Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus

The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporu s cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840 bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes we re identified. The cDNA of lcc3-2 contains an open reading frame of 1563 bp . The deduced mature laccase protein consisted of 498 amino acids and was p receded by a signal peptide of 23 amino acids. The sequence of lcc3-2 revea ls 73% similarity on the protein level to the previously characterized lcc3 -1. The new laccase gene shares highest similarity to lcc1 from Trametes vi llosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (pl) of 6.1 for the gene product LCC3-2 w as in good agreement with the experimentally determined pi of a laccase sec reted by P. cinnabarinus grown on cellulose. Transcription analysis using c ompetitive reverse transcription (RT)PCR showed that lcc3-2 was expressed i n glucose and cellulose containing cultures. However, in contrast to lcc3-1 , lcc3-2 transcription was not increased in response to 2,5-xylidine. (C) 1 999 Elsevier Science B.V. All rights reserved.