- 學位論文
探討不同β-葡聚醣酶進行羅漢果皂苷轉醣作用
Effect of β-glucanases on transglycosylation of mogrosides
摘要
羅漢果 (Luo Han Kuo, LHK)中主要的甜味物質為Mogroside V (MG V)，為一帶有五個葡萄糖的皂苷結構。而iso-mogroisde V (IMV)是較MG V甜度更高的皂苷同分異構物。兩者差異是在皂苷元三號碳上雙醣之間的鍵結不同。MG V為β-1,6鍵結，而IMV為β-1,4鍵結。因為自然界中IMV含量非常稀少，且無法透過化學合成產生，因此希望透過微生物或轉醣酵素產生IMV。實驗中Saccharomyces cerevisiae野生型菌株會將羅漢果皂苷萃取物中所含微量的IMV水解，無法達到產生IMV的目的，但產生未知的MG V 同分異構物 (compound 𝓧)，為了瞭解compound 𝓧的甜度、結構和功效，需要產生大量的compound 𝓧。首先，透過基因缺陷菌株轉換羅漢果皂苷，並且與S. cerevisiae野生型菌株比較發酵結果的差異，篩選出可能的轉醣基因。將可能的轉醣基因放回原本的缺陷菌株中進行確認，證實 compound 𝓧 的生成與外切β-1,3葡聚醣酶Exg1有關。之後利用大量表現EXG1基因的菌株和純化的ScExg1酵素轉換羅漢果希望得到大量的compound 𝓧，但產量仍然很少。實驗中也利用Dekkera bruxellensis的外切葡聚醣酶 (DbExg1)進行相同反應，然而DbExg1則沒有產生compound 𝓧和IMV的活性。因此在本研究中雖未能找到可轉換IMV的酵素，但也證實S. cerevisiae Exg1具有微弱的compound 𝓧轉換能力。未來也期望利用其他β-1,4葡聚醣酶轉換出IMV。
並列摘要
Mogroside V (MG V), a mogrol pentaglucoside, is the major sweet saponin in the fruit of Siraitia grosvenori Swingle (Luo Han Kuo, LHK). Recently, a rare isomer of MG V (iso-mogroside V, IMV) has been identified with a higher potency sweet taste than MG V. Interestingly, the difference between MG V and IMV is the linkage of disaccharide at C3 of mogrol. The linkage of MG V is β-1,6, while that of IMV is β-1,4. To produce IMV, Saccharomyces cerevisiae containing many glucosyltransferase and glucosidases was employed. Unexpectedly, we found that wild-type strain could produce an unknown mogroside V isomer-compound 𝓧 but hydrolyze IMV. To further understand the sweet potency, structure, and biological effects of compound 𝓧, the yield of compound 𝓧 should be increased. However, the gene for transglycosylation was unknown. We screened wild-type cells and genetic mutant strains with defects of glucosidases or glucosyltransferases to explore the conversion phenotypes, and the confirmation test was then conducted by the strain complemented with the candidate gene. EXG1 encoding exo-1,3-β-glucanase was confirmed to be responsible for the production of compound 𝓧. Next, the strategies to increase the production of compound 𝓧 were bioconversion of Luo Han Kuo by the strains with overexpression of ScEXG1 and by ScExg1 in vitro transglycosylation assay. However, the yield of compound 𝓧 wasn’t improved. As such, we wonder whether the exo-glucanase from Dekkera bruxellensis (DbExg1) presents the transglucosidase activity, and the result demonstrated that DbExg1 has almost no activity to produce compound 𝓧 and IMV. Although we didn’t identify the enzyme for producing IMV, we found that ScExg1 has transglucosidase activity to produce compound 𝓧. It is hoped that we can explore other β-1,4-glucanases to bioconvert IMV in the future.
參考文獻
延伸閱讀
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