## 3 Results  
Cloning, Purification, and Molecular Mass Determination of \(\beta\)-Galactosidase from T. uzoniensis  
A glycoside hydrolase family 1 (GH1) protein from _T. uzoniensis_ showed high hydrolytic activity on glucosides such as ginseng [19], but it has not yet been characterized and applied to hydrolyzed isoflav. Therefore, the 1482 bp gene encoding the protein from _T. uzoniensis_ DSM 5263 with the same sequence in GenBank (Accession No., AEA12778; gene name, TUZN_RS06265) was cloned and expressed in _E. coli_ BL21 (DE3) in a soluble form. The amino acid sequence of the expressed enzyme showed a 62.1%, 61.9%, 56.9%, and 55.2% identity with those of the GH1 glycosides from _Sulfolobus slibatae_ (AAA79030) [29], _S. solfataricus_ (AAA72843) [30], _Sulfolobus acidocalarius_ (AAY81155) [31], and _P. furious_ (AAL80197) [32], respectively. \(\beta\)-Galactosidase from _T. uzoniensis_ was purified using HisTrap affinity chromatography into a soluble protein with a 10.5-fold purification, a final yield of 21%, and a specific activity for \(p\)NP-\(\beta\)-D-galactopyranoside of 1103 umol/min/mg. The molecular mass of the purified enzyme was approximately 57 kDa on SDS-PAGE (Figure 1a), which was consistent with the calculated value of 57,131 Da based on 493 amino acids combined with six histidine residues. The native enzyme was determined to be a homodimer with a molecular mass of 113 kDa by gel-filtration chromatography (Figure 1b). The GH1 glycosides from _S. sibutae_, _S. solfataricus_, _S. acidocalarius_, and _P. furious_ with high sequence identity (>50%) to the _T. uzoniensis_\(\beta\)-galactosidase are tetramers, whereas those from _Calcidiculospirutor ovensensis_[27], _Calcidiculospirutor saccharolyticus_[33], and _Therntoga petrophila_[34] with low sequence identity (<30%) exist as dimers. These results indicate that within the GH1 family, the level of sequence identity is not critical in determining the association form. Figure 1: Molecular mass determination of \(\beta\)-galactosidase from _T. uzoniensis_. (**a**) SDS-PAGE analysis. Lane 1, molecular weight size marker; lane 2, cell debris; lane 3, crude extract; and lane 4, purified enzyme. (**b**) Determination of native molecular mass using gel-filtration chromatography. The reference proteins ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa), and purified \(\beta\)-galactosidase from _T. uzoniensis_ are represented by filled circles and open circles, respectively. Effects of pH and Temperature on the Hydrolytic Activity of \(\beta\)-Galactosidase from T. uzoniensis  
The hydrolytic activity of \(\beta\)-galactosidase from _T. uzoniensis_ was examined in the pH range 4.0 to 7.0 and the maximal activity was observed at pH 5.0 (Figure 2a). At pH 4.5 and 6.0, the hydrolytic activity was approximately 70% of the maximum. The temperature was varied from 65 to 95 \({}^{\circ}\)C at pH 5.0, where the maximal activity was observed, to investigate its effect on the hydrolytic activity of \(\beta\)-galactosidasease from _T. uzoniensis_, and the activity was maximal at 90 \({}^{\circ}\)C (Figure 2b). Thermophilic \(\beta\)-glucosidase from _M. thermotolerans_[11], _T. lyeettanus_[15], _N. fischeri_[12], _D. turgidum_[10], _T. pendens_[16], _S. solfataricus_[14], and _P. furious_[13], which were applied to the hydrolysis of isoflavynes glycosides, showed maximal activities at 50, 65, 80, 85, 90, 90, and 95 \({}^{\circ}\)C, respectively. The thermal stability of the enzyme was examined in the temperature range of 75-95 \({}^{\circ}\)C. First-order kinetics were displayed for thermal inactivation and the half-lives of \(\beta\)-galactosidase from _T. uzoniensis_ were 235, 122, 68, 46, and 26 h at 75, 80, 85, 90, and 95 \({}^{\circ}\)C, respectively (Figure 3). Among the enzymes applied to the hydrolysis of isoflavynesosides, \(\beta\)-glucosidase from _P. furious_[13] and _S.