Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF- 1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized R chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000 +/- 1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30degreesC, respectively. This enzyme was stable in the pH range 4.5-6.0, at 25 degreesC and also up to 35 degreesC at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN3, ascorbic acid, beta-mercaptoethanol and dithreitol. The K-m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenot were 71.4 and 28.57 muM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated. (C) 2003 Elsevier Ltd. All rights reserved