3.1 Selection of the mutagenesis sites for improving thermostability of sucrose isomerase
In order to rationally design the most promising mutants, we used FoldX to screen natural hot spots that may be related to thermostability. ΔΔG values of all 10982 possible single point mutations were first calculated to predict the possibility of point mutations affecting protein thermostability. Among them, the mutation site with ΔΔG>0 will be excluded. To further improve the prediction accuracy by the FoldX algorithm, an additional conservation analysis was performed to avoid point mutations that would lead to loss of enzyme activity.[33] The amino acid marked with f or s was highly conserved amino acid and further excluded (Figure S2). Finally, 10 point mutations were selected for the next experimental study: E76R, A100E, G152P, I205M, V280L, S328F, S499F, S563R, S563L, N578M (Table 1). The distribution of the mutation points is shown in the Figure 1A.
To test whether these substitution mutations improved the thermostability of SI, each single point mutation was individually expressed in C. glutamium 13032 (Figure 1B). The thermostability of mutants was evalutated by determining the residual activity of enzyme after heat treatment at 45°C for 20 min. As shown in Figure 1C, WT retained 15.7% of its initial activity, whereas two positive mutants V280L, and S499F retained 49.1% and 43.2% of their individual original activity, respectively. However, the thermostability of other mutants did not change or decreased significantly. These results demonstrated that V280L and S499F show better thermostability than WT. To assess the possible interaction between these two single point mutations, double mutant V280L/S499F was further constructed and investigated. V280L/S499F retained 93.3% of its initial activity after incubating at 45°C for 20 min.