Extraction and purification of a plant peroxidase by aqueous two-phase extraction coupled with gel filtration

Abstract

A plant peroxidase extracted from leaves of Ipomoea palmetta was partially purified by extraction using a polyethylene glycol (PEG)/ammonium sulphate aqueous two-phase system (ATPS), followed by gel filtration on a Sephadex G-100 column. Variation of phase composition and NaCl concentration resulted in the desired reduction in volume of the extract and selective partitioning of the enzyme. PEG/ammonium sulphate/NaCl (24/7.5/2.0%, w/v) system gave a partition coefficient of 0.042, purification factor of 2.18, volume reduction of 57.5% and bottom phase recovery of 91.5%. The enzyme was further concentrated by anti-dialysis and was finally purified about 49-fold by gel filtration on a Sephadex G-100 column, with a recovery of 75.3%. The enzyme was stable at 60°C with an optimum activity at 50°C. The enzyme was stable at pH range 5–11 and had an optimum pH at 6.0. The enzyme showed a K_m of 18.15 mM and V_max of 27.1 μmol/min when guaiacol was used as substrate.