To isolate laccase from Indian milky mushroom Calocybe indica and evaluated its potential in dye decolorization
Methods: Quantitative assay of laccase activity in broth was done spectrophotometrically using guaiacol broth as a substrate. Laccase enzyme was partially purified from C. indica by ammonium sulfate fraction. The fractions were collected and assayed for laccase activity and for protein concentration. Protein concentration was determined by the method of Brad-ford (1976) using bovine serum albumin (BSA) standard. After protein estimation, the purified sample subjected to SDS –PAGE analysis. Laccase is one of the major sources used for the dye decolorization. Decolorization of dye using laccase enzyme from C. indica was studied at different concentration (mg/ml) spectrophotometrically. The maximal toxic free concentration of laccase enzyme was evaluated on Vero cell line.
Result: The reddish brown color developed due to oxidation of guaiacol by laccase which was measured at 450 nm. The partially purified enzyme, when subjected to SDS PAGE analysis, revealed a molecular weight of 45kDa. HPLC analysis was performed which showed laccase at RT 4.572. The enzyme from C. indica was able to decolorize synthetic dyes malachite green to 83% and congo red 79% after 78 hrs of incubation. The maximal toxic free concentration evaluated on Vero cell line found it to be nontoxic at the concentration tested.
Conclusion: From the present study it can be concluded that the C. indica is a promising source for extracellular laccase production.