Isolation, Molecular Characterization and Reactivity with 2,6 Dichlorophenol of a Laccase and Isolation of Laccase Gene Specific Sequences from Lignin Degrading Basidiomycete Phanerochaete chrysosporium (TL 1)
A new lignin degrading basidiomycete, Phanerochaete chrysosporium (TL 1) was isolated from pulp and paper mill effluent enriched soil samples can be induced to produce high level laccases when grown on a cellobiose-asparagine liquid medium with 150 μM CuSO4. The fungus grown under static conditions had 70% of total extra cellular laccase protein and about 2.5 fold purification with a final yield of 13.2% of protein purification by Sephadex G-100 column and FPLC. The resultant enzyme pool of the purification process is found to contain a single polypeptide, which produced a single band on an SDS-PAGE. The purified protein showed a specific activity of 106 U mg-1 and the molecular mass (Mr) of native laccase was 65 kDa. The purified laccase has an isoelctric point of 4.0, it is stable in pH range from 4.0 to 6.0 and its optimum pH is 4.5. The optimal reaction temperature is 60°C and stable at 70°C for more than 1 h. Degenerative primers corresponding to the consensus sequences of the copper binding regions in the N-terminal domains of known basidiomycete laccase were used to isolate laccase gene specific sequences from this strain and the laccase gene gave PCR product of about 150 bp and cloned product gave 85% similarity with laccase from T. villosa LCC 2 (L49377).
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