酚偶合反應。
研究結果發現海藻酸鈉及幾丁聚醣固定化條件中利用海藻酸鈉濃度2%及氯化鈣濃度為0.1M;戊二醛濃度1%及酵素鍵結時間12小時所固定之酵素有最佳活性。不同pH值及溫度的活性測定,游離及海藻酸鈉固定化酵素之最佳pH值皆為4,幾丁聚醣固定化酵素則以pH3有最高的活性。游離及固定化酵素在20℃~50℃間有最大活性且差異不大,但固定化酵素之熱穩定性及儲存穩定性皆較游離酵素佳。
氯酚偶合實驗中,酵素添加量越多、反應時間越長、過氧化氫添加量越少,酵素可催化氯酚偶合產生新物質且偶合產物量也越多。固定化酵素能重複催化偶合氯酚,固定載體上之酵素活性顯示海藻酸鈉大於幾丁聚醣,但幾丁聚醣催化偶合效果則較穩定。酵素應用於不同溫度及pH值條件下偶合氯酚,固定化酵素在溫度20℃至60℃催化偶合效果較游離酵素穩定,且較能忍受高pH值的變異。
摘要(英) In this study, chlorophenol removal by enzyme was carried out. Oxidative coupling of chlorophenol was catalyzed by enzyme to form eco-friendly polymer. The enzyme was immobilized by sodium alginate and chitosan for the convenience of experimental design.
The optimal activity of the immobilized enzyme was found when the concentrations of the sodium alginate, calcium chloride, and flutaraldehyde were 2%, 0.1 M, and 1%, respectively, with 12 hours of immobilization. The optimal acidity for the mobilized enzyme and the one immobilized by sodium alginate was found at pH 4; optimal acidity of the enzyme immobilized by chitosan was found at pH 3. Both the mobilized and the immobilized enzymes had the optimal activity between 20 to 50°C. However, the immobilized enzyme possened better thermal stability and life time.
In the oxidative coupling, there was a positive correlation between the enzyme amount and the yield as well between the reaction time and the yield; a negative correlation was found between amount of hydrogen peroxide and the yield. Enzyme immobilized by sodium alginate was more active than that of immobilized by chitosan, but the coupling was more stable with the chitosan-immobilized enzyme. Between 20 to 60°C, the coupling of chlorophenol by the immobilized enzyme was more stable than the coupling by the mobilized one. Higher coupling yield was found with lower acidity.
關鍵字(中) ★ 酵素固定化
★ 海藻酸鈉
★ 幾丁聚醣
★ 氯酚偶合
關鍵字(英) ★ immobilized enzyme
★ sodium alginate
★ chitosan
★ chlorophenol coupling
論文目次 目錄 I
圖目錄 V
表目錄 VIII

第一章 前言 1

1-1 研究緣起 1
1-2 研究目的 2
第二章 文獻回顧 3

2-1 氯酚類化合物簡介 3
2-1-1 氯酚化合物之來源及性質 3
2-1-2 氯酚化合物的毒性與危害 4
2-1-3 含氯酚類化合物之處理方法 5
2-2 酵素 7
2-2-1 特性與種類 7
2-2-2 漆氧化酵素介紹( Laccases ) 9
2-2-3 過氧化酵素介紹( Peroxidase) 10
2-2-4 酵素在環工上之應用 11
2-3 酵素固定化 12
2-3-1 固定化方法 14
2-3-2 固定化載體選擇 19
2-4 偶合反應相關介紹 22
2-4-1 偶合反應( oxidative-coupling ) 22
2-4-2 傅里德-克拉夫茨反應( Friedel–Crafts reaction ) 23
2-4-3 酵素於偶合作用扮演之角色 24
2-4-4 酵素於酚類偶合作用過程中可能之機制 26
第三章 實驗內容、方法與設備 29

3-1 實驗內容 29
3-2 實驗方法 29
3-1-2 酵素活性分析 29
3-2-2 固定化酵素製備 30
3-2-2-1 海藻酸鈉 (Alginate) 30
3-2-2-2 幾丁聚醣 (Chitosan) 30
3-2-3 酵素最適反應條件 31
3-2-3-1 酵素活性作用之最適pH 31
3-2-3-2 酵素活性作用之最適溫度 32
3-2-3-3 海藻酸鈉固定化酵素 32
3-2-3-4 幾丁聚醣固定化酵素 32
3-2-3-5 酵素熱穩定性 33
3-2-3-6 酵素儲存穩定性 33
3-2-4 Laccase酵素與氯酚偶合實驗 34
3-2-4-1 游離Laccase酵素量對不同氯酚之偶合反應 34
3-2-4-2 不同時間游離Laccase對氯酚之偶合反應 34
3-2-4-3 固定化Laccase酵素對氯酚偶合重複批次實驗 34
3-2-4-4 Laccase於不同溫度及pH值之氯酚偶合反應 34
3-2-5 Peroxidase酵素與氯酚偶合實驗 35
3-2-5-1 過氧化氫量對不同氯酚之偶合反應 35
3-2-5-2 游離Peroxidase酵素量對不同氯酚之偶合 35
3-2-5-3 不同時間游離Peroxidase對氯酚之偶合反應 35
3-2-5-4 固定化Peroxidase酵素對氯酚偶合重複批次實驗 36
3-2-5-5 Peroxidase於不同溫度及pH值之氯酚偶合反應 36
3-3實驗設備 36
3-3-1 電子天平 36
3-3-2 水平震盪器 37
3-3-3 分光光譜儀 37
3-3-4 高效能液相層析儀(HPLC) 37
3-3-5 烘箱 38
3-3-6 pH計 38
3-3-7 高速攪拌器 38
3-3-8 蠕動幫浦 38
3-3-9 控溫水槽 39
3-3-10 SEM (Scanning electron microscopy) 39
3-3-11 液相層析串聯質譜儀(Q-Tof LC/MS/MS) 40
3-4 實驗材料 42
第四章 結果與討論 46

4-1 游離酵素之活性性 46
4-2 海藻酸鈉固定化酵素之活性 50
4-2-1 固定化酵素之最佳pH值及最佳溫度 50
4-2-2 海藻酸鈉濃度對酵素活性之影響 53
4-2-3 氯化鈣濃度對酵素活性之影響 54
4-3 幾丁聚醣固定化酵素之活性 55
4-3-1 固定化酵素之最佳pH值及最佳溫度 59
4-3-2 戊二醛濃度對酵素活性之影響 62
4-3-3 酵素鍵結時間對酵素活性之影響 63
4-4 游離及固定化酵素穩定性探討 64
4-4-1 熱穩定性 64
4-4-2 儲存穩定性 65
4-5 酵素對氯酚偶合反應 66
4-5-1 不同時間游離酵素對氯酚之偶合反應 70
4-5-2 游離酵素量對氯酚偶合反應之影響 74
4-5-3 過氧化氫量對氯酚偶合反應 80
4-5-4 固定化酵素對氯酚偶合重複批次實驗 84
4-5-5 溫度及pH值對氯酚偶合反應 89
4-5-6 氯酚偶合產物之鑑定 93
第五章 結論與建議 98

5-1 結論 98
5-2 建議 100
參考文獻 101
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