Abstract Efficient biological carbon dioxide sequestration for slowing down the climate changes requires a highly active and stable carbonic anhydrase (CA). A CA from Mesorhizobium loti (i.e., MlCA) was first over-expressed in E. coli via different vectors and a concentration of up to 5 g/L was obtained with pET32a(+). The whole cell biocatalyst was stable between pH 4.0 and pH 9.0 and maintained 82% activity within 6% salinity. The cations NH4+, K+, Na+, Mg2+, Ca2+ and Mn2+ did not affect the catalytic activity, while Co2+, Cu2+, and Zn2+ inhibited >70% biocatalyst activity at 5 mM. The Kcat and Km of whole cell CA was observed to be 1.76 × 106 /s and 0.0133 mM, while that of the crude enzyme was of 2.35 × 106 s−1 and 0.0364 mM, respectively. The effective diffusivity coefficient (De) of whole cell CA was 3.98 μ m2/s. Whole cell biocatalyst immobilized in 2% (w/v) agar could be reused for 6 times for CO2 sequestration and remained stable for 40 days. A novel two-column system with immobilized whole cell biocatalyst and recombinant MlCA effectively converted 100% CO2 to CaCO3 within 3.5 min. This provides an alternative, eco-friendly and low-cost process for carbon capture and storage (CCS) in the future.