Overexpression and characterization of a thermostable, pH-stable and organic solvent-tolerant Ganoderma fornicatum laccase in Pichia pastoris

Abstract

The newly defined laccase gene, lac1, from the medicinal white-rot fungus Ganoderma fornicatum 0814 was cloned and expressed in Pichia pastoris. The corresponding open reading frame has 1563 nucleotides and encodes a protein of 521 amino acids, including a 21-residue secretion signal peptide. The absorption spectrum of the purified laccase, rLac1, indicated that it is a yellow laccase rather than the common blue laccase. The optimum pH of rLac1 was 2.5, 3.0 and 3.0 for ABTS, DMP and guaiacol, respectively. The kinetic parameter K m of rLac1 was 103.9, 276.7 and 1263.4 μM for ABTS, DMP and guaiacol, respectively. The optimal temperature for rLac1 was 55 °C for all of the substrates. rLac1 is stable up to 60 °C and in the pH range 2.5–10.0. Sodium azide and thioglycolic acid strongly inhibited rLac1 activity, but it was not affected by EDTA. rLac1 retained more than 80% activity in 50% (v/v) ethanol, methanol, DMF and DMSO after a 3-h incubation at 25 °C. Less than 20% of activity was found in acetone and acetonitrile. The results show that rLac1 is thermostable, pH-stable and tolerant to organic solvents. These properties suggest that rLac1 has potential uses in industrial applications.