利用乳酸菌及酵母菌之酵素轉換人參皂苷

Abstract

人參皂苷為人參裡的主要活性成分，水解其上醣基能增加生理功效，透過具專一性且作用條件溫和的生物方法能轉換出特定的人參皂苷。過去文獻指出Bifidobacterium longum具有豐富的醣苷水解酵素且於發酵過程中似乎能轉換帶有不同種醣基的人參皂苷，因此本實驗欲探討其中參與的酵素。首先製備B. longum的粗萃酵素並測定活性，結果發現菌株本身 (whole cell fraction) 能水解簡單受質 (p-nitrophenyl glycoside) 上的葡萄糖、阿拉伯吡喃糖及阿拉伯呋喃糖，然而於人參皂苷的轉換上，僅能水解Rb1上20號碳以β-1,6鍵結的外側葡萄糖以及人參皂苷Rc的阿拉伯呋喃糖。欲進一步探討B. longum裡多個阿拉伯呋喃糖基水解酶是否會轉換人參皂苷，後續利用cloning技術及Escherichia coli系統製備重組酵素並純化以得到單一酵素，結果發現，測試的六個阿拉伯呋喃糖基水解酶中，只有一個 (Araf0101) 能有效將人參皂苷Rc轉換成Rd，其餘則利於水解含阿拉伯糖之多醣。Araf0101不僅能成功地被大量表現並純化，也能搭配先前實驗室發現來自酵母菌Kluyveromyces marxianus的葡萄糖基水解酶 (KmBgl1)，應用於提高人參裡的活性皂苷compound K之含量。於反應2小時後，單一KmBgl1可產出13.29±3.87 μmole/g compound K，而同時添加Araf0101和KmBgl1，則能將含量提升至20.91±3.74 μmole/g。

Biological conversion is a useful way to transform the major ginsenosides to minor deglycosylated ginsenoside with more pharmacologic activity. Bifidobacterium longum has the abundant glycosyl hydrolases and seems to transform some ginsenosides with various glycosides during fermentation. The purpose of this research is to explore the possible enzymes from B. longum for transforming the ginsenosides. The results demonstrated the whole cell of B. longum showed glucoside-releasing, arabinofuranose-releasing and arabinopyranose-releasing activity using p-nitrophenyl glycosides as the substrate. However, it had limited ability to hydrolyze the C-20 outer glucoside on Rb1 and arabinofuranoside on Rc. By comparison of candidate enzymes, we discovered that the one of α-L-arabinofuranosidase (Araf0101) can perform the effective biotransformation of ginsenoside Rc to Rd though it had mild activities on hydrolyzing arabinose-containing polysaccharides. Araf0101 was not only overexpressed and purified successfully but also applied to combine with glucosidase from Kluyveromyces marxianus to enhance the concentration of bioactive ginsenoside compound K in ginseng. After incubation for 2 hr, KmBgl1 produced 13.29±3.87 μmole/g compound K while Araf0101 and KmBgl1 added simultaneously could rise the yield to 20.91±3.74 μmole/g.