枯草桿菌BCRC 80517對大豆異黃酮生物轉換之研究

存在於黃豆中的異黃酮(isoflavones)是黃豆的二級代謝物，其化學結構與人體雌激素的雌二醇(estradiol)相似，具有雌激素活性，被認為是植物雌激素(phytoestrogen)。異黃酮依化學結構可分成四大類: malonyl-glucosides、acetyl-glucosides、glucosides和aglycones。在此四大類中，aglycones被認為生理活性最佳，因此許多研究致力於將帶有醣基形態的異黃酮經由去醣基作用(deglycosylation)而轉換成aglycones。近年來，許多文獻指出aglycones的daidzein和genistein具有預防骨質疏鬆、心血管疾病、乳癌及前列腺癌等生理活性。然而，根據Merck Index及眾多文獻記載，daidzein和genistein幾乎不溶於水，生物可利用率(bioavailability)亦不佳。本研究室先前自市售納豆產品中篩選出B. subtilis BCRC 80517，可將glucosides轉換成aglycones，再轉換成水溶性高的代謝物。本論文以此為基礎，進行後續的研究。第一部分探討B. subtilis BCRC 80517對不同種類異黃酮之生物轉換與代謝途徑。結果發現BCRC 80517可將aglycone (daidzein和genistein)及glucosides (daidzin和genistin)轉換成daidzein 7-O-phosphate (D7P)和genistein 7-O-phosphate (G7P)，且BCRC 80517對genistein和genistin的生物轉換率皆大於daidzein和daidzin；malonyl-glucosides則會經由去醣基作用產生aglycones，卻無法進一步被磷酸化。BCRC 80517對異黃酮的生物轉換過程中，大部分的daidzein和genistein被磷酸化生成D7P和G7P，伴隨著微量的daidzein 4’-O-phosphate和genistein 4’-O-phosphate產生。同時，少部分的aglycones也會被醣基化生成glucosides，再進一步轉換成sucinnyl-glucosides。第二部分探討B. subtilis BCRC 80517生產異黃酮磷酸酯之最適培養條件。結果顯示，BCRC 80517生產異黃酮磷酸酯的最適生產培養基為2% (w/v) sucrose，1% (w/v) (NH4)2HPO4，1.5% (w/v) K2HPO4/KH2PO4 (pH 7.0)，0.05% (w/v) MgSO4•7H2O和0.025% (w/v) MnSO4•H2O；最適培養條件為接種5% (v/v)種菌，同時投入2 g/L (1.2 mM daidzein, 2.1 mM genistein)生物轉換基質至生產培養基，於37℃，150 rpm以Hinton’s flask振盪培養48小時後，D7P和G7P生成濃度為0.9 mM和2.0 mM，達到最高生物轉換率，分別為80%和97%。上述培養液經離心除菌、乙酸乙酯萃取、濃縮、正己烷沉澱、HP-20疏水性樹脂層析、濃縮和凍乾後，可得異黃酮磷酸酯混合物，純度為83% (29% D7P, 54% G7P)，回收率為94%。D7P和G7P在25℃純水中的溶解度分別為17.9%和10%，比daidzein和genistein的溶解度提高10萬倍之多。第三部分首先建立測定磷酸化酵素活性的分析方法，再由B. subtilis BCRC 80517的菌體分離與純化出異黃酮磷酸酯合成酶(Isoflavone phosphate synthetase, IFPS)。IFPS屬於胞內酵素，且不會因菌體與異黃酮共培養而使酵素表現量增加。將菌體破菌、離心並收集上清液作為粗酵素液，經硫酸銨沉澱(飽和度40-60%)、DEAE FF、Q HP陰離子交換層析、Phenyl HP疏水性層析及Superdex 75膠體過濾層析等分離純化步驟後，IFPS比活性為128 unit/mg，活性回收率為0.11%，酵素純化倍率為183倍。SDS-PAGE和LC-MS/MS分析純化後之酵素，發現IFPS有839個胺基酸，分子量為95.4 kDa，pI值為4.86，酵素分類為EC 2.7.9.x。IFPS只能以ATP作為磷酸根轉移之來源，Mg2+和Mn2+存在時，酵素才有活性；酵素最適反應溫度為40℃，最適反應pH為7.5，酵素在40℃以下或pH值中性(pH 6.5-8.5)靜置一小時，可維持80%以上酵素活性。

關鍵字

納豆菌；異黃酮；生物轉換；異黃酮磷酸酯；異黃酮磷酸酯合成?

