不同酵母菌株對羅漢果皂苷轉化之影響

Abstract

先期研究發現不同種酵母菌(Saccharomyces cerevisiae 和Dekkera bruxellensis) 分泌的Exg1酵素具有不同生物轉換羅漢果(Siraitia grosvenorii Swingle)皂苷的能力。因此本實驗首先利用Exg1蛋白親緣性分析探討不同酵母菌株生物轉換粗羅漢果皂苷【mogroside V (MG V)、siamenoside I (S I)、mogroside Ⅳ (MG Ⅳ)和mogroside ⅢE (MG ⅢE)】的特性。實驗初步結果顯示Exg1親緣性相近的18株酵母菌水解羅漢果皂苷的能力不完全相同，並依羅漢果皂苷的生物轉換結果將菌株分成三群。第一群菌株主要生成MG ⅢE；第二群可觀察到生物轉換效率低，四種皂苷皆存在；第三群則是S I與MG ⅢE，隨發酵時間增加，S I有降低的趨勢，MG ⅢE隨之上升。我們進一步將隸屬第三群菌株以MG V標準品進行發酵，發現該群菌株生物轉換產物為S I和MG ⅢE，說明此類群菌株生成的酵素較傾向水解MG V三號碳端以β-1,6鍵結的醣基。據上述結果，由於Dekkera bruxellensis生物轉換羅漢果皂苷的效率低且欲提升Saccharomyces cerevisiae轉化羅漢果皂苷的效率，因此接著探討在序列相似度高的蛋白質中，影響皂苷轉換的關鍵胺基酸。初步結果顯示點突變酵素的生物轉換羅漢果皂苷速度與未修飾原酵素相比，ScExg1 I230L降低，ScExg1 A301S不變、ScExg1 Q304E提升、DbExg1 L207I不變、DbExg1 S278A和DbExg1 E281Q降低，說明經特定修飾後雖無法改變酵素專一性，但卻影響對於皂苷的轉換速率。綜合上述，我們以不同菌株發酵羅漢果皂苷並襯托出DbExg1基因能專一性將粗羅漢果皂苷中的MG V完全轉成S I，表示其獨特性與高度商業價值，期望未來可用作專門生產S I的最佳酵素，同時，我們也發現新一類群的菌株轉換羅漢果皂苷的機制與ScExg1和DbExg1截然不同。另外，目前無直接的證據證明氨基酸與轉換羅漢果皂苷的關係，但可依據可能的線索於未來做更深入的探討，期望對商業市場及酵素研究領域有所貢獻。

The previous studies found Exg1 was secreted from various yeasts (Saccharomyces cerevisiae and Dekkera bruxellensis) had different characteristics to bioconvert Siraitia grosvenorii Swingle mogrosides. Therefore, we investigated the bioconversion of crude Luo Han Guo (LHK) extract which contained mogroside V (MG V), siamenoside I (S I), mogroside Ⅳ (MG Ⅳ), and mogroside ⅢE (MG ⅢE) by different yeasts based on Exg1 evolutionary relationship. The results showed that 18 strains which had closly Exg1 evolutionary relationship to each other had different abilities to bioconvert mogrosides, and the strains were divided into three groups according to the bioconversion of LHK. The strains in first group mainly produced MG ⅢE; there are four kinds of compounds in the second group due to low bioconversion efficiency; the products of the third group were S I and MG ⅢE, with the fermentation time increased, the percent of S I ratio decreased and MG ⅢE ratio raised. MG V standard compound was further fermented with strains which belonged to third group, and found the products were S I and MG ⅢE. It indicated the enzyme produced by strains in third group tend to hydrolyze C3 end β-1,6 glycosidic bond of MG V. In order to improve low efficiency of mogrosides conversion by Dekkera bruxellensis and enhance the efficiency of mogrosides bioconversion by Saccharomyces cerevisiae, we used highly identitied Exg1 sequences to explore the key amino acids of LHK conversion. The results of point mutation showed that the rate to convert mogrosides by ScExg1 I230L was decreased, ScExg1 A301S was unchanged, ScExg1 Q304E was increased, DbExg1 L207I was unchanged, and DbExg1 S278A and DbExg1 E281Q were decreased compared with each unmodified enzyme. Although our specific modifications can’t change the enzyme specificity, they affect the conversion rate of mogrosides. To sum up, we used the different strains to ferment mogrosides can bring out the uniqueness and high commercial value of DbExg1 which can specifically convert MG V to S I. Hope it can be used as the best enzyme to produced S I in the future, and we also found the new mechanism of mogroside bioconversion by new group in our experiment which is different from ScExg1 and DbExg1. In addition, there is no direct evidence to prove the relationship between amino acids and conversion of mogrosides, but we can do more in-depth experiment based on possible clues, and contribute to commercial market and enzyme research.