Partitioning and purification of menadione induced NAD(P)H oxidase from Phanerochaete chrysosporium in aqueous two-phase systems

The partitioning behavior of menadione-induced NAD(P)H oxidase from the white rot fungus Phanerochaete chrysosporium has been studied in various poly(ethylene glycol) (PEG)-salt aqueous two-phase systems (ATPSs). The effects of the molecular weight of the PEG, system composition, and pH of the system on NAD(P)H oxidase partitioning were investigated at 25°C. The best partition parameters were observed in the PEG 2000 18%–K₂HPO₄ 13%–NaCl 0.5% (w/w, pH 7.0) system; the partition coefficient of total NAD(P)H oxidase activity (K_e) of the system was 0.232. While the recovery in the bottom phase (R_b) was 87.644%, this value was 20.350% in the top phase of the optimized system. The purification fold was found as 8.331 in the bottom phase. NAD(P)H oxidase purified from P. chrysosporium by the PEG–K₂HPO₄ two-phase system was determined to be a homodimer in its native state with a molecular weight of 116kDa.