3.6 MD simulation analysis for improved thermostability
In order to further clarify the overall structural rigidity of enzyme and the fluctuation changes of each amino acid residue, we conducted MD of WT and three mutants (V280L, S499F, V280L/S499F) at 318K for 30ns in this study. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) are used to represent the degree of molecular structure change and freedom of movement of individual atoms in a molecule respectively. As shown in Figure 5A, the RMSD of all systems no longer fluctuates drastically after 13 ns, and then RMSD varied around 1.5 nm. After equilibration at 318 K, the average values of WT was 1.659 nm, whereas the average values of three mutants (V280L, S499F, V280L/S499F) declined to 1.457 nm, 1.520 nm and 1.353 nm, respectively. Since the thermostability of protein is not positively correlated with its RMSD value, the lower RMSD value of mutants indicated the mutated structure was relative stable than that of the WT.
Similarly, RMSF could also reflect the local flexibility of protein. The higher RMSF value of one region, indicating that conformation of this region was more unstable. As shown in Figure 5B, some regions around residue V280, S499 showed great fluctuations in RMSF values of WT at 318 K. Generally, these amino acids were thought to be thermo-unstable. On the contrary, RMSF of three mutants (V280L, S499F, V280L/S499F) showed mild fluctuations in the same areas of WT mentioned above. In conclusion, mutations in these sites (V280, S499) contribute greatly to improve the stability of SI.
Concluding remarks
In this work, the thermalstability of SI from Pantoea dispersa UQ 68J toward sucrose isomerization was greatly improved via rational engineering by utilizing computer-aided design coupled with conservation analysis and functional region assessment. We obtained a robust variant V280L/S499F, displayed a 3.6 ℃ increase in apparent melting temperature, an 8.9-fold longer of half-life at 45 ℃. Moreover, the recombinant C. glutamicum /pXMJ19/pdsi V280L/S499F whole-cell exhibited robust and the continuous operational stability for recyclable synthesis of isomaltulose, the conversion rate remained 83.2 ± 2.1% even after 15 continuous rounds of biocatalysis. More importantly, we further characterized the reason underlying increased thermostability in detail. These results showed that the comprehensive strategies are a universal and efficient method that can improve the thermostability of enzyme without a lot of experiment.