Purification and characterization of laccase from Pleurotus tuber-regium and its application in dye decolourization

Pleurotus tuber-regium is a unique sclerotium-forming white rot fungus. It is edible and has medicinal value and a high potentials of application in various industries. However, to date, there is no information on the production of laccase by this fungus, the purification and determination of decolourization potential of laccase have also not been reported. In this study, purification of laccase from P. tuber-regium is demonstrated for the first time. Laccase was purified from the submerged culture of P. tuber-regium using ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. The molecular weight of laccase was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase was characterized to determine its optimum pH and temperature, substrate specificity, Michaelis-Menten constant, and inhibitors. The decolourization potential of the crude laccase was evaluated using Congo red, trypan blue, textile dyes and textile effluent. The purified laccase had a molecular weight of 52 kDa, optimum pH and temperature were 4 and 60EC, respectively. The best substrate for laccase was found to be 2,2N-azino-bis (3-ethylbenzothiazoline-6-sulfonate), with a Michaelis-Menten constant (Km) of 7.82 μM. Laccase activity was mildly inhibited by ethylenediamine tetraacetic acid (EDTA) and L-cysteine, but strongly inhibited by sodium azide. The activity of the purified laccase was enhanced by more than two fold by Na+ , Ca2+ and Ba2+, and slightly enhanced by Hg2+ and Mn2+. The crude laccase was able to efficiently decolourize trypan blue and Congo red dyes and red and blue textile dyes. Notably, on solid agar, trypan blue was completely degraded by intracellular laccase.