Cascade Kinetics in an Enzyme-Loaded Aqueous Two-Phase System

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 mechanism 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)−dextran 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 because of 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 the ATPS and therefore provides substantial implications en route toward dynamic regulation of reactivity in synthetic protocells.

Polysaccharide nanoparticles: from fabrication to applications

“…7,198 In recent years, ATPS have been utilised frequently for the separation of biomacromolecules as well as nanoparticles [199][200][201] or as environment for enzymatic catalysis. 202 In addition to macroscopic ATPS, dispersed systems, i.e. water-in-water emulsions, have been investigated as well 135,136 that can be employed as template for the formation of microparticles in aqueous environment.…”

Exploring New Horizons in Liquid Compartmentalization via Microfluidics

“… 88 − 91 Other interesting studies are the accumulation of different cargoes in different phases of complex emulsions for drug delivery purposes or incorporation of different catalysts for reaction cascades. 92 …”

Cascade communication in disordered networks of enzyme-loaded microdroplets

“…[5][6][7][8][9] Also, rivalling reaction centers have to be spatially separated from one another in order to control their destructive interference. [10][11][12] Whereas a direct mimicking of the complexity of living cells cannot yet be realized in artificial assemblies, a dissection and emulation of these individual capabilities within a simplified synthetic system that shows similarly sophisticated behavior would be of large scientific and technological value.…”