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Abstract

Macromolecular crowding plays a critical role in the kinetics of enzymatic reactions. Dynamic compartmentalization of biological components in living cells due to liquid-liquid phase separation represents an important cell regulatory mech-anism that can increase enzyme concentration locally and influence the diffusion of substrates. In the present study, we probed partitioning of two enzymes (horseradish-peroxidase and urate-oxidase) in a poly(ethylene glycol) (PEG) – dextran (Dex) aqueous two-phase system (ATPS) as a function of salt concentration and ion position in the Hofmeister series. Moreover, we investigated enzymatic cascade reactions and their kinetics within the ATPS, which revealed a strong influence of the ion hydration stemming from the background electrolyte on the partitioning coefficients of proteins following the Hofmeister series. As a result, we were able to realize cross-partitioning of two enzymes due to different protein net charges at a chosen pH. Our study reveals a strong dependency of the enzyme activity on the substrate type and crowding agent interaction on the final kinetics of enzymatic reactions in ATPS and therefore provides substantial implications en route towards dynamic regulation of reactivity in synthetic protocells.