3.6. Thermostability study
Thermostability of DA11 and DAwild was studied under 50 ℃ (Fig. 5). The deactivation kinetic parameters was obtained, and the half-life of DA11 was 11.2 h, almost 2-fold of DAwild (Table S1). Thermostability of DA11 was further studied under 60, 70, 80 ℃ (Figure S2). DA11 retained 52% activity under 80 ℃ after 10 h. The result indicated interfacial interactions enhancement greatly improved thermostability. Mutations of DA11 on the subunit interface were marked in Fig. 6. MDS result indicated the salt bridges formed between Lys300-Glu482, Lys341-Asp407, Arg324-Asp407, Lys346-Asp410, Lys439-Glu375, Arg351-Asp410 indicated the enhanced thermostability by interfacial interactions of subunits of DA11(Table S3). Therefore, co-evolutionary analysis are sufficient to identify higher-than-pairwise mutation patterns of DAADH, which can both increase the activity and thermostability.