Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica.
A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETalphaA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae alpha-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the alpha-factor signal peptide was 9.79 U ml(-1). The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.
Guo
Mei
M
Department of Food Science, Tianjin Agriculture College, People's Republic of China.
Lu
Fuping
F
Pu
Jun
J
Bai
Dongqing
D
Du
Lianxiang
L
D015153:Blotting, Western; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D042845:Laccase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D003300:Copper; D042845:Laccase