並列摘要

Isoflavones are a group of plant secondary metabolites that occur mostly in the subfamily Papilionoideae of the Leguminosae. Soybean (Glycine max) is the most abundant source of isoflavones. To date, 12 natural isoflavones have been found in soybeans, consisting of 3 types of isoflavone aglycones (namely daidzein, genistein and glycitein), and their corresponding conjugates with glucose, acetylglucose and malonylglucose. Due to the similarity of chemical structure between the metabolites of isoflavone and human estrogen-estradiol, daidzein and genistein were found to bind to estrogen receptors. Therefore, aglyconic forms of soy isoflavones are commonly referred to as phytoestrogens and bioactive isoflavones. Hence, most of the relevant studies were devoted to the conversion of glucosidesinto their corresponding aglycones. Recent studies indicated that isoflavones may have health benefits such as prevention of breast cancer, prostate cancer, decreasing the risk of cardiovascular diseases, increasing bone mass density to prevent osteoporosis and reducing menopause symptoms. However, according to the Merck Index and literatures, aglycones are partically water insoluble and show poor bioavailability as well. In previous study, we isolated an FC-10 strain from commercial natto products, which can convert glucosides into their corresponding aglycones, and then further convert them into two unknown metabolites with higher polarity. In the first part of this thesis, we generated two water soluble isoflavone derivatives, daidzein 7-O-phosphate (D7P) and genistein 7-O-phosphate (G7P), by incubating Bacillus substilis BCRC 80517 with daidzein and genistein. These two isoflavone derivatives were characterized by HPLC-ESI-MS/MS, 13C NMR and 31P NMR. Genistein was found to be phosphorylated more rapidly than daidzein. In addition, this bacterial strain could transform glucosides via aglucones into the corresponding 7-O-phosphate conjugates. However, BCRC 80517 could not transform malonyl glucosides into 7-O-phosphate conjugates. We concluded that during the bioconversion of aglycones, daidzein and genistein were predominantly phosphorylated into D7P and G7P, respectively, meanwhile, a small amount of daidzein 4’-O-phosphate and genistein 4’-O-phosphate were also generated by BCRC 80517. In addition, small amount of aglycones were glycosylated into glucosides, respectively, and were then further converted into 6’’-O-succinyl-glucosides. In the second part of this thesis, we investigated the optimal production medium and culture condition of biotransformation for the production of isoflavone phosphate conjugates by BCRC 80517. The optimal composition of production medium was composed of 2% (w/v) sucrose, 1% (w/v) (NH4)2HPO4, 1.5% (w/v) K2HPO4/KH2PO4 (pH 7.0), 0.05% (w/v) MgSO4•7H2O and 0.025% (w/v) MnSO4•H2O with 2 g/L (1.2 mM daidzein, 2.1 mM genistein) isoflavone substrates. The optimal culture condition was that BCRC 80517 was cultivated with inoculum size of 5% (v/v) seed culture, at 37℃ and 150 rpm in a 500-mL Hinton’s flask containing 100 mL production medium. At the end of 48 h incubation, the bioconversion rates of daidzein and genistein were 80% and 97%, respectively, and the concentration of D7P and G7P in the harvested broth were 0.9 mM and 2.0 mM, respectively. Moreover, the process for recovering D7P and G7P from the culture broth was also investigated in this work. After remove the biomass by centrifugation, supernatant was extracted with an equal volume of ethyl acetate by 4 times, and then n-hexane was added to make the precipitation out of D7P and G7P from the solvent. The precipitate was then dissolved by a small amount of water and further purified with HP-20 resin. The purity and recovery of purified products containing isoflavone phosphate conjugates were 83% (29% D7P, 54% G7P) and 94%, respectively. The water solubility at 25℃ of D7P and G7P were 17.9% and 10%, respectively. Both of the water solubility of these two phosphorylated isoflavone were 100,000-fold higher than their corresponding aglycones. The third part of this thesis is to purify and characterize the biochemical properties of the enzyme acting on the isoflavone phosphorylation. An enzyme assay was developed firstly, and then, the enzyme assay-guided protein purification was conducted to obtain the purified isoflavone phosphate synthetase (IFPS) from BCRC 80517. As a result, the IFPS is an intracellular and constitutent enzyme of BCRC 80517. The enzyme was purified to homogeneity through ammonium sulfate fractionation, DEAE FF, Q HP anion-exchange chromatography, Phenyl HP hydrophobic interaction chromatography and Superdex 75 gel filtration chromatography with the enzyme activity recovery of 0.11% and a 183-fold purification efficiency. The purified isoflavone phosphate synthetase exhibited a specific activity of 128 unit/mg. The molecular mass of IFPS was estimated around 90 kDa by gel filtration and 95 kDa by SDS-PAGE. The protein sequence was identified by LC-MS/MS, which showed that this enzyme contained 839 amino acids, and has a molecular mass was 95.4 kDa with a theoretical pI value was 4.86. This novel enzyme is is categorized into EC 2.7.9.x.The IFPS can use only ATP as the phosphate donor, and Mg2+ is a dominant factor on the activity rather than Mn2+. The optimal conditions for temperature and pH were 7.5 and 40℃ on the activity, respectively. The enzyme was stable in the pH range of 6.5-8.5 with the temperature lower than 40℃.