納豆菌NTU-18對大豆異黃酮去醣基轉化之研究

Abstract

本研究由市售納豆產品中篩選分離出一株具有高 β-葡萄糖苷酶活性並且能高效率地將帶醣基之大豆異黃酮進行去醣基化之納豆菌 Bacillus subtilis natto NTU-18。將此納豆菌於 5% 黑豆奶中進行批式培養，發現 β-葡萄糖苷酶活性於第 8 小時開始測得，並於第12小時達到最高，且酵素活性位於菌體之表面。第 24 小時培養基中所含具醣基大豆異黃酮 daidzin 與 genistin 分別有 100% 與 75% 成功的被去醣基化。與去醣基化進行之同時，發酵液呈現對 CHO-K1 雌激素受體 ERβ 高結合度、ERα 低結合度之功效。關於納豆菌之 β-葡萄糖苷酶性質探討，我們進一步的經由比對枯草桿菌 Bacillus subtilis 168，由此納豆菌株中選殖出四種可能具 β-葡萄糖苷酶序列的基因 (bglA, bglH, yckE 和 ydhP)，並分別表現於 E. coli M15 中進行 β-葡萄糖苷酶重組蛋白質特性之研究。經由胺基酸序列分析比對，此四種 β-葡萄糖苷酶 (BglA, BglH, YckE 和YdhP) 均屬於 BGA-subfamily。在我們所使用的基質測定中，表現的重組蛋白質以 BglH 與 YckE 具有β-葡萄糖苷酶的活性，而另兩者則無。在重組β-葡萄糖苷酶 BglH 和YckE的特性研究，當以p-nitrophenyl-β-D-glucoside 為基質，兩者最適反應溫度均介於 37-45 °C， 而 BglH 比 YckE 有更高的耐熱安定性。兩者最適反應 pH 均為 6.0，而 YckE 比 BglH 有較廣的 pH 安定性 (pH 6.0-9.0)。在金屬離子抑制活性方面，當重組 BglH 分別與1 mM Cd2+、Fe2+ 和Cu2+ 作用後其活性分別下降了 73%、63% 和 43%，當與其它二價金屬離子 (Ca2+, Mg2+ 和 Mn2+) 作用後，其活性大約下降 0-23%；而重組 YckE與前述二價金屬離子作用後其活性下降幅度均小於20%。在我們所測試的基質中，重組 BglH 與重組 YckE 分別與 genistin 和 p-nitrophenyl-β-D-fructopyranoside 有最高的親和性。而此二重組蛋白質均具有將帶醣基的大豆異黃酮 daidzin 和 genistin 進行去醣基化的能力，其中以重組 BglH 之去醣基效率較佳。將 B. subtilis homo-BglH 培養於5%豆粕培養基中，在第9小時到第12小時其對大豆異黃酮的轉化率較其他三株基因突變者低。我們認為，B. subtilis 中的 BglH 在大豆異黃酮去醣基過程中可能扮演著重要的角色。本研究之主要目的在建立一個高效率的大豆異黃酮去醣基的轉化生產程序，考慮 β-葡萄糖苷酶活性存在位置，以及納豆菌生長快速之特性。本研究嘗試結合菌體培養、β-葡萄糖苷酶活性誘導以及大豆異黃酮連續轉化之程序於一體，亦即發展菌體在批次培養後，以一次或連續饋料的方式在液態培養過程中添加含醣基的大豆異黃酮抽出物進行基質去醣基化反應。結果顯示，在批次培養中，當起始培養液中所添加的帶醣基大豆異黃酮濃度太高會抑制納豆菌的生長，延長菌體於遲滯期的時間。在連續饋料培養中，先將納豆菌培養於 2 L 的 5% 豆粕培養基 8 小時後，開始饋入帶醣基大豆異黃酮溶液(daidzin 與 genistin 濃度分別為 3.0 mg/mL 與 1.0 mg/mL)，當饋料流速控制於 1.5 mL/min，總饋入體積為 3 L，有最佳的去醣基轉化率，在第 42 小時 daidzin 和 genistin 被轉化率分別為 97.7% 和 94.6%，發酵液所含 daidzein 和 genistein 濃度可達 4228 μM 和 1314 μM，其轉換速率分別為 25.6 mg/L/h 與 8.5 mg/L/h。我們認為這個生產程序可以實際應用於工業量產，而此發酵液則可做為開發選擇性雌激素受器調節劑之用。

