Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions

Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unclear. Multiple laccase isoforms produced by the same organism imply a greater flexibility and/or adaptation to constantly changing environments. In this study, we explore and discuss the laccase isoform diversity of Trametes versicolor under submerged culture conditions. Oak sawdust was used as a natural, abundant and cheap source of laccase inducers. Two laccase bands (lcc1 and lcc2) were detected by SDS-PAGE zymography. Lcc2 exhibited an apparent molecular mass of 100 kDa, and was purified to its characteristic blue color for biochemical characterization. Lcc2 displayed high affinities toward all the substrates used in this study (0.01 ± 0.0043, 0.5 ± 0.053, 2.23 ± 0.53 and 3.77 ± 0.85 mM, for ABTS, DMP, GUA and SYR, respectively). Isoelectric focusing analysis of this band revealed the presence of 5 distinct laccase isoforms. The suggested existence of different laccase heterodimers in lcc2 may have had an influence on its catalyzing capacity. Laccase isoform diversity was corroborated by molecular characterization of 5 allelic mRNA sequences. Multiple alignment analysis, 3-dimensional mapping and theoretical sequence-structure-function assumptions suggests that the newly characterized alleles would most likely display diversity in their biochemical behavior if functionally expressed.