3.5. Experimental verification by mutation
Activity of mutations were tested. The activity of DAwild and DA11 at 25 °C were 2.46 and 2.47 U/mg, respectively, while the activity of DA06 was only 0.27 U/mg. This result indicated the DA11 of interfacial interactions would not affect the enzyme activity. Normally, engineering enzyme with improved the rigidity for thermostability enhancement cause the decrease of activity. Multi-site mutation for enzyme activity improvement can affect the stability, which is a compromise between enzyme activity and stability [28].
Interaction analysis indicated that the 12 mutations of DA06 (L133W-V136I-F305C-F309I-G310W-S313Q) led to the formation of salt bridges between 346LYS-408GLU, 349LYS-375GLU, 349LYS-408GLU (Table S2). New formed salt bridges elucidated the mechanism of a key amino acid residue mutation enhanced thermostability [17]. However, to much extra salt bridges and hydrogen bonds of DA06 decreased enzyme activity due to the effect of activity-stability trade-off by enhanced the rigidity[32].
Effect of pH on DAADHs was studied. Among them, DA11 is with the widest adaptable pH range, especially in alkaline solutions (Fig. 4a). Catalytic properties the designed enzyme were verified. The effect of temperature on DAwild, DA06 and DA11 activity was studied in the range of 30 to 80 °C. The optimal temperature for DA06 and DA11 was 60 °C, while it was only 50 °C for DAwild (Fig. 4b). DA11 can maintain 93% activity in 80 °C, while it was only about 40% for DAwild. This result indicated the interifical mutations of subunits enhanced thermostability of DA11 without decreasing the activity.