Extraction And Characterization Of The Enzymes Involved In The Metabolism Of Daidzein

Soy isoflavones are secondary metabolites, being formed in the growing process of Soybean.Equol is metabolized from daidzein, the mian ingredient of glycosides, by intestinal bacteria, havebetter physiology effects than oter soy isoflavones. To date, several bacteria capable of producingequol have been isolated from human or animal feces. Many of these strains are suggested to firstmetabolize daidzein as a substrate to dihydrodaidzein and to then metabolize dihydrodaidzein toequol. However, the details of the enzymes involved in the production of equol from daidzein, andof the genes encoding them, remain largely unknown. A Slackia HY-2was isolated form hunmanfeces by our lab, it can directly produce equol from daidzein. In this study, both the two enzymaticreactions form daidzein to dihydrodaidzein and dihydrodaidzein to equol wre primarily studied.We investigated the properties of the crude enzyme. In the reaction, daidzein reductase can convertdaidzein to dihydrodaidzein, and dihydrodaidzein-equol reductase can convert dihydrodaidzein toequol.(1)we study the metabolism of isoflavones afer ingestion of soybean foods in healthy adults.The twelve subjects consumed soybean foods, which have different aglycone content.Concentration of isoflavones and their metabolites in the urine were measured by HPLC in order toknow the metabolism of isoflavones is affected by the concomitant ingestion of glucosidase oraglycones. The result is the excretion of isoflavones wre no significant differences. It isdemonstrated that the metabolism of isoflavones may be not altered by the chemical compositionof the isoflavones ingested.(2)The results through the study indicated: daidzein reductase that converted daidzein todihydrodaidzein was detected mianly in the culture supernatant, which required induced bydaidzein. Based on the results of single factor experiments, orthogonal experiments have been usedto optimed the fermentation parameters. The optimal levels of the mian factors are asfollows:intitial pH7.5, inoculum amount6%, daidzein75μg/mL, cultivation temperature34℃witha fermentation time of12h. Under this condition, the levelof dihydrodaidzein increased by20.9%more than that of initial level. Analyzing the properties of crude enzyme produced by strain HY-2,when daidzein was the reaction substrate, it showed that:the optimum temperature was35℃, theoptimum pH was6.5. The crude enzyme could maintain high reduction activity if the pH is6.0~7.5.Most metal ions is not have no effect on the activity of the crude enzyme. (3)The results through the study on characteristic of HY-2dihydrodaidzein-equol reductaseindicated: the crude enzyme of this strain that converted dihydrodaidzein to equol was detectedmianly in the cell extract, which also required induced by daidzein. Based on the results of singlefactor experiments, orthogonal experiments have been used to optimed the extaction conditionsand the salting conditions. The optimal levels of the mian factors are as follows:work time15min,work/rerral time2s/8s, cell concentration is1/3g/mL. Analyzing the properties of crude enzymeproduced by strain HY-2, when dihydrodaidzein was the reaction substrate, it showed that: theoptimum temperature was30℃, the optimum pH was7.0. The crude enzyme could maintain highreduction activity if the pH is6.0~7.5. Most metal ions is not have no effect on the activity of thecrude enzyme.