Purification and characterization of a thermotolerant laccase isoform in Trametes trogii strain and its potential in dye decolorization

Abstract

Using response surface methodology, the maximum laccase activity of 122.9 U ml−1 was obtained in Trametes trogii S0301. The major isoform of the laccase secreted in the optimized medium was purified by 4-fold to a specific activity of 352.1 U mg−1 protein. The laccase (a molecular mass of 56 kDa), acted optimally at pH 3.0 and exhibited an optimum temperature of 45 °C using ABTS as substrate, with the half-life at 60 °C and 75 °C for 3 h and 10 min, respectively. The purified laccase was highly resistant to Co2+, Cu2+, Zn2+ and Mn2+ (100 mM). In addition, the purified laccase was effective to decolorize malachite green, bromophenol blue, crystal violet and acid red without the addition of redox mediators. Peptide-mass fingerprinting analysis by MALDI-TOF MS showed the purified laccase of T. trogii S0301 was a typical laccase isoform, which shared 99.3% identity with a laccase from Coriolopsis gallica. Further, the full-length DNA of the laccase was cloned based on the highly conservative copper-binding domains using degenerate PCR and TAIL-PCR, and the deduced amino acid sequence of the matured protein matched exactly with the peptides of the purified laccase.