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The effect of lignin containing natural substrates corn-cob, coir-dust, saw-dust, wheat straw and bagasse particles on the extracellular secretion of laccase in the liquid culture growth medium of Pleurotus sajor-caju MTCC 141 has been studied. The culture conditions for maximum secretion of laccase by Pleurotus sajor-caju MTCC 141 have been optimized. Homogeneous preparation of laccase from the culture filtrate of the fungus has been achieved using ammonium sulphate precipitation, anion exchange chromatography on DEAE and gel filtration chromatography on Sephadex G-100. The purified enzyme preparation gave a single protein band in SDS-PAGE analysis indicating a molecular weight of 90 kD. The enzymatic characteristics Km, kcat, pH and temperature optima of the purified laccase have been determined using 2, 6-dimethoxyphenol as the substrate and have been found to be 35μmol/L, 0.30 min-1, 4.5 and 37℃ respectively. The Km values for the other substrate like catechol, m-cresol, pyrogallol and syringaldazine have also been determined which were found to be 216 μmol/L, 380 μmol/L, 370 μmol/L and 260 μmol/L respectively.