Chimeric protein of internally duplicated α-type carbonic anhydrase from Dunaliella species for improved expression and CO2 sequestration

Abstract An internally duplicated α-type carbonic anhydrase (CA) from Dunaliella species (Dsp-CA) has been expressed as single-domain derivatives, N- and C- half domains. Although both derivatives have structures similar to that of a known CA (PDB ID: 1y7w ), only the C-half domain (Dsp-CA-c) exhibited enzymatic activity, albeit with low solubility. In order to increase the solubility of Dsp-CA-c, its proximal N-terminal amino acids 1–11 were substituted with VSEPHDYNYEK (NT11) of the N-half domain (Dsp-CA-n) which exhibits a high soluble expression yield. The new chimeric gene products, Dsp-nCA-c displayed approximately 2-fold the solubility and activity shown by Dsp-CA-c. Dsp-nCA-c showed increased thermal stability ( ΔT m  > 10 °C) by multimerization compared to Dsp-CA-c. Finally, the chimeric protein effectively catalyzed the conversion of CO 2 into its calcite form in the presence of CaCl 2 . These findings indicate that the substituted NT11 may contribute to soluble expression and enhanced activity of Dsp-nCA-c and cause multimerization which can confer increased thermostability to Dsp-nCA-c. Therefore, N-terminal engineering can be an effective strategy for improving soluble production yield and thermostabillity of CA without disrupting catalytic activity, and the engineered CA could be usefully employed for the development of an efficient enzymatic CO 2 sequestration system.