Equilibrium data and thermodynamic studies of α-amylase partition in aqueous two-phase systems

Abstract

Liquid-liquid equilibrium data (LLE) were obtained for aqueous two-phase systems (ATPS) composed of polyethylene glycol (average molar mass of 1500 g mol−1), sodium citrate/citric acid and water, at different pH (4.0, 5.0, 6.0 and 7.0) and temperatures T = (303.15 and 313.15) K. The thermodynamic study of the phase equilibrium was performed. The experimental results of the LLE were correlated using the UNIversal Functional Activity Coefficient (UNIFAC) model. The salting-out effect evaluated using the model of the effective excluded volume (EEV). Partitioning of the α-amylase (EC 3.2.1.1) was investigated as a function of pH, temperature, and tie line length (TLL). It was observed that the temperature had no influence on the formation of the biphasic region. Increments in pH value resulted in the raising of biphasic region. Isothermal titration calorimetry (ITC) assays was performed in order to deeply understanding the intermolecular interactions involved in the α-amylase partition and the driving forces that govern this process. It was measured the Gibbs free energy transfer (ΔtrG), transfer enthalpy variation (ΔtrH) and transfer entropy variation (ΔtrS) during the enzyme partition, as a function of the pH, temperature and TLL. For α-amylase partition, it was verified that at pH 4.0 the enzyme had a strong tendency to transfer to the polymer-rich phase. At pH 5.0 α-amylase showed affinity for the salt-rich bottom phase, while at pH 6.0, the partition coefficient of the enzyme increased as temperature was reduced to 303.15 K. The opposite behavior was noted for α-amylase at pH 7.0. Thermodynamic analysis based on isothermal titration microcalorimetry indicated the partition of α-amylase partition in the ATPS at pH 4.0 and 7.0 and 313.15 K was accompanied by endothermic heat and was entropically driven to the upper phase. Results of yield (95.373%) parameter indicated the applicability of ATPS for amylase purification.