經由微生物生物轉化程序生產橙皮素磷酸酯之研究

Abstract

橙皮素(hesperetin)為類黃酮化合物，存在於柑橘屬果實之植物次級代謝物，具有預防心血管疾病、提升免疫力、抗癌症等生理活性。然而橙皮素的水溶性不佳，生物可利用率(bioavailability)極低，使其在應用上受到許多限制。本研究室先前篩選出Bacillus subtilis BCRC 80517菌株中具有類黃酮磷酸酯合成酶(flavonoid phosphate synthetase)可將類黃酮磷酸酯化以增加其溶解度。本研究以此為基礎，利用Bacillus subtilis BCRC 80517菌株進行生物轉化橙皮素以生產高水溶性之橙皮素磷酸酯，以期提升生物可利用率。 第一部分為探討搖瓶實驗之生物轉化，並進行衍生物的結構鑑定與溶解度測試。結果顯示，經Bacillus subtilis BCRC 80517 生物轉化後的橙皮素衍生物以ESI-MS/MS與NMR鑑定其化學結構，主產物以2D-HMQC 1H−31P NMR 鑑定為橙皮素磷酸酯hesperetin 7-O-phosphate 與hesperetin 3’-O-phosphate ([M+H]+ m/z 383.06) ; 少量副產物經2D-HSQC 1H-13C NMR鑑定為葡萄糖苷橙皮素hesperetin 7-O-glucoside ([M+H]+ m/z 465.15)、hesperetin 3’-O-glucoside ([M+H]+ m/z 465.15)與6’’-O-succinyl hesperetin 7-O-glucoside ([M+H]+ m/z 565.15)，並由衍生物推測出橙皮素於Bacillus subtilis BCRC 80517之代謝路徑。溶解度測試結果顯示，hesperetin、hesperetin 7-O-phosphate 與hesperetin 3’-O-phosphate 在25 oC下水中的溶解度分別為40.2 mg/L、2.6×104 mg/L與 2.8×104 mg/L，顯示橙皮素磷酸酯之溶解度比Hesperetin 高出650倍之多。 第二部分為利用5 L通氣攪拌式發酵槽放大生物轉換以生產橙皮素磷酸酯。結果顯示，發酵初期在OD600為4的時候饋入轉換基質Hesperetin進行饋料式(fed-batch) 發酵生產，可降低轉換基質抑制之影響。最後在二次性饋料( twice fed-batch)發酵策略中，最高投入17.6 g/L之轉換基質hesperetin，可於發酵時間40小時達到84.1%之轉換率，生成 18.7 g/L 之橙皮素磷酸酯。

Hesperetin, one of the polyphenolic secondary metabolites in Citrus fruit, has a number of pharmacological and biological activities, such as ameliorating cardiac inflammation, increasing intact immunity systems, activities of anti-cancer. However, its application is restricted because of its low water solubility. In our previous studies, we found the enzyme, flavonoid phosphate synthetase isolated from B. subtilis BCRC 80517, can phosphorylate some selected flavonoid compounds to produce the correspoiding phosphate conjugates that are water-soluble. In this work, we investigated the biotransformation strategies of B. subtilis BCRC 80517 with hesperetin to produce water-soluble hesperetin phosphate conjugates. In the first part of this thesis, we experimented the feasibility of biotransformation of B.subtils BCRC 80517 with hesperetin, and identified the hesperetin derivates in culture broth. The results of biotransformation indicated that hesperetin 7-O-phosphate ([M+H]+m/z 383.06) and hesperetin 3’-O-phosphate ([M+H]+m/z 383.06) were the predominant products of biotransformation. Furthermore, we demonstrated some minor peaks of high-performance liquid chromatograms which were also related to the biotransformation of hesperetin 7-O-glucoside ([M+H] + m/z 465.15), hesperetin 3’-O-glucoside ([M+H] +m/z 465.15) and 6’’-O-succinyl hesperetin 7-O-glucoside ([M+H] + m/z 565.15) according to the corresponding spectra of UV absorption, ESI-MS/MS and NMR spectral. The minor hesperetin derivatives were also supposed to be water-soluble than hesperetin. With the growth of these peaks and decline during the biotransformation, we concluded that B. subtilis BCRC 80517 transforms hesperetin into hesperetin 7-O-phosphate and hesperetin 3’-O-phosphate majorly and into hesperetin 7-O-glucoside hesperetin 3’-O-glucoside and 6’’-O-succinyl hesperetin 7-O-glucoside inevitably with little amount. The water solubility at 25oC of hesperetin, H7P and H3’P were 40.2 mg/L, 2.6 x104 mg/L and 2.8 x 104 mg/L, respectively. The water solubility of hesperetin phosphate conjugates were 650 times higher than that of hesperetin. In the second part of this thesis, a fermentation process for H7P and H3’P production by Bacillus subtilis BCRC 80517 was successfully scaled up from 500 mL shake flask to 5 L stirred tank bioreactor. We have developed a fed batch fermentation strategy, which can decrease the inhibition effect of feeding hesperetin through the optimal time of feeding hesperetin substrate at OD600 = 4. The conversion rate of hesperetin phosphate conjugates was 84.1% and the biotransformed products were 18.7 g/L at the end of 40 h incubation time.