Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many laccase isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have been purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative gel electrophoresis, and electroelution. Their estimated molecular weights are 65-68 kDa, and they contain 7-10% N-linked carbohydrates. The three isozymes have identical N-terminal amino acid sequences: G-I-G-P-T. The optimum pH and temperature both for each isozyme singly and the isozyme mixture are pH 3.5 and 20 degrees C, respectively. One isozyme (GaLc 3) is quite stable at pH 4.0-10.0, and shows good stability when incubated at temperatures lower than 40 degrees C. The Km values of GaLc 3 for o-tolidine and 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) are 401.6 microM and 3.7 microM respectively, and the Vmax of GaLc 3 for these substrates is 0.0198 OD min(-1) unit(-1) and 0.0142 OD min(-1) unit(-1), respectively.