Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium.
The wood-degrading fungus Pleurotus pulmonarius produces at least two laccase isoforms, Lcc1 and Lcc2, when grown on wheat bran solid state medium. The main laccase, Lcc2, was purified to apparent electrophoretic homogeneity by using acetone precipitation, anion-exchange chromatography and gel filtration. Lcc2 had been purified 5.9-fold with a yield of 49%. A specific activity of 19750 U/mg protein was found using syringaldazine as a substrate under standard assay conditions. The enzyme is a homodimeric glycoprotein containing 44% glycosilation and an apparent molecular mass of 46 kDa. Type I and type III Cu(2+) centers were identified by spectrophotometry. The laccase showed optimal activity at pH 6.2-6.5, 4.0-5.5, and 6.0-8.0 with syringaldazine, ABTS and guaiacol as substrates, respectively. For all substrates, the highest oxidation rates were obtained at 50 degrees C. The enzyme was stable over a large range of pH (4.5-8.0) and at temperatures up to 50 degrees C. Under standard assay conditions, the apparent K(M) values were 12, 210 and 550 microM for syringaldazine, ABTS and guaiacol, respectively. Purified Lcc2 was strongly inhibited by sodium azide, 2-mercaptoethanol and Hg(2+), and slightly inhibited by Mn(+2) and the chelant agents, EDTA and EGTA. The enzyme was activated by Cu(2+) and it retained a high percentage of its activity in the presence of organic solvents, such as acetonitrile and acetone.
Marques De Souza
Cristina Giatti
CG
Departamento de Bioquímica, Universidade Estadual de Maringá, 87020-900, Maringá, PR, Brazil.
Peralta
Rosane Marina
RM
D005656:Fungal Proteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D042845:Laccase; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D020076:Pleurotus / Q000201:enzymology*; D014908:Triticum / Q000382:microbiology*
D005656:Fungal Proteins; D007527:Isoenzymes; D010088:Oxidoreductases; D042845:Laccase