Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica.
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.
Dong
Jia Li
JL
College of Life Science, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Sichuan University, Chengdu, PR China.
Zhang
Yi Zheng
YZ
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
D002241:Carbohydrates; D006023:Glycoproteins; D007527:Isoenzymes; D008031:Lignin; D042845:Laccase