## 3 Results
### Substrate Specificity of \(\beta\)-galactosidase from _T. uzoniensis_  
The specific activity of \(\beta\)-galactosidase from _T. uzoniensis_ was investigated using aryl glycosides and isoflavque glycosides. The enzyme exhibited specific activities on aryl-glycosides as substrates in the order \(\alpha\)NP-\(\beta\)-D-galactopyranoside \(>\)\(p\)NP-\(\beta\)-D-galactopyranoside \(>\)\(p\)NP-\(\beta\)-D-glucopyranoside \(>\)\(p\)NP-\(\beta\)-D-glucopyranoside \(>\)\(p\)NP-\(\beta\)-D-xylopyranoside (Table 1). However, no activity was observed on \(p\)NP-\(\alpha\)-L-arabinopyranoside and \(p\)NP-\(\alpha\)-L-rhamnopyranoside. These results indicated that this enzyme is a \(\beta\)-galactosidase with higher specificity for the \(\beta\)-1,2 linkage than for the \(\beta\)-1,4 linkage. The substrate specificity of \(\beta\)-galactosidase from _T. uzoniensis_ for isoflavque glycosides followed the order: genistin \(>\) daidzin \(>\) ononin \(>\) glycitin (Table 1). Most other glycosidases from _Dalbergia cohinchinensis_[35], _D. turgidum_[10], _P. furiosus_[13], _Paecilomyces thermophila_[36], _T. leycettanus_[15], and _T. maritima_[17] also showed a higher specific activity for genistin than for daidzin as a substrate. Conversely, the substrate specificity of \(\beta\)-glucosidase from _N. fischeri_[12] was higher for daidzin than for genistin. \begin{table}
\begin{tabular}{c c} \hline \hline \multicolumn{1}{c}{**Substrate**} & **Specific Activity** \\
**Substrate** & **(umol/min/mg)** \\ \hline pNP-\(\beta\)-D-glucuronoside & 1021 \(\pm\) 10 \\ pNP-\(\beta\)-D-galactopyranoside & 1103 \(\pm\) 13 \\ pNP-\(\beta\)-D-xylopyranoside & 105 \(\pm\) 2.7 \\ pNP-\(\alpha\)-L-arabinopyranoside & ND \\ pNP-\(\alpha\)-L-rhamnopyranoside & ND \\ oNP-\(\beta\)-D-glucopyranoside & 830 \(\pm\) 8.1 \\ oNP-\(\beta\)-D-galactopyranoside & 1208 \(\pm\) 16 \\ daidzin & 304 \(\pm\) 9.6 \\ genistin & 405 \(\pm\) 5.5 \\ ononin & 197 \(\pm\) 3.5 \\ glycitin & 96 \(\pm\) 1.3 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Substrate specificity of \(\beta\)-galactosidase from _T. Uuzoniensis_a. Figure 3: Thermal inactivation of \(\beta\)-galactosidase from _T. uzoniensis_. The enzyme was incubated at 75 (closed triangle), 80 (open square), 85 (closed square), 90 (open circle), and 95 \({}^{\circ}\)C (closed circle) in 50 mM CPB (pH 5.0) for different time periods and the residual activity was determined. Data are represented as the means of triplicate experiments and error bars represent the standard deviation. Among the enzymes used for the hydrolysis of isoflavene glycosides, \(\beta\)-glucosidase from _N. fischeri_ showed the highest specific activity (2189 umol/min/mg) for _p_NP-\(\beta\)-D-glucopyranoside [12], followed by \(\beta\)-glucosidase from _P. furiosus_ with a specific activity of 912 umol/min/mg [13], which was 2.14-fold higher and 1.12-fold lower than that of \(\beta\)-galactosidase from _T. uzoniensis_, respectively. Nevertheless, the specific activities of \(\beta\)-galactosidase from _T. uzoniensis_ were highest for daidzin and genistin and were 1.69- and 1.23-fold higher, respectively, than those of \(\beta\)-glucosidase from _P. furiosus_, with the highest activity so far [13]. These results indicate that \(\beta\)-galactosidase from _T. uzoniensis_ is an enzyme suitable for the hydrolysis of isoflavene glycosides. Conversion of Reagent Grade Isoflavone Glycos into Isoflavone Aglycones by \(\beta\)-Galactosidase from T. uzoniensis  
The time-course reactions for the conversion of daidzin and genistin, isoflavone glycosides abundantly expressed in soybeans, into daidzein and genistein were carried out under the conditions of pH 5.0, 90 \({}^{\circ}\)C, 2 mM substrate, and 0.2 mg/mL \(\beta\)-galactosidase from _T. uzoniensis_. Conversions were investigated by varying the enzyme concentration from 0.05 to 1 mg/mL for 2 mM reagent grade daidzin or genistin for 60 min.