臺灣博碩士論文加值系統

銀杏 (Ginkgo biloba) ，其種子稱白果，常作傳統藥材；銀杏葉萃取物亦廣泛應用於臨床治療；然而銀杏葉和白果種皮中含有大量銀杏酸（ginkgolic acid），具有嚴重過敏性。因此德國 Commission E 規定，其驗出量不得高於 5 ppm。
本研究以酵素固定化的方式，將漆氧化酶 (laccase) 固定於玻璃微球、纖維素顆粒和尼龍載體，方便反應完後可回收重複利用，解決酵素成本高昂的缺點。將載體與酵素進行交聯反應後，以掃描式電子顯微鏡觀察，發現固定化處理前後載體表面具有明顯差異；且於原子能譜 (electron spectroscopy for chemical analysis, ESCA) 發現，固定化處理後載體表面均出現 C=N 鍵結，佐證酵素確實與載體結合。並量測各載體上酵素固定率分別為：玻璃微球 88.0%、纖維素顆粒 18.3%、尼龍載體 74.1%。進一步將各載體上之酵素與游離態酵素活性比較，玻璃微球活性可達游離酵素之90.9%，而纖維素顆粒與尼龍載體分別僅有65.8% 及 60.4%；酵素動力學測試中，玻璃微球具有最小的 Km (0.06) 與最大的 Vmax (30.5)，對比纖維素顆粒 0.21、30.0 與尼龍載體 0.22、29.6。證實玻璃微球具有最好的固定率與反應活性，因此選用玻璃微球為載體，進行銀杏酸的降解反應。
進一步進行反應條件最適化，發現溫度於 40 – 50oC、pH於4 – 5的環境中，固定化 laccase 可達最高活性。但是當溫度超過 40 oC 後，反應時間拉長，固定化 laccase 活性隨之下降；因此將溫度設定於25 oC、pH = 4進行反應。以高效液相層析儀 (high performance liquid chromatography, HPLC) 偵測銀杏葉萃取物中銀杏酸含量，經固定化 laccase 催化降解反應後，銀杏葉萃取物中的總銀杏酸量可降解至低於最小偵測極限 ( 2.12 ppm )。於穩定性實驗中，固定化 laccase 可回收重複使用達 25次；亦可長時間貯存達10週後再使用；確認其活性都仍可有效進行降解反應。證實固定化 laccase 可重複使用以降低生產成本，且耐久存，具有產業應用價值。
關鍵字：銀杏酸、酵素固定化、漆氧化酶、酵素動力學

Ginkgo biloba is known as the ginkgo tree. The tree is widely cultivated in northeast Asia and its fruit has various uses in traditional medicine. Ginkgo biloba leaf extract also have been proved to have wide biological activities. However, ginkgolic acid is a toxic compound in the leaf and the fruit of Ginkgo biloba and it should be less than 5 ppm in the ginkgo product in the guide of Commission E.
Laccase was immobilized on solid carriers by crosslinking reaction for the degradation of ginkgolic acid in this study. The binding ratio of laccase could reached 88% on the carrier. In SEM image, carriers with immobilized treatment had significant difference compared with carriers before immobilized in surface morphology. ESCA image also shown that the signal of C=N double bond appeared after carrier with immobilized treatment. These results proved that laccase immobilized on the carriers after crosslinking reaction.
Degradation efficiency of immobilized laccase was also well researched in this study. The activity of immobilized laccase could be 25.0 ± 3.06 compared with free laccase (27.5 ±1.67). Moreover, the Km value of immobilized laccase is 0.06 compared with free laccase (0.10). The complete reaction can be less than 1 hour and the reusability and storage stability were also further verified. The enzymatic system can be used more than 25 cycles and 10 weeks which still retained 73.8% of enzyme activity. The results indicated that the immobilized laccase system exhibited a significant efficiency for ginkgolic acid degradation in Ginkgo biloba leaf extract. It is capable of being further applied to prepare large scale of ginkgolic acid free ginkgo product.

Key words : ginkgolic acid, enzyme immobilization, laccase and enzyme kinetic.

