Integration of ultrafiltration into an aqueous two-phase system in the keratinase purification

• The keratinase purification was maximized using ATPS two-stage process.
• A keratinase purification protocol using ATPS two-stage process combined ultrafiltration was established.
• The polyethylene glycol was separated from the protein solution by ultrafiltration.

Keratinases are proteases that have several important applications in the daily life. Although they have attracted the attention of researchers in the field of biotechnology, their use in industrial processes requires efficient purification techniques allowing scaling-up. Among these techniques, the aqueous two-phase system and ultrafiltration stand out. This study reports the purification of keratinase from Bacillus sp. P45 by aqueous two-phase system two-stage process integrated to ultrafiltration. In the aqueous two-phase system, the addition of sodium chloride in polyethylene glycol-potassium phosphate system favored the partitioning of keratinase. The keratinase purified by an aqueous two-phase system two-stage process integrated to ultrafiltration in diafiltration mode presented purification factor of 6.1-fold and enzymatic recovery of 56.3%, and the enzyme was separated from polyethylene glycol. The purified keratinase was capable of hydrolyzing both soluble and insoluble protein substrates more efficiently than the crude enzyme, evidencing the beneficial effect of enzyme purification on its catalytic activity.