Aqueous two-phase extraction in combination with ultrafiltration for downstream processing of Ipomoea peroxidase

Abstract

The partition behaviour of a plant peroxidase extracted from the leaves of Ipomoea palmetta was studied in aqueous two-phase systems. The influence of various parameters such as system pH, phase composition, phase polymer molecular weight and concentration of neutral salt on differential partitioning of the enzyme and cell debris into opposite phases were evaluated. Desirable conditions for differential partitioning were found in systems having polyethylene glycol of lower molecular weight 1550, where the enzyme partitioned to the bottom phase and the cell debris partitioned to the top phase. Optimum conditions for extraction of the enzyme were observed in a polyethylene glycol (PEG-1550)/KH₂PO₄ system having 2% NaCl. Hence, using this system, extraction, enrichment and purification of the enzyme was performed on a larger-scale (1-l scale) in combination with ultrafiltration to obtain about 76% recovery, 5.9-fold purity and 9.7-fold activity concentration of the enzyme.