Screening of white rot basidiomycetes for novel laccases

Basidiocarps (fruiting bodies) of wood rotting fungi were inoculated into mycological agar slants and incubated for 20 days at 30°C along with the available isolates. Microscopic observations revealed few characteristic features belonging to basidiomycetes. Twenty of these novel isolates were screened for laccase production after growing them on Potato Dextrose Agar, Malt Extract and Mycological Agar for seven days at 30°C. Three different indicator compounds viz., 0.1% Syringaldehyde; 0.1% Guaiacol and 0.1% (w/v) ABTS were added to the solid media in order to detect ligninolytic enzymes. Eight strains produced detectable amounts of laccase in culture supernatant using Malt extract broth and Kirk's Basal medium, as detected by activity on ABTS. Evaluation of the selected eight cultures, using seven different buffers (Sodium acetate buffer pH - 4.5, pH - 5.0 and pH - 5.2; Glycine –HCl buffer; Succinate buffer; Sodium tartrate buffer and Sodium dihydrogen phosphate-citric acid buffer) failed to show significant variation in the activity (P>0.05). Though sonication of fungal biomass yielded higher enzyme activity, it was not significant (P>0.05). Based on the intensity of the color formation and Quantitative ABTS enzyme assay of the 8 positive isolates, four isolates, NI-04, NI-07, NI-09 and NI-03 showed 1843, 1383, 1530 and 386 units of laccase activity with the corresponding intensity of the color developments. Based on the results of the complete screening, NI-09, NI-04 and NI-07 proved to be the most promising white rot basidiomycetes strains. Results obtained were very encouraging. The enzyme produced can be purified and used for heterologous vector mediated transformation, and thus available for industrial and biological bioprocess use