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Separation and purification of proteins, organic compounds and other biomolecules are feasible by liquid–liquid extraction based on phase separation in aqueous micellar solution. This work aimed to optimize the extraction of the pectinase enzyme produced by Aspergillus oryzae in a passion fruit peel media, using a micellar Triton X-114/sodium phosphate buffer system. Best condition for this extraction was evaluated in a 2³ experimental factorial design with a centered face and three repetitions at the central point. The variable analyzed was the pectinase activity, and in this design, it varied from 0.233 to 0.601 U mL⁻¹. The highest activity was obtained in the top phase at conditions 25 °C, 8% Triton X-114 (wt/wt) and 20% concentrated crude extract (wt/wt). The results indicated that this aqueous two-phase micellar system (ATPMS) was able to remove visible pigments and possible pectinase inhibitors present in …