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.