Stabilized and Immobilized Carbonic Anhydrase on Electrospun Nanofibers for Enzymatic CO₂ Conversion and Utilization in Expedited Microalgal Growth

Carbonic anhydrases convert CO₂ to bicarbonate at a high turnover rate up to 10⁶ s^-1, but their actual applications in CO₂ conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO₂ to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO₂ reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO₂ conversion and utilization technologies.