Carbonic anhydrase (CA) is a diffusion‐limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO2). CA has been proposed as an eco‐friendly yet powerful catalyst for CO2 capture and utilization. A bacterial whole‐cell biocatalyst equipped with periplasmic CA provides an option for a cost‐effective CO2‐capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole‐cell catalyst: a CA‐coding gene, a signal sequence, and a ribosome‐binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec‐dependent pathway. The engineering of RBS strength further enhanced whole‐cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7‐fold higher activity and 780‐fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO2 capture and utilization.

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