Intracellular Self-Immobilization of Carbonic Anhydrase Enables Carrier-Free Recovery and Stable Operation under Direct Air Capture Conditions

Efficient deployment of carbonic anhydrase (CA) in direct air capture (DAC) is constrained by the lack of enzyme durability under alkaline, chemically complex, and long-term operating conditions. A carrier-free self-immobilization strategy based on encapsulin-mediated intracellular assembly was proposed to address these challenges. The fusion of Sulphurihydrogenibium azorense carbonic anhydrase (SazCA) with the encapsulin EncMh from Mycolicibacterium hassiacum enables spontaneous self-assembly into supramolecular particles during expression in host cells, allowing high-yield production and low-energy recovery by simple centrifugation. The resulting SazCA-EncMh assemblies exhibit high thermal stability and broad tolerance toward alkaline and ion-rich environments. Notably, their supramolecular state can be reversibly switched between nano- and microscale assemblies by pH modulation without loss of catalytic activity over multiple cycles. Under DAC, free SazCA-EncMh significantly enhances long-term Chlorella cultivation over 21 days, indicating sustained extracellular catalytic function and improved inorganic carbon availability. In addition, SazCA-EncMh exbibits improved stability relative to ferritin-based assemblies in representative DAC absorbents, including amino acid salts and tertiary amines. Overall, encapsulin-enabled self-immobilization offers a practical and scalable strategy for stabilizing CA under DAC-relevant conditions, supporting its integration into biological and hybrid carbon capture systems.