Precipitation-Based Nanoscale Enzyme Reactor with Improved Loading, Stability, and Mass Transfer for Enzymatic CO₂ Conversion and Utilization

Enzymatic CO₂ conversion has gathered a growing attention due to its fast kinetics in converting CO₂ to bicarbonate, but the carbonic anhydrase enzymes easily lose their activities in CO₂ conversion processes. Here, we propose a "precipitation-based nanoscale enzyme reactor (p-NER)" approach, which stabilizes the activity of carbonic anhydrase, prepared via the two steps of enzyme adsorption into magnetic mesoporous silica and simultaneous enzyme precipitation/cross-linking. The simple addition of enzyme precipitation during cross-linking step resulted in the formation of cross-linked enzyme aggregates (CLEAs) not only inside the mesopores but also on the surface of mesoporous silica. External CLEAs of p-NER contributed to the improvement of enzyme loading (32.9% (w/w)) and mass transfer (K_M = 3.68 mM) compared to those of NER (20.1% (w/w) and 4.29 mM, respectively), prepared without enzyme precipitation step and showing no external CLEAs. p-NER was stable under vigorous shaking (200 rpm) with no activity decrease for 160 days after the inactivation of 25% labile enzyme population at the initial stage of incubation. It suggests that external CLEAs were tightly bound on the surface of mesoporous silica by having roots of CLEAs in the internal mesopores. p-NER of carbonic anhydrase was used to convert CO₂ to bicarbonate, and the resulting bicarbonate was further utilized for the generation of calcium carbonate. The addition of p-NER into the CO₂ bubbling reactor resulted in 6.5-fold higher production of calcium carbonate than the control with no enzyme, revealing the accelerated kinetics of CO₂ conversion in the presence of p-NER. p-NER can be easily recycled via magnetic separation, and retained 89% of initial activity after 10 recycled uses. This study has demonstrated great potential of p-NER not only for enzymatic CO₂ conversion but also in various other applications where the short lifetimes of enzymes hamper their practical applications.