目錄

摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 x
壹、 前言 1
貳、 文獻回顧 3
2.1 銀杏簡介 3
2.1.1 銀杏傳統功效 3
2.1.2 銀杏有效成分 3
2.1.3 銀杏葉近代醫學研究 6
2.1.4 銀杏專利與產品市場價值分析 7
2.1.5 銀杏酸 9
2.1.5.1 銀杏酸常見降解方法 10
2.1.5.2 酵素降解法 10
2.2 漆氧化酶 10
2.2.1 漆氧化酶結構 12
2.2.2 漆氧化酶作用機制 13
2.2.3 漆氧化酶的應用 14
2.3 酵素固定化 15
2.3.1 酵素固定化方法 16
2.3.1.1 吸附法 16
2.3.1.2 共價結合法 16
2.3.1.3 交聯法 18
2.3.1.4 酵素包埋法 18
2.3.2 載體材料 21
2.3.2.1 無機載體 21
2.3.2.2 有機高分子載體 21
2.3.2.3 複合載體 22
2.3.2.4 廢棄物載體材料 22
參、 材料與方法 23
3.1 研究目的 23
3.2 實驗架構 24
3.3 實驗材料 24
3.3.1 銀杏葉萃取物 24
3.3.2 實驗藥品 24
3.3.3 儀器設備 26
3.4 實驗方法 27
3.4.1 固定化laccase置備 27
3.4.1.1 尼龍 (Nylon pellets) 27
3.4.1.2 纖維素顆粒 (Cellulose beads) 27
3.4.1.3 玻璃微球 (Glass microspheres) 27
3.4.1.4 將載體與laccase共價結合 27
3.4.2 Laccase含量檢測 28
3.4.3 Laccase 活性檢測 28
3.4.4 載體表面型態分析 29
3.4.5 載體表面共價鍵分析 29
3.4.6 最佳反應條件測試 29
3.4.6.1 溫度 29
3.4.6.2 pH 30
3.4.6.3 酵素動力學參數測定 30
3.4.7 銀杏酸反應液製備 30
3.4.8 銀杏酸定量分析 30
3.4.9 統計分析 31
肆、 結果與討論 32
4.1 Laccase固定化系統建立 32
4.1.1 Laccase吸光值標準曲線製作 32
4.1.2 載體上酵素固定量測試 34
4.1.3 Laccase固定前後載體表面微觀結構分析 36
4.1.3.1 尼龍載體固定化處理前後表面微觀結構分析 36
4.1.3.2 纖維素顆粒載體固定化處理前後表面微觀結構分析 36
4.1.3.3 玻璃微球載體固定化處理前後表面微觀結構分析 37
4.1.4 固定化laccase與載體表面元素和化學鍵結分析 41
4.1.4.1 尼龍載體表面元素和化學鍵結分析 42
4.1.4.2 纖維素顆粒表面元素和化學鍵結分析 42
4.1.4.3 玻璃微球載體表面元素和化學鍵結分析 43
4.1.5 固定化 laccase系統反應活性測試 47
4.1.6 固定化 laccase系統反應pH和反應溫度測試 49
4.1.7 固定化 laccase系統酵素動力學參數 52
4.1.8 玻璃微球酵素固定系統穩定性測試 56
4.1.8.1 溫度穩定性測試 56
4.1.8.2 貯存穩定性測試 58
4.1.8.3 重複操作利用穩定性測試 58
4.2 固定化酵素系統對於銀杏酸之降解 61
4.2.1 HPLC-UV對銀杏酸分析與定量 61
4.2.2 銀杏酸經 laccase酵素系統降解 65
4.2.3 銀杏葉萃取液經由 laccase酵素降解之結果分析 69
4.2.4 玻璃微球固定化 laccase系統之穩定性測試 72
4.2.4.1 固定化 laccase進行銀杏酸降解反應連續27個批次之結果分析 72
4.2.4.2 固定化 laccase貯存穩定性測試 74
伍、 結論與展望 76
陸、 參考文獻 77
柒、 附錄 91
7.1 個人簡歷 91

