Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1

A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200-700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 degrees C and retained 61 % activity at 50 degrees C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest Km value being for ortho-dianisidine and highest K-cat and K-cat/K-m for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.