| 초록 |
Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide (CO2) hydration, is an attractive route for carbon sequestration. However, the industrial applications of CA are hampered by the unstable nature of enzymes. We introduced in silico designed, de novo disulfide bond in α-type CA from Neisseria gonorrhoeae (ngCA) to enhance thermostability. Three ngCA variants (T133C-D197C, P56C-P156C, and N63C-P145C) were selected and expressed in Escherichia coli. The thermostability of N63C-P145C was greatly increased, showing 8-fold higher half-life at 70 ˚C. The melting temperature of N63C-P145C was 7.8 °C higher than that of the wild-type. This difference could be attributed to the loss of conformational entropy of the unfolded state. N63C-P145C showed an upward-shifted optimal temperature and was thermoactivated, which compensated for the lowered activity at 25 ˚C. Collectively, de novo disulfide engineering was successfully applied to improve the thermostability of CA and the constructed ngCA variant N63C-P145C can be used as an efficient biocatalyst for CO2 sequestration under high temperature conditions. |
