利用醣基水解酶進行羅漢果皂苷轉醣作用

Abstract

Mogroside V (MG V) 與Iso-mogroside V (IM V)為具甜味的羅漢果三萜類皂苷。MG V甜度為蔗糖的300倍，但具有明顯的餘後味；IM V 甜度為蔗糖的500倍，無不良風味，但含量相當稀少，因此若能將結構相似之MG V轉變為IM V，將可增加羅漢果皂苷作為代糖的使用。而MG V與IM V之間的差異在於醣苷配基mogrol在三號碳處雙醣的鍵結方式，因此如何選擇具有良好專一性的酵素和糖基轉換方法將MG V轉變為IM V是目前最大的挑戰。醣基水解酶（Glycosyl hydrolases, GHs）對於受體和供體具有廣泛的靈活性，且在特定條件下可以將糖轉移到多種生物分子上，因此利用GHs對羅漢果皂苷進行轉醣是個有潛力的方式。 在我們的研究中，利用Kluyveromyces marxianus 的BGL1酵素(KmBGL1)、Trichoderma Reesei的纖維素酶、與Dekkera bruxellensis 的 EXG1 酵素 (DbEXG1)對羅漢果皂苷進行轉糖，然後以薄層層析(Thin layer chromatography, TLC)和高效能液相層析串聯質譜進行分析轉醣結果。在三種酵素中僅DbEXG1在0.02 U酵素濃度下，以MG V與羅漢果皂苷siamenoside I (S I)作為醣基受體，以pNP-glucose作為醣基供體時，能夠產生IM V，其中以MG V作為受體時轉換出的IM V產量 (2.99%) 較S I作為受體時 (0.22%) 高；而DbEXG1除了轉醣形成IM V外，亦能夠以β-1,6與β-1,2醣苷鍵的方式將醣基接於羅漢果皂苷上，形成多樣的醣基化羅漢果皂苷，如MG VI、MG VI a與MG VI b。綜上所述，DbEXG1的多醣基化能力具有合成新的羅漢果皂苷的潛力。

Mogroside V (MG V) and Iso-mogroside V (IM V) are sweet-tasting triterpene glycosides found in monk fruit. MG V has a sweetness that is 300 times that of sucrose, but with a noticeable aftertaste; IM V has a sweetness that is 500 times that of sucrose, without any unpleasant flavor, but it is present in very small quantities. Therefore, if the structurally similar MG V can be transformed into IM V, it would increase the usage of monk fruit mogrosides as sugar substitutes. The main challenge in this transformation is the difference in the way the glycosyl moieties is bound to the mogrol at C3 position in MG V and IM V. Finding enzymes with high specificity and glycosylation methods to convert MG V into IM V is currently a significant challenge. Glycosyl hydrolases (GHs) have broad flexibility for both acceptors and donors, and under certain conditions can transfer sugars onto various biomolecules, making them a promising method for glycosylation of mogrosides. In our research, we utilized the enzymes BGL1 from Kluyveromyces marxianus (KmBGL1), cellulase from Trichoderma Reesei, and EXG1 from Dekkera bruxellensis (DbEXG1) to perform glycosylation on mogrosides, and then analyzed the glycosylation results using thin-layer chromatography (TLC) and high-performance liquid chromatography coupled with mass spectrometry. Among the three enzymes, only DbEXG1, at a 0.02 U enzyme concentration, was able to produce IM V using MG V or siamenoside I (SI) as glycosyl acceptors and pNP-glucose as the glycosyl donor. The yield of IM V produced using MG V as an acceptor (2.99%) was higher than when SI was used as an acceptor (0.22%). Furthermore, DbEXG1 was not only able to form IM V through glycosylation but could also attach sugar mogrosides in a β-1,6 and β-1,2 glycosidic linkage manner, forming various glycosylated mogrosides such as MG VI, MG VI a, and MG VI b. In conclusion, the glycosylation ability of DbEXG1 has the potential for synthesizing new mogrosides.