Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri.
Cyathus bulleri, a bird's nest fungus, known to decolorize polymeric dye Poly R-478, was found to produce 8 U ml(-1) of laccase in malt extract broth. Laccase activity appeared as a single band on non-denaturing gel. Laccase was purified to homogeneity by anion exchange chromatography and gel filtration. The enzyme was a monomer with an apparent molecular mass of 60 kD, pI of 3.7 and was stable in the pH range of 2-6 with an optimum pH of 5.2. The optimal reaction temperature was 45 degrees C and the enzyme lost its activity above 70 degrees C. Enzyme could oxidize a broad range of various phenolic substrates. K(m) values for ABTS, 2,6-dimethoxyphenol, guaiacol, and ferulic acid were found to be 48.6, 56, 22, and 14 mM while K(cat) values were 204, 180, 95.6, and 5.2, respectively. It was completely inhibited by KCN, NaN(3), beta-mercaptoethanol, HgCl(2), and SDS, while EDTA had no effect on enzyme activity. The N-terminal amino acid sequence of C. bulleri laccase showed close homology to N-terminal sequences of laccase from other white-rot fungi. A 150 bp gene sequence encoding copper-binding domains I and II was most similar to the sequence encoding a laccase from Pycnoporus cinnabarinus with 74.8% level of similarity.
Vasdev
Kavita
K
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
Dhawan
Shikha
S
Kapoor
Rajeev Kumar
RK
Kuhad
Ramesh Chander
RC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology*; D001717:Birds / Q000382:microbiology; D002413:Cations, Divalent / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D003300:Copper; D042845:Laccase; D008628:Mercury