Bacillus subtilis var. natto BCRC 80517對丙二醯葡萄糖苷異黃酮生物轉換成磷酸酯衍生物之研究

Abstract

大豆異黃酮(isoflavone)為存在於黃豆中的二次代謝物，因其具有雌激素活性而被廣泛研究，依結構可分為四大類，malonyl glucosides、acetyl glucosides、glucosides和aglycones，其中aglycones之生理活性最佳，但其水溶性差且生物可利用率(bioavailability)不佳。本研究室先前發現Bacillus subtilis var. natto BCRC 80517可將配醣基異黃酮(aglycones)轉換成水溶性高的異黃酮磷酸酯(isoflavone 7-O-phosphate)；亦可將glucosides去醣基(deglycosylation)產生aglycones再轉換成isoflavone 7-O-phosphate；malonyl glucosides僅能被去醣基生成aglycones，無法進一步生成isoflavone 7-O-phosphate。為了闡明malonyl glucosides無法被轉換生成異黃酮磷酸酯的原因，本研究首先探討malonyl glucosides是否影響菌體的生長，結果顯示當菌體和250 μM malonyl glucosides共培養時，對菌體的生長並無顯著影響；接著探討是否為malonyl glucosides經由β-葡萄糖苷酶作用的水解產物會抑制BCRC 80517對aglycones的磷酸化作用，結果顯示malonic acid和glucose 6-malonate皆不會抑制反應系統中isoflavone 7-O-phosphate產生；BCRC 80517和malonyl glucosides共培養的其他代謝物中，也未發現有抑制aglycones磷酸化作用之物質。探討BCRC 80517在不同生長階段對aglycones生物轉換效率的結果顯示，在生長初期之生物轉換效率最佳，隨著菌體生長進入停滯期(stationary phase)後，生物轉換效率逐漸下降，且觀察到菌體之內孢子(endospore)逐漸生成。另外探討BCRC 80517之β-葡萄糖苷酶對不同帶醣基異黃酮的去醣基效率，在反應24小時後，glucosides和malonyl glucosides的去醣基率分別為100%和19%，顯示BCRC 80517對glucosides的水解效率顯著大於malonyl glucosides。由於malonyl glucosides在生物轉換過程中，BCRC 80517對malonyl glucosides之醣苷鍵水解效率不佳，且生物轉換速率隨著菌體生長過程逐漸下降，僅有少量在菌體生長初期生成之aglycones被進一步磷酸化，導致B. subtilis BCRC 80517無法有效將malonyl glucosides轉換生成isoflavone 7-O-phosphate。

Isoflavones are a group of plant secondary metabolites that occur mostly in soybean (Glycine max). There are 4 types of isoflavones found in soybeans, consisting of isoflavone aglycones and their corresponding conjugation forms (malonyl glucosides, acetyl glucosides and glucosides). Due to the similar chemical structure between isoflavone aglycones and estradiol, aglycones were referred to be a class of phytoestrogens. Among all types of soy isoflavones, aglycones have been reported as the bioactive forms. However, aglycones have shown low bioavailability owing to their poor water solubility. In our previous study, Bacillus subtilis var. natto BCRC 80517 showed the capability to convert isoflavone aglycones into isoflavone 7-O-phosphates which are highly water-soluble. Glucosides would be deglycosylated into their corresponding aglycones then further converted into isoflavone 7-O-phosphates. However, malonyl glucosides could only be deglycosylated into their corresponding aglycones without forming phosphate conjugates. The aim of this work was to figure out the reason why malonyl glucosides contribute to the failure of biotransformation by BCRC 80517. Firstly, we found that there was no significant effect on the bacterial growth while BCRC 80517 cultivated with malonyl glucosides. Then, we explored whether the hydrolysate of malonyl glucosides by β-glucosidase impact on the phosphorylation of aglycones by BCRC 80517. The results revealed that neither malonic acid nor glucose 6-malonate could inhibit the production of isoflavone 7-O-phosphates during the biotransformation. Moreover, the metabolites from the culture broth of BCRC 80517 with malonyl glucosides showed no inhibition on the phosphorylation of aglycones, either. And meanwhile, we found that the biotransformation performance varied considerably by the growth status of BCRC 80517 cells. The biotransformation rate was the most efficient when the bacteria grew at the exponential phase of growth, and the performance of biotransformation decreased gradually along with the bacterial growth. Besides, the results from comparing the deglycosylation efficiency between malonyl glucosides and glucosides of soy isoflavone by Bacillus subtilis var. natto BCRC 80517 revealed that malonyl glucosides were carried out more slowly than glucosides. Therefore, we concluded the dominant factors of the phosphate esters of isoflavone not being generated by the biotransformation with malonyl glucosides of soy isoflavone would be highly related to inferior substrate specificity with deglycosylation and the attenuation of phosphorylation along with the bacterial growth.