## 3 Results
### Substrate Specificity of \(\beta\)-galactosidase from _T. uzoniensis_
The production of daidzein or genistein increased with increasing enzyme concentrations up to 0.2 mg/mL and reached a plateau above that concentration (data not shown). Therefore, the optimal enzyme concentration for 2 mM reagent grade daidzein or genistein was 0.2 mg/mL. The enzyme completely hydrolyzed 2 mM reagent grade daidzin and genistin to daidzein and genistein within 90 min and 80 min, respectively (Figure 4a,b). The HPLC profiles for the conversion of daidzin and genistin into daidzein and genistein are shown in Figure 4c. The productivities for daidzein and genistein corresponded to 1.33 and 1.50 mM/h, which were 3.1- and 2.5-fold higher than those achieved with \(\beta\)-glucosidase from _P. furiosus_ for isoflavene aglycones from soybean flour extract [13]. Therefore, \(\beta\)-galactosidase from _T. uzoniensis_ was further applied to the production of isoflavene aglycones from extracts of Korean wild soybean. Figure 4: _Cont._  
Production of Ioflavone Aglycones from Isoflavone Glycosides in Seed and Root Extracts of Korean Wild Soybeans by \(\beta\)-Galactosidase from T. uzoniensis  
Isoflavones were extracted from the seeds, stems, and roots of Korean wild soybeans and the amount of isoflaves was measured (Table 2). In Table 2, statistical steps through parametric or non-parametric tests were not performed because it was only to indicate the content of isoflaves required for biotransformation. Large amounts of daidzin (20,580 mg/g) and genistin (3600 mg/g) were found in the root and seed extracts, whereas no glycitin was found in any of the extracts. The root extract not only contained a 1.7-fold higher isoflave glycoside content than that of the seed extract but also contained a high content of isoflave aglycones, including 7560 mg/g of daidzein and 1770 mg/g of genistein. In contrast, the stem extract contained only genistin as an isoflave glycoside without daidzin, which was only about 16% of the genistin contained in the seed extract. Therefore, we applied \(\beta\)-galactosidase from _T. uzoniensis_ to the production of daidzein and genistein from daidzin and genistin in the seed and root extracts of Korean wild soybeans. The production was investigated by varying the enzyme concentration from 0.1 to 2 mg/mL for 20% (\(w/v\)) seed or root extracts for 60 or 90 min. The daidzein or genistein yield increased with increasing enzyme concentrations up to 0.4 mg/mL and decreased considerably at higher enzyme concentrations (data not shown), indicating that the optimal enzyme concentration for 20% (\(w/v\)) seed or root extracts was 0.4 mg/mL. Figure 4: Time-course reactions for the biotransformation of reagent grade daidzin and genistin into daidzein and genistein by \(\beta\)-galactosidase from _T. Uzoniensis._ (**a**) Biotransformation of daidzin (open circle) into daidzein (closed circle). (**b**) Biotransformation of genistin (open square) into genistein (closed square). (**c**) HPLC profiles for the biotransformation of daidzin and genistin into daidzein and genistein. The reactions were performed at 90 \({}^{\circ}\)C in 50 mM CPB (pH 5.0) containing 3 mM isoflave substrate and 0.2 mg/mL enzyme. Data are represented as the means of thriplicate experiments and error bars represent the standard deviation. The time-course reactions for the production of daidzein and genistein from daidzin and genistein in the extracts were carried out at pH 5.0 and 90 \({}^{\circ}\)C with 20% (\(w/v\)) seed or root extract, and 0.4 mg/mL \(\beta\)-galactosidase from _T. uozniensis_. The seed and root extracts at 20% (\(w/v\)) contained 6.62 mM and 11.56 mM isoflavure glycosides (2.77 mM daidzin and 3.85 mM genistin, and 9.89 mM daidzin and 1.67 mM genistin) as substrates and 0.53 mM and 6.08 mM of the corresponding aglycones (0.26 mM daidzein and 0.27 mM genistein, and 5.95 mM daidzein and 0.13 mM genistein), respectively. Daidzin and genistin in the seed extract were completely hydrolyzed to daidzein and genistein within 80 and 70 min, corresponding to the productivities of 1.86 and 3.30 mM/h, respectively.