Driving forces for chymosin partitioning on the macromolecule-salt aqueous two phase system

Abstract Aqueous two-phase systems (ATPSs) are strategic liquid–liquid systems for extraction and purification of compounds. However, only a few studies have evaluated the thermodynamic parameters that allow comprehension of the partition process of different molecules. Here, we investigated the chymosin (Chy) partitioning behavior in macromolecule + salt + water ATPSs by obtaining the partition coefficient ( K C h y ), Gibbs free energy change of transference ( Δ G t r , C h y ∞ ), enthalpy change of transference ( Δ H t r , C h y ∞ ), and entropy change of transference ( Δ S t r , C h y ∞ ), at infinite dilution, and their dependence on the ATPS properties. Chy transfer from the bottom to the top phase of the ATPS was enthalpically driven, with −4.84 kJ mol −1 Δ H t r , C h y ∞ −1 and −11.69 J mol −1  K −1 Δ S t r , C h y ∞ −1  K −1 characterizing an enthalpy–entropy compensation process; −1.36 kJ mol −1 Δ G t r , C h y ∞ −1 . Δ H t r , C h y ∞ became more negative as the tie-line length increased, showing that specific macromolecule–Chy interactions determine the enzyme concentration in the top phase. The nature of the cation/anion, hydrophobic/hydrophilic balance of the top phase, and macromolecule molar mass influence the intermolecular interaction between Chy and top phase components, changing the enzyme partition behavior. Negative Δ S t r , C h y ∞ parameters were attributed to the Chy transfer from a higher (bottom phase) to the lower (top phase) configurational entropy region.