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

- Thermodynamic of chymosin partition in macromolecule-salt ATPS was determined.
- Transfer parameters (ΔGtr,Chy∞, ΔHtr,Chy∞ and ΔStr,Chy∞) were obtained by nanocalorimetry.
- Chymosin transfer was enthalpically driven, with enthalpy-entropy compensation.
- ΔHdil,Chy∞ was used to ascertain the interactions involved in the enzyme partition.
- Chymosin ATPS components interaction competition driven the enzyme partition.

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 (KChy), Gibbs free energy change of transference (ΔGtr,Chy∞), enthalpy change of transference (ΔHtr,Chy∞), and entropy change of transference (ΔStr,Chy∞), 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 < ΔHtr,Chy∞ < −170.34 kJ mol−1 and −11.69 J mol−1 K−1 < ΔStr,Chy∞ < −558.95 J mol−1 K−1 characterizing an enthalpy-entropy compensation process; −1.36 kJ mol−1 < ΔGtr,Chy∞ < −3.77 kJ mol−1. ΔHtr,Chy∞ 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 ΔStr,Chy∞ parameters were attributed to the Chy transfer from a higher (bottom phase) to the lower (top phase) configurational entropy region.

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