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

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 Km 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.