以固定化漆氧化酶分解銀杏葉萃取物中銀杏酸之研究

Study on Immobilized-Laccase for Ginkgolic Acid Degradation in Ginkgo biloba Leaf Extract

吳鳴展(Ming-Chan Wu)

指導教授 ： 鄭光成

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703481

摘要

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

關鍵字

銀杏酸 ； 酵素固定化 ； 漆氧化酶 ； 酵素動力學

並列摘要

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.

並列關鍵字

ginkgolic acid ； enzyme immobilization ； laccase ； enzyme kinetic

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