Cloning and heterologous expression of the gene of laccase C from Trametes sp. 420 and potential of recombinant laccase in dye decolorization

A new laccase gene (lacC) was cloned from the genomic DNA isolated from Trametes sp. 420, a new laccase-producing fungus, using the degenerate primers based on the conserved copper-binding regions in fungal laccases. Long distance-inverse PCR (LD-IPCR) was used to amplify the flanking sequences of the gene. The lacC DNA sequence obtained was 3640 base pairs (bp), including the entire open reading frame (2263bp) and the 5′-and 3′-noncoding regions. The lacC cDNA sequence is 1560bp, encoding a 519 amino acid protein. The deduced peptide sequence of LacC contains ten putative N-glycosylation sites and four conserved copper-binding regions. The lacC cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The positive transformant was cultured at 20℃ in BMM medium containing 0.3mmol/L CuSO_4 and 0.8% alanine, with the yield of the recombinant laccase rLacC being 1.62×10~4 U/L after a 9-day cell growth. Furthermore, the crude enzyme was used to decolorize several synthetic dyes at a final concentration of 50mg/L. The results showed that rLacC (6U/L) possessed the valuable ability to decolorize dyes of triarylmethane and azo types tested. The presence of low molecular weight redox mediators of ABTS and HBT increased the efficiency and velocity of dye decolorization significantly.