In this study, strain Bacillus subtilis natto NTU-18 with high isoflavone glycoside-hydrolyzing β-glucosidase activity was isolated from commercial natto product. During the batch fermentation of 5% black soymilk by B. subtilis natto NTU-18, the activity of β-glucosidase appeared at 8 h after inoculation and reached maximum (3.3 U/ml) at 12-h, and the β-glucosidase was found cell associated. Deglycosylation of isoflavone glycosides was observed at the same period, the deglycosylation rate of daidzin and genistin at 24-h was 100 % and 75%, respectively. In accordance with the deglycosylation of isoflavone glycosides, the estrogenic activity of the 24-h fermented black soymilk broth bind to and induce transcription of hERβ to a higher extent than of hERα. To clinically important issues, the ERα is highly expressed in breast and uterine tissue and the ERβ refers to such as bone stability and cardiovascular health. The biochemical properties of β-glucosidases in Bacillus subtili natto NTU-18 were studied basing on the genomic sequence of Bacillus subtilis 168, four β-glucosidase genes (bglA, bglH, yckE and ydhP) from B. subtilis natto NTU-18 were cloned and the characteristics of the recombinant enzymes expressed in E. coli were investigated. Compare the amino acid sequences, these four β-glucosidases belonged to the BGA subfamily. The recombinant protein of BglH and YckE showed β-glucosidase activity using substrates of used, and another two did not. In the biochemical properties of recombinant β-glucosidases BglH and YckE, the optimal temperature for p-nitrophenyl-β-D-glucoside (pNPG) hydrolyzing activity of both enzymes was between 37 and 45 °C, but BglH had a higher thermal stability than that of YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than that of BglH (pH 6.0-9.0). Activities of recombinant BglH was inhibited 73%, 63%, and 43% by 1.0 mM Cd2+, Fe2+, or Cu2+, respectively, while other divalent metal ions (Ca2+, Mg2+ and Mn2+) resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BglH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-β-D-fructopyranoside (pNPF). Both BglH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BglH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher kcat). The insertion mutant strain B. subtilis homo-BglH showed lower isoflavone deglycosylation rate than other gene mutant strains during 9-h to 12-h in 5% defatted-soy medium fermentation. According to these results, BglH may play a more important role than others in the deglycosylation of isoflavone glucosides in B. subtilis natto during fermentation. Main of this study is to establish a highly efficient isoflavone deglycosylation process. In batch fermentation, high concentration soy isoflavone glucosides (SIG) of the initial culture medium inhibited the growth of B. subtilis natto. In a continuous feeding process (the feeding rate 1.5 mL/min) and totally 3 L of soy isoflavone glucosides (SIG) solution (daidzin 3.0 mg/mL and genistin 1.0 mg/mL) was to be fed into after 8 h of batch fermentation at the beginning contained 2 L of 5% defatted-soy medium inoculated with B. subtilis natto NTU-18, our results showed the highest isoflavones deglycosylation rate of daidzin and genistin (97.7% and 94.6%, respectively). The maximum concentration of daidzein and genistein in broth reached 4227.8 μM and 1314.3 μM, respectively, and the productivity of daidzein and genistein was 25.6 mg/L/h and 8.5 mg/L/h, respectively, during 42 h of fermentation. This process showed that it is an efficient bioconversion process for continuously production of selective estrogen receptor modulator (SERM).