Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molec ular oxygen. In this study, a full-length cDNA coding for laccase (lac1) fr om Pycnoporus cinnabarinus I-937 was isolated and characterized. The corres ponding open reading frame is 1557 nucleotides long and encodes a protein o f 518 amino acids. The cDNA encodes a precursor protein containing a 21 ami no-acid signal sequence corresponding to a putative signal peptide. The ded uced amino-acid sequence of the encoded protein was similar to that of othe r laccase proteins, with the residues involved in copper coordination shari ng the greatest extent of similarity. The cDNA encoding for laccase was pla ced under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficien tly directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The i dentity of the recombinant product was further confirmed by protein immunob lotting. The expected molecular mass of the mature protein is 81 kDa. Howev er, the apparent molecular mass of the recombinant protein is 110 k Da, thu s suggesting that the protein expressed in P. pastoris may be hyperglycosyl ated.