## 3 Results
### Enzymatic Properties of Recombinant Man26E  
The properties of recombinant \(\beta\)-mannanase were investigated under different pHs (3-10) and temperature (40-70 \({}^{\circ}\)C) conditions. The recombinant Man26E had the maximal activity at pH 6.0 and kept over 75% of the highest activity at 5-9. The recombinant Man26E still showed more than 60% relative activity at pH 4.0 and pH 10.0, but no activity at pH 3.0 (Figure 6a). The enzyme activities at other pH values were significantly different from that at pH 6.0 (\(p<0.01\)). Figure 5: The activity analysis of positive colonies dyed by Congo-red and SDS-PAGE analysis. (**a**) 1 and 2: _Escherichia coli_ BL21(DE3) harboring pET-28a (+)-_Man26E_; 3: _Escherichia coli_ BL21(DE3) harboring pET-28a (+); 4: _Escherichia coli_ BL21(DE3). (**b**) SDS-PAGE analysis. Lane M: Marker; Lane 1: _Escherichia coli_ BL21(DE3) control; Lane 2: _Escherichia coli_ BL21(DE3) harboring pET-28a (+)-_Man26E_ Lane 3, Lane 4 and Lane 5: The purified Man26E. Figure 6: Influence of pH and temperature on the enzyme. (**a**) enzyme activity at different pH values; (**b**) pH stability of the enzyme; (**c**) enzyme activity at different temperatures; (**d**) thermostability of the enzyme. In addition, the recombinant Man26E exhibited a good pH stability and over 80% of the optimal activity was maintained after incubation at 50 \({}^{\circ}\)C and pH 5-8 for 60 min. Its stability rapidly decreased when pH values was below 5.0 and gradually declined in the range of pH 8.0-10.0, but remained about 40% residual activity at pH 3.0 and 65% residual activity at pH 10.0 after incubation at 50 \({}^{\circ}\)C for 60 min (Figure 6b). The optimal temperature for most \(\beta\)- mannanases is between 40 and 75 \({}^{\circ}\)C. As shown in Figure 6c, Man26E had the optimal temperature at 55 \({}^{\circ}\)C. The recombinant Man26E was active at 40-70 \({}^{\circ}\)C, and kept more than 60% relative activity in the temperature range of 45 to 65 \({}^{\circ}\)C. Man26E still remained about 40% relative activity at 70 \({}^{\circ}\)C. The enzyme activities at other temperatures were significantly different from that at 55 \({}^{\circ}\)C (\(p<0.01\)). For testing thermostability, the purified enzyme was first pretreated at different temperatures for 60 minutes, and then the enzyme activity was determined. The enzyme was stable up to 40 \({}^{\circ}\)C for 60 min, and maintained more than 80% of its activity. At temperatures above 50 \({}^{\circ}\)C, its stability decreased gradually, but remained about 60% residual activity at 50 \({}^{\circ}\)C and nearly 40% residual activity at 60 \({}^{\circ}\)C for 60 min (Figure 6d). Different kinds of metal ions (1, 5 and 10 mmol L\({}^{-1}\)) were respectively added into the solution of the purified enzyme and preincubated at 55 \({}^{\circ}\)C for 10 min, and the residual activity was measured according to the standard method. Among these additives, the action of metal cations on the activity of the recombinant Man26E was quite interesting, as shown in Figure 7.  
For example, Co\({}^{2+}\) and Mn\({}^{2+}\) enhanced remarkably the \(\beta\)-mannanase activity, especially 5 mmol L\({}^{-1}\) of Co\({}^{2+}\) and Mn\({}^{2+}\) increased enzyme activity by 72% and 60%, respectively. Fe\({}^{2+}\) at 1 mmol L\({}^{-1}\) and 5 mmol L\({}^{-1}\) showed activation on the recombinant Man26E, but at 10 mmol L\({}^{-1}\) had no effect Ni\({}^{2+}\), K\({}^{+}\) and Ca\({}^{2+}\) at 5 mmol L\({}^{-1}\) had activation on the recombinant Man26E, whereas at 1 and 10 mmol L\({}^{-1}\) displayed varying degrees of inhibition effect. All three concentrations of Mg\({}^{2+}\) and Cu\({}^{2+}\) showed inhibition on the recombinant Man26E. Zn\({}^{2+}\) at 1 mmol L\({}^{-1}\) reduced the enzyme activity of the recombinant Man26E, but at 5 and 10 mmol L\({}^{-1}\) had little impact.