固定化葡萄糖苷酵素應用於羅漢果皂苷去糖基之研究

Abstract

羅漢果皂苷為一群具有高度甜味的三萜類糖苷分子，已經被證實具有廣泛的生理功能，分子結構上以帶有五個β-D-葡萄糖基的Mogroside V為主要天然羅漢果的皂苷形式。有文獻研究指出羅漢果皂苷為麥芽糖酵素抑制劑，可以有效地控制餐後血糖濃度的上升，在不同的羅漢果皂苷中，Mogroside IIIE較Mogroside V具有更好的抑制小腸麥芽糖酵素活力的功能。利用酵素催化的方法可將五醣皂苷Mogroside V轉化為Mogroside IIIE，然酵素生產成本高，酵素本身容易失活，且不能重複使用，因此在實際應用中具有一定的侷限性。本實驗利用固定化酵素的方式，將葡萄糖苷酵素固定於載體上進行Mogroside V的轉化反應，此一方法的重複利用性可克服酵素高昂生產成本的問題。實驗中利用HPLC-UV系統可以有效地分離和定量酵素反應系統中四種不同的羅漢果皂苷，進一步利用HPLC/ESI tandem MS建立對於羅漢果皂苷的定性分析方法。以此觀察到在酵素反應的實驗中Mogroside V含量逐漸下降；兩種類型的四醣皂苷Siamenoside I和Mogroside IV含量先增長後下降；而Mogroside IIIE（三個醣基）含量逐漸上升；結果顯示葡萄糖苷酵素可選擇性水解Mogroside V的β-1,6糖苷鍵結，並經由五醣皂苷到四醣皂苷，最後到三醣皂苷 (Mogroside IIIE) 的轉換過程。

Mogrosides, a group of triterpene glycosides from the fruit of Siraitia grosvenorii (Swingle), have been proved to have wide biological activities. Mogroside V (with five glucose residues) and mogroside IIIE (with three glucose residues) are the major components of mogrosides in Lo han ko fruit. It has been demonstrated that mogroside IIIE exerts anti-hypoglycemic effect by inhibiting intestinal maltase activity than mogroside V. In this study, enzymatic hydrolysis was utilized for the enrichment of mogroside IIIE by β-glucosidase from Aspergillus niger that can hydrolyze glycosidic bonds to release the non-reducing terminal glycosyl residues from glycosides and oligosaccharides. Nevertheless, suspended enzyme catalysis exhibits some limitations in practical applications by economic concerns. β-Glucosidase was then immobilized on solid carriers for the catalysis of mogroside V with reusability for reducing costs. During the enzymatic reaction, mogroside V was transformed to mogroside III E via siamenoside I and mogroside IV. The results indicated that the immobilized glucosidase system enables to selectively hydrolyze the β-1,6-glycosidic linkage of mogrosides. Moreover, we attempt to establish HPLC-ESI tandem MS analytical method for qualitative and quantitative analysis of mogrosides. The development of the immobilized enzyme system achieved the conversion of mogroside V into mogroside IIIE, which is capable of being further applied to prepare large scale of mogroside IIIE.