Expression and characterization of a recombinant multi-copper oxidase:: laccase IV from Trametes versicolor

A cDNA encoding LccIV, a previously uncharacterized laccase isozyme of the white-rot basidiomycete Tramete) versicolor, was expressed in the methylotrophic yeast Pichia pastoris. The LccIV isozyme is not expressed by T. versicolor under normal culture conditions and the enzyme was, therefore, investigated to determine whether it had any unusual properties. The native signal peptide of LccIV directed efficient secretion and correct proteolytic processing of LccIV to the mature form, whereas. substitution with the Saccharomyces cerevisiae alpha-mating factor signal peptide led to retention of an additional tetrapeptide at the amino-terminus of the secreted enzyme and similar to25% lower specific activity in fermentor medium. Active LccIV was purified to homogeneity by sequential steps of ion-exchange. size-exclusion and hydrophobic interaction chromatography. The enzyme contains similar to25% N-linked glycans (similar to40% total carbohydrate) and has an apparent molecular mass of similar to85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and similar to100 kDa by size-exclusion chromatography, indicating a monomeric structure. A pH of 5.5 was optimal for oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Thus, the LccIV isozyme appears to be similar in these respects to the laccase isozymes constitutively expressed by T. versicolor. (C) 2002 Published by Elsevier Science B.V.