Biocatalyzed mineralization in an aqueous two-phase system: effect of background polymers and enzyme partitioning

The formation of minerals in living organisms occurs in crowded microenvironments generated by the organization of soft matter. Here, we used a biphasic aqueous polymer medium to mimic the macromolecular crowding and compartmentalization of intracellular environments. Mineralization was performed in an aqueous two-phase system (ATPS) containing two nonionic polymers, poly(ethylene glycol) (PEG, 8 kDa) and dextran (Dx, 10 kDa). The enzyme urease was used to catalyze CaCO₃ formation by hydrolyzing urea to produce CO₃ ^2-, which reacted with Ca²⁺ already present in solution. Urease partitioning into the Dx-rich phase provided a mechanism for localizing the hydrolysis reaction, which consequently restricted mineral formation to this phase, despite the initially equal concentration of Ca²⁺ in both phases. Spatially confined mineralization was quantified by sampling the phases during bulk reactions and also directly observed in microscale systems by optical microscopy. Decreasing the volume of the Dx-rich phase relative to that of the PEG-rich phase significantly enhanced the local urease concentration in the Dx-rich phase, increasing local reaction rates. The PEG and Dx polymers, though present at up to 30 wt% in the ATPS, did not strongly influence the morphology of CaCO₃(s) observed. However, addition of ovalbumin (1.5 wt%) caused marked changes in crystal morphology. The PEG/dextran ATPS reaction medium captured several key aspects of the biological environment including macromolecular crowding, localized reagent production via enzymatic activity, and reaction compartmentalization while not precluding the use of structure-directing additives such as proteins.