Surface display of carbonic anhydrase on Escherichia coli for CO₂ capture and mineralization

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO₂. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-I_RLS (11.43 U·mL^-1OD₆₀₀ ^-1) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL^-1OD₆₀₀ ^-1) under optimized induction conditions. Compared with free SazCA, E-22b-I_RLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO₂ mineralization, the amount of CaCO₃ deposition catalyzed by the strain E-22b-I_RLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO₂ capture and mineralization.