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

Ganoderma lucidum, a medicinal white-rot basidiomycete, produces many lacca se isozymes in liquid culture. Three laccase isozymes (GaLc 1, 2, 3) have b een purified 32.4-fold from the crude enzyme protein through anion exchange chromatography, preparative get 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 seq uences: G-I-G-P-T. The optimum pH and temperature both for each isozyme sin gly and the isozyme mixture are pH 3.5 and 20 degreesC, respectively. One i sozyme (GaLc 3) is quite stable at pH 4.0-10.0, and shows good stability wh en incubated at temperatures lower than 40 degreesC. The K-m values of GaLc 3 for o-tolidine and 2.2'-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) are 401.6 muM and 3.7 muM respectively. and the V-max of Gal-c 3 for these substrates is 0.0198 OD min(-1)unit(-1) and 0.0142 OD min(-1)unit(-1), res pectively.