title: Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica.
abstract: Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.
authors.affiliation: College of Life Science, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Sichuan University, Chengdu, PR China.
authors.forename: Jia Li
authors.initials: JL
authors.lastname: Dong
authors.forename: Yi Zheng
authors.initials: YZ
authors.lastname: Zhang
mesh_terms: D001487:Basidiomycota / Q000201:enzymology* / Q000254:growth & development; D002241:Carbohydrates / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification*; D008031:Lignin / Q000737:chemistry; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D006023:Glycoproteins; D007527:Isoenzymes; D008031:Lignin; D042845:Laccase