Identification, Purification and Characterization of a Novel Extracellular Laccase from Cladosporium Cladosporioides

Cladosporium cladosporioides was identified as a laccase producer when grown on malt extract media supplemented with 0.0 2% ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid). Extracellular laccase was purified to homogeneity from culture supernatant of Cladosporium cladosporioides by acetone precipitation, gel filtration chromatography and anion exchange chromatography, and was also characterized. Purified laccase (specific activity 1785.71 U·mg −1 ) is a monomeric protein with molecular weight of 50 kDa on SDS-PAGE. Purified laccase showed maximum activity at pH 5 and optimal temperature was observed 40°C. The enzyme showed a high relative activity over a wide range of pHfrom 3 to 6 and was stable in a temperature range of 40° to 70°C. The K m value of the purified enzyme was found to be 15 μmol·L −1 , while the Vmax value was observed to be 4612 μmol·L −1 ·min −1 with ABTS as a substrate. The catalytic activity of laccase was completely inhibited by dithiothreitol, sodium azide and L-cysteine at a final concentration of 5 mmol·L −1 , and slightly inhibited by EDTA and SDS at a final concentration of 10 mmol·L −1 . The laccase was activated by Cu 2+ and Mg 2+ , and certain metal ions such as Ni +2 , Ba +2 and Ag +2 inhibit the catalytic activity of enzyme at a final concentration of 1.0 mmol·L −1 . To the best of our knowledge this is the first report on the purification and characterization of laccase from Cladosporium cladosporioides.