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

Abstract

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 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest K_m 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.

Keywords: ABTS; Laccase; N-terminal sequencing; Pleurotus ostreatus; Purification.

Fig. 1

a The SDS-PAGE (A) indicating single band of protein with molecular weight of approximately 66,000 Da which corresponds to the single band showing blue color of laccase activity in activity staining (B). b The peak of MALDI-TOF indicating the actual molecular weight of the purified laccase

Fig. 2

Effect of different temperature 30 °C wide diamond, 40 °C square, 50 °C triangle, 60 °C diamond and 70 °C cross on stability of laccase

Fig. 3

Effect of different pH of the buffer on oxidation of different substrates by purified laccase. filled circle, open circle—dianisidine (U ml⁻¹), open circle guaiacol (U ml⁻¹), inverted filled triangle ABTS (U ml⁻¹), inverted open triangle DMP (U ml⁻¹)