Purification and characterization of an extracellular laccase of a fungus (family Chaetomiaceae) isolated from soil

A laccase-producing fungus was newly isolated from soil and shown to belong to family Chaetomiaceae. The extracellular laccase was purified to electrophoretic homogeneity from coffeic acid-induced culture medium by ammonium sulfate precipitation and anion exchange column chromatography. The enzyme was determined to be a monomeric protein with an apparent molecular mass of approximately 73–80 kDa and an isoelectric point (pI) of 3.5. One of its peptide fragments derived by endopeptidase digestion had a 68.75% amino acid sequence homologous to that of laccase I of Botryotinia fuckeliana. Spectroscopic analysis revealed that the enzyme has four bound copper atoms, a type I Cu(II), a type II Cu(II), and a type III binuclear Cu(II). The optimum pH for the oxidation of syringaldazine was 7.0 and the optimum temperature was 42 °C. The laccase was stable for up to 288 h at 4 °C and its respective half-life times at 25 and 40 °C were estimated to be 150 and 20 h. The enzyme reaction was inhibited by l-cysteine, dithiothreitol (DTT), p-coumaric acid, kojic acid, and thioglycolic acid. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for 4-hydroxyindole, and highest kcat/Km for 2,6-dimethoxy-phenol. In addition to these substrates, this laccase was effective for the biodegradation of endocrine-disrupting chemicals such as bisphenol A and nonylphenol (NP).