2.5.2 Binding free energy analysis
To gain more insights of the effects of point mutations on the interactions between α-L-rhamnosidase and substrate, the binding free energies and individual energy components were calculated by using MM-GBSA method. The results showed that A355N, S356Y and D525N mutations enhanced the binding affinity between α-L-rhamnosidase and substrate, which were consistent with our experimental results. Comparing the individual components contributing to the binding free energy (Table 3), it can be concluded that the electrostatic and Van der Waal interaction contributed the most in the changes of the binding strength of A355N and S356Y, while in the case of D525N, besides electrostatic and Van der Waal interaction, polar salvation also plays a significant role. Overall, MM/GBSA binding free-energy analyses corroborated perfectly with the outcome of molecular docking and dynamics analyses, and revealed paramountly lower binding and energy for optimum activity temperature which indicated stably mutate enzyme substrate complex.51, 52