Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties, Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively, A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH less than or equal to 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively, The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined, The genes for lcc1 and lcc2 have 8 and 10 introns, respectively, The predicted proteins are 79% identical at the amino acid level, From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.