3.3. Design of interfacial interactions
Double mutations of V137I-S314L, F306H-G311I, L134W-F310I were designed to enhance the hydrogen bonds and salt bridges, which aim to promote structure rigidity of enzyme. Calculation of mutation energy was carried out by the webserver Calculate Mutation Energy of Discovery Studio 2016 Client. All mutation energy of double mutated enzymes was negative, which indicated the enhanced thermostability. Therefore, positive mutations were further combined and the designed enzymes DA06 to DA11 were obtained. None of mutated sites is located within active sites, which would not arise activity change from binding substrate. Mutation energy for the combined mutations indicated the cooperative effect on the improvement of thermostability (Table 2).
Thermodynamic parameters of all designed enzymes were calculated by Scoop and were listed in Table 3 [25]. Parameters such as change in enthalpy (ΔHm), change in heat capacity (ΔCp), melting temperature (Tm) and difference in Gibbs free energy of stability (ΔGr), were calculated. These thermodynamic parameters which have important implications for enzyme thermostablility[26].