探討酵母菌中β-葡萄糖苷酶之特性及受質水解特異性

Abstract

葡萄糖苷水解酶普遍存在於生物體中，可從多醣或醣苷的非還原端水解醣苷鍵，常被食品工業用來水解帶醣基的物質，提升產品的生理活性及改善風味。皂苷為植物的二級代謝產物，通常以醣苷形式存在，過去文獻提及水解醣苷上葡萄糖基能改善風味及提升皂苷的生理活性。先前實驗室利用多株酵母菌與乳酸菌篩選具有苦瓜皂苷能力之菌株，發現酵母菌Kluyveromyces marxianus具有轉換苦瓜中苦味皂苷Momordicoside L及Momordicoside K能力，同時提升具有降血糖功效成分3β, 7β, 25-trihydroxycucurbita-5, 23 (E)-dien-19-al之含量，並觀察到K.marxianus中β-glucosidase (Bgl1) 蛋白可能為轉換苦瓜皂苷之目標蛋白。本研究利用基因突變菌株更進一步驗證Bgl1為K.marxianus中主要轉換苦瓜皂苷之蛋白，並以Escherichia coli BL21 (DE3)大量表現Bgl1，利用親和性管柱純化，將純化蛋白質進行受質水解特異性探討，結果顯示從結構簡單的單環之硝基苯酚 (nitrophenyl)、二環之香豆素 (coumarins)衍生物及三環之黃酮類 (flavonoids)化合物，至結構較複雜的四環三萜類化合物苦瓜皂苷及羅漢果皂苷，Bgl1能水解不同環狀結構上O-linked醣基，也能水解羅漢果皂苷上β-1,6鍵結之醣鏈，但無法水解β-1,2鍵結之醣鏈。了解Bgl1在水解位點上具有特殊性，不同於文獻中指出β-glucosidase對於皂苷上O-linked糖基水解能力差之特性，顯示Bgl1未來可應用於水解不同苷元型態之植物功效性成分。

Saponins are secondary metabolites of plants, and commonly exist as glycosyl forms. Microbial biotransformation is a useful way to remove the glycosyl group for improving the flavor and producing the bioactive compounds in foods. Our previous studies indicated that yeasts and lactic acid bacteria had limited capacity to convert the bitter melon saponins. In contrast, yeast Kluyveromyces marxianus could be able to transform momordicoside L to antidiabetic compound 3β, 7β, 25 -trihydroxycucurbita-5,23(E)-dien-19-al (THC). Besides, it was observed that β-glucosidase (Bgl1) in K.marxianus was the putative protein for conversion. Here, we provide further evidence to demonstrate that Bgl1 is the target protein for bitter melon saponins transformation. We also characterize substrate specificity by purified Bgl1 protein. The results demonstrate that Bgl1 hydrolyzes broad spectrum of glycones, including coumarins derivative, flavonoids and tetracyclic triterpenes. In addition, this enzyme performs the hydrolysis on both the β-linked O-glycosyl bond of above stated glycosides and β-1,6 glycosidic bond in mogrosides. In conclusion, we characterized the function and substrate specificity of Bgl1 enzyme which may be valuable for bioactive compound production in the near future.