建立固定化β-葡萄糖苷酶系統生產賽門苷Ⅰ和羅漢果苷Ⅳ

Abstract

羅漢果 (Siraitia grosvenorii) 為中國廣西省桂林市特產之一，可作為中藥材使用，具有潤肺、舒緩咳嗽及預防便祕等功效。近年來，羅漢果皂苷相關的研究日漸增加，其中指出siamenoside Ⅰ具有比5%蔗糖水溶液563倍的甜度，且相較於其他羅漢果皂苷風味較佳，更適合作為甜味劑使用。mogroside Ⅳ可減緩肺纖維化症狀與抑制癌細胞增殖，在生醫藥產業中極具發展潛力。於先前的研究中常以化學水解的方式進行皂苷的轉換，但使用化學水解有環境危害及產量低的問題，故本研究目的為建立一固定化酵素的連續式生物反應器以生產siamenoside Ⅰ和mogroside Ⅳ，降低環境危害及有效提高產量並增加酵素再利用性。結果顯示固定化β-葡萄糖苷酶在戊二醛濃度1.5%、載體活化時間1小時及結合酵素時間12小時有最佳的相對活性，並且在60oC、pH 5的環境下有最佳反應活性，後續填充已固定β-葡萄糖苷酶之玻璃微粒（glass microspheres）至反應器中並維持最佳活性環境，利用控制流速的方式通入羅漢果萃取物水溶液，以0.3 mL/min的流速下可得最高濃度之siamenoside Ⅰ；0.2 mL/min的流速下可得最高濃度之mogroside Ⅳ。藉由開發此種固定化酵素系統以製備大量且純度高之siamenoside Ⅰ與mogroside Ⅳ。

Siraitia grosvenorii (Swingle) is abundant in Guangxi province, China. Several studies have reported its benefits, such as moisturizing lungs, soothing coughs, reducing blood pressure and preventing constipation. Recently, research related to mogroside is increasing. Siamenoside I has been reported to be 563 times sweeter than 5% sucrose solution without bitterness compared to others; Mogroside IV has also been found several positive effects for attenuating liver fibrosis and suppressing the proliferation of cancer cells. Mogroside IV shows high potential to be applied in pharmaceutical industry. Traditionally, the most common conversion method is acid hydrolysis which has some drawback, such as low efficiency and polluted environment. On the contrary, immobilized enzyme shows high specificity, reusability and stability. Therefore, our objective is to develop immobilized enzyme bioreactor system to produce the siamenoside Ⅰ and mogroside Ⅳ as well as improving its productivity. The results show that the optimal conditions of immobilization with glutaraldehyde concentration 1.5%, activation time 1 hour and coupling time 12 hours. Moreover, immobilized enzyme has the highest relative activity at 60℃ and pH 5. Subsequently, the glass microsphere with immobilized β-glucosidase was filled into the reactor and the optimal environment was maintained. We found that the most content of siamenoside I was transformed in feed rate 0.3 mL/min and mogroside IV was in 0.2 mL/min through introducing the solution of Luo Han Kuo extract.