3.2 Enzymatic properties and Kinetic Analysis of mutants
The thermostabilities and catalytic properties of the WT and mutants were further characterized. The optimal pH value of three mutants was 5.5, which was similar to that of the WT (Figure 2A). Consistent with WT, the optimal temperature of these three positive mutants were also 30°C, while they exhibited higher relative activity at the same temperature (Figure 2B). At 45°C, WT retained 58.1% of its maximum activity, whereas mutants V280L, S499F and V280L/S499F retained 61.8%, 59.4%, and 74.1% of its maximum activities, respectively.
Then, we evaluated the changes in thermostability by assessing residual activities of these three mutants and WT after different incubation times at 45°C. As shown in Figure 2C, the thermostability of mutants was better than that of WT, and V280L/S499F displayed the greatest improvement in thermostability. At 45°C, the t1/2 of WT was 11.2 min. In contrast, the t1/2 of V280L, S499F and V280L/S499F were 25.4, 21.5 and 100 min, which are 2.26, 1.91 and 8.9 times better than that of WT. These findings thus indicated that two of amino acid substitutions (V280L and S499F) were beneficial to improve thermostability of SI.
Kinetic parameters of the WT and its mutants were measured using different concentrations of sucrose as substrate. As listed in Table 1, three mutants showed little differences in catalytic activity with WT. Km and kcat/Km of these mutants also changed slightly, indicated that point mutations has little influence on enzyme properties while improving the thermostability.
To further evaluate thermodynamic stability of WT and its variants, melting temperature (Tm ) was measured by DSF. As shown in Figure 2D, the Tm of WT was 50.6°C, while the Tm values of V280L, S499F, and V280L/S499F mutants was 52.7, 51.8 and 54.2°C, respectively. These results are consistent with the thermostability studies of the three positive mutants.