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.