 
圖目錄

圖 1 銀杏葉與銀杏果。 4
圖 2 銀杏黃酮結構圖。 5
圖 3 神經認知領域，銀杏專利年申請案量。 8
圖 4 漆氧化酶結構圖。 12
圖 5 漆氧化酶作用機制。 13
圖 6 H2O2分解過程能量變化。 15
圖 7 物理吸附法。 17
圖 8 共價結合法。 17
圖 9 交聯法。 19
圖 10 包埋法。 19
圖 11 實驗架構流程圖 24
圖 12 Laccase 酵素蛋白濃度標準曲線。 33
圖 13 尼龍載體酵素固定化前 (A-1) 30x (A-2) 10000x 和固定化後 (B-1) 30x (B-2) 10000x 表面微觀結構圖。 38
圖 14 纖維素顆粒載體酵素固定化前 (A-1) 30x (A-2) 10000x 和固定化後 (B-1) 30x (B-2) 10000x 表面微觀結構圖。 39
圖 15 玻璃微球載體酵素固定化前 (A-1) 10000x (A-2) 20000x 和固定化後 (B-1) 9500x (B-2) 20000x 表面微觀結構圖。 40
圖 16 尼龍載體酵素固定前 (A) 和酵素固定化後 (B) 之ESCA圖譜。 44
圖 17 纖維素顆粒酵素固定前 (A) 和酵素固定後 (B) 之ESCA圖譜。 45
圖 18 玻璃微球酵素固定前 (A) 和酵素固定化後 (B) 之ESCA圖譜。 46
圖 19 ABTS氧化反應結構圖。 47
圖 20 各酵素系統於不同pH值之反應活性。 50
圖 21 各酵素系統於不同溫度之反應活性。 51
圖 22 反應受質ABTS濃度對 (A)free laccase (B) 玻璃微球 (C) 纖維素顆粒 (D)尼龍 等固定方式催化反應之影響。 53
圖 23 反應受質ABTS濃度對 (A)free laccase (B) 玻璃微球 (C) 纖維素顆粒 (D)尼龍 等固定方式催化反應之影響雙倒數圖。 54
圖 24 (A) Free laccase (B) 玻璃微球固定化酵素 溫度穩定性測試。 57
圖 25 Laccase貯存穩定性測試。 59
圖 26 玻璃微球固定化 laccase重複使用反應能力測試。 60
圖 27 銀杏酸HPLC層析圖 (A) 銀杏酸C15和C17；(B) 銀杏葉粗萃液 (內含200 ppm之總銀杏酸)；(C) 於40°C 反應 30 分鐘之銀杏葉粗萃液。 63
圖 28 銀杏酸 C15 標準曲線。 64
圖 29 銀杏酸 C17 標準曲線。 64
圖 30 銀杏葉萃取物水溶液經由free laccase 和固定化laccase系統轉化銀杏酸 (A) C15 (B) C17 濃度隨時間的變化。 68
圖 31 不同濃度之銀杏酸對 (A)游離laccase (B)固定化laccase 降解催化反應之影響。 70
圖 32 不同濃度之銀杏酸對 (A)游離 laccase (B)固定化 laccase 降解催化反應之影響雙倒數圖。 71
圖 33 固定化 laccase於銀杏葉萃取液連續27個批次的重複試驗。 73
圖 34 固定化 laccase系統保存十週貯存穩定性測試。 75


表目錄

表 1 銀杏萜內脂結構。 5
表 2 各研究領域銀杏專利分布表。 8
表 3 銀杏酸結構。 9
表 4 固定化酵素在工業上固定方法與應用。 20
表 5 玻璃微球、纖維素和尼龍載體上固定化laccase含量。 35
表 6 游離態和玻璃微球、纖維素和尼龍載體固定化 laccase 活性比較。 48
表 7 Free laccase、玻璃微球、纖維素顆粒和尼龍等固定方式酵素動力學參數值。 55
表 8 銀杏酸C15、C17之偵測極限和定量極限 (ppm)。 63
表 9 Laccase酵素系統銀杏酸降解率。 66
表 10 游離 laccase和固定化 laccase擬合動力參數。 67
表 11 Free laccase、固定化laccase系統酵素動力學參數值。 70

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