Gene Cloning and Characterization of a Novel Laccase from the Tropical White-Rot Fungus Ganoderma weberianum TZC-1

The novel laccase gene GwLac1 was cloned from Ganoderma weberianum TZC-1 by reverse transcriptase PCR with degenerate primers on the basis of conserved copper-binding regions of known laccases and the rapid amplification of cDNA ends technique. GwLac1 contains 9 introns and its open reading frame is 1566 bp long. The coding domain sequence of GwLac1 was cloned into the vector pPICZB and expressed in Pichia pastoris strain GS115, resulting in the highest yield of laccase, 2.26 U/mL, when the transformants had been cultivated at 20 degrees C for 8 days. The molecular mass of recombinant GwLAC1 was a little more than 50 kDa as estimated by SDS-PAGE and exhibited catalytic properties with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate. rGwLAC1 was stable at pH of 5.0-7.0 and at temperatures < 45 degrees C. The pH and temperature optima and K-m of the enzyme for ABTS oxidation were 2.2, 35 degrees C and 85.5 mu M, respectively.