Effect of phenolic monomers on the production of laccases by the edible mushroom Pleurotus sajor-caju, and partial characterization of a major laccase component

Abstract

The effects of several phenolic monomers on the growth and laccase production by the edible mushroom Pleurotus sajor-caju have been compared. Of the phenols tested, 4-hydroxybenzaldehyde and vanillin were most inhibitory to fungal growth, and cultures supplemented with these two compounds also exhibited the largest increases in laccase specific activity compared with unsupplemented controls. Up to five laccase isoforms were detected in culture fluids when P. sajor-caju was grown in submerged culture in the presence of several phenolic monomers. A major laccase component (laccase IV) present in P. sajor-caju culture supernatants was purified 152-fold with an overall recovery of 4.9%. Laccase IV was shown to be homogeneous by SDS-PAGE and isoelectric focusing, and had a molecular weight of 55 kD and an isoelectric point of 3.6. It displayed a maximum reaction velocity at pH 2.1 and 45 C, and the activation energy of the enzyme reaction between 25 and 45 C as determined by an Arrhenius plot was 12.4 kJ mol-1. The Km of laccase IV, as determined using ABTS as the substrate, was 0.092 mM. Laccase IV was glycosylated with mannose as the major sugar component together with galactose, glucosamine and fucose. Comparison of up to thirteen N-terminal amino acids of laccase IV with those of other fungal laccases revealed the highest similarity (85%) with a laccase from Pleurotus ostreatus. Considerable similarity (46-50%) was also observed with laccases from Coriolus versicolor, Pycnoporus cinnabarinus, Phlebia radiata, Coriolus hirsutus and Ceriporiopsis subvermispora but not Agaricus bisporus.