Liquid–liquid equilibrium data of aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) 8000 + sodium sulfate + water, PEG 8000 + ammonium sulfate + water, and PEG 8000 + magnesium sulfate + water at T = 298.15, 308.15, and 318.15 K and pH 2.0 were determined. The universal function activity coefficient (UNIFAC) model was correlated to the experimental tie-line data, and the root-mean-square deviations (RMSDs) between experimental and calculated data were considered in the calculation. The effects of temperature and cation type on the spinodal curves were evaluated. The salting-out effect was studied by the determination of the effective excluded volume (EEV). Rising temperature resulted in an increase in the biphasic region and tie-lines. Regarding the cation type, the phase-separation abilities followed the order Mg2+ > Na+ > (NH4)+. Increases in the overall composition of the systems resulted in increased tie-line lengths. The tie-line slope absolute values also tended to rise with increasing temperature. Increasing the ATPS constituent concentrations resulted in an increase in the phase densities. The increase in temperature resulted in an increase in the salting-out effect. The exclusion volume values increased in the following order: (NH4)+ < Na+ < Mg2+. The overall mean deviation between the experimental and correlated LLE compositions for the PEG 8000 + ammonium sulfate + water, PEG 8000 + magnesium sulfate + water, and PEG 8000 g mol–1 + sodium sulfate + water was 3.901%. The results of the UNIFAC model agree with the experimental tie-line values. This allows the use of this model to obtain reliable data for this system, thus reducing the number of experiments to be performed for a processing design.

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This article studies the influence of pH and extraction solution, time, and solid/liquid ratio on the extraction of protease from ora‐pro‐nobis. Aqueous two‐phase systems (ATPS) formed by polyethylene glycol 1,500 or 4,000 and phosphate were investigated as a purification step for these proteases. The best results were with sodium phosphate pH 7.0, 60 min of extraction, and at the liquid/solid ratio of 21 ml.g−1. ATPS formed by polyethylene glycol and phosphate were obtained. The increase in molar mass resulted in the expansion of the biphasic region, exclusion volume, and the free volume of the upper phase. The proteases were preferentially partitioned to the upper phase. The free energy indicates that the partition of proteases to the upper phase is a spontaneous process. High selectivity, activity recovery, and the purification factor indicate that the studied systems can be used as a good alternative for the purification of this enzyme. Practical applications An alternative plant protease source obtained from ora‐pro‐nobis leaves has been investigated for the first time. The results show that experimental conditions established in this work allow the efficient extraction and partial purification of protease.

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