Carbonic anhydrase is a valuable and efficient catalyst for CO2 hydration. Most often the free enzyme is employed which complicates catalyst recycling, and can increase cost due to the need for protein purification. Immobilization of the enzyme may address these shortcomings. Here we report the development of whole-cell biocatalysts for CO2 hydration via periplasmic expression of two forms of carbonic anhydrase in Escherichia coli using two different targeting sequences. The enzymatic turnover numbers (kcat) and catalytic efficiencies (kcat/KM) were decreased by an order of magnitude as compared to the free soluble enzyme, indicating the introduction of transport limitations. However, the thermal stabilities were improved for most configurations (>88% activity retention up to 95 degrees C for three of four whole-cell biocatalysts), operational stabilities were more than satisfactory (100% retention after 24h of use for all four whole-cell biocatalysts), and CO2 hydration was significantly enhanced relative to the uncatalyzed reaction (approximate to 50-70% increase in CaCO3 precipitate formed). A significant advantage of the whole-cell approach is that protein purification is no longer necessary, and the cells can be easily separated and recycled in future applications including biofuel production, biosensors, and carbon capture and storage. Biotechnol. Bioeng. 2013; 110: 1865-1873. (c) 2013 Wiley Periodicals, Inc.