大豆之isoflavone conjugates及其相關水解酵素之研究

Abstract

大豆異黃酮為植物雌激素重要來源之ㄧ。目前已有不少研究指出大豆異黃酮具有保健生理活性功效。異黃酮素主要有12種衍生物，於大豆中含量最高的為malonylglucosides形式之異黃酮，其次為β-glucosides形式之異黃酮，然而此兩類結構並非人體所能吸收之結構，若能將此類異黃酮轉換成人體較易吸收利用之aglyocnoes，則可提高大豆異黃酮的利用性。 根據本研究室先前關於大豆異黃酮之研究中發現，凍乾大豆粉於40℃熱反應，malonylglucosides含量會明顯減少，而aglycones含量則有上升的趨勢，因此推測大豆中存在著可水解malonylglucosides之相關酵素，因而著手進行對大豆異黃酮相關水解酵素之研究。 欲進行相關水解酵素之研究，需製備可供酵素作用之異黃酮素為反應基質，因此本論文亦包括精製純化malonylglucosides與glucosides等兩類異黃酮素的方法。 本研究第一部分為isoflavone malonylglucosidess及isoflavone glucosides之分離純化。結果發現，將大豆粉以60%甲醇萃取大豆異黃酮，將其濃縮去除溶劑後通過HP-20疏水性管柱，水洗去除雜質，然後以40%甲醇洗出malonylglucosides部份；接著改以70%甲醇洗出殘餘malonylglucosides及glucosdies，於95℃下加熱迴流8小時，使malonylglucosides全部轉變成glucosides，即得isoflavone glucosides。 另一方面，本研究初步試驗結果發現β-glucosidase存在於豆渣(okara)中。將分離洗淨之豆渣以3% NaCl(aq)萃取、將萃取物以UF濃縮、以50-60%丙酮沉澱、經DTT(1,4-Dithioerythritol)還原後進行CM-Sepharose CL-6B陽離子交換層析及膠體過濾而得純化之β-glucosidase。此β-glucosidase之分子量為67kD之monomer，最適溫度為45℃，最適pH為4.5；溫度穩定性佳，於70℃加熱1小時仍有85%之酵素活性，而pH穩定性以接近pH 6.0的情況最好。在醣類與金屬離子抑制方面， 1 mM Hg2+能完全抑制此酵素之活性。此純化之β-glucosidase可將glucosides全部轉換為algycones，對malonylglucosides則沒有作用。顯示此酵素為對glucosides具有專一性之酵素。

Soybean isoflavones, a kind of phytoestrogen, have been reported to possess a variety of physiological activities and a potential health benefit. Malonylglucoside is the most abundant isoflavone derivatives in soybeans, and glucoisde is the second abundant group. However, aglycones were reported as the most easily absorbed among the various isoflavone derivatives. Therefore, malonylglucosides and glucosides were desired to be converted to aglycones before consumption. According to our previous studies on soybean isoflavones, we found that the content of malonylglucosides were decreased and aglycones were increased obviously when ground soybean suspension incubated at 40℃. This result suggested that the enzyme existing in soybean could hydrolyze malonylglucosides. Consequently, the aim of this study was to investigate the isoflavone-related hydrolyzing enzymes. In this study, isolation and purification of isoflavones was also carried out to prepare the substrates for enzyme reactions. Isoflavones in soybeans were extracted with 60% methanol. After removing methanol by cacuum evaporation , the aqueous was applied to a HP-20 column, After washing with water, the counn was eluted with 40% methanol solution to recover malonylglucosides. Afterwards, the residual malonylglucosides and glucosides were eluted with 70% methanol solution, followed by heating at 95℃ for 8 hour to convert malonylglucosides to glucosides completely. β-glucosidase discovered in okara was extracted with 3% sodium choloride solution, followed by subjecting to UF concentration, precipitation with 50-60% acetone , DTT reduction, cation exchange absorption and gel filtration. The β-glucosidase was estimated to be a monomer with a mass of 67kD. The optimum temperature was 45℃, and the optimum pH was 4.5. Thermal stability was measured at 40-70℃. The enzyme maintained its activity by 85% at 70℃. It was relatively stable at pH 6.0. The enzyme activity was completely inhibited by 1 mM Hg+. The purified β-glucosidase could completely convert glucosides to their corresponding aglycones. While it could not catalyze the hydrolysis of malonylglucosides. The result indicated that the β-glucosidase from soybean okara was specific to glucosides but not to malonylglucosides.