title: Purification and characterization of laccase from basidiomycete Fomitella fraxinea.
abstract: A laccase was isolated from the culture filtrate of basidiomycete Fomitella fraxinea. The enzyme was purified to electrophoretical homogeneity using ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography. The enzyme was identified a monomeric protein with a molecular mass of 47 kDa sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography, and had an isoelectric point of 3.8. The N-terminal amino acid sequence for the enzyme was ATXSNXKTLAAD, which had a very low similarity to the sequences previously reported for laccases from other basidiomycetes. The optimum and temperature for 2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulfonate) (ABTS) were 3.0 and 70 degrees C, respectively. The enzyme also showed a much higher level of specific activity for ABTS and 2,6-dimethoxyphenol (DMP), where the values of the enzyme for ABTS and 2,6-DMP were 270 and 426 microM, respectively, and the Vmax values were 876 and 433.3 microM/min, respectively. The laccase activity was completely inhibited by L-cysteine, dithiothreitol (DTT), and sodium azide, significantly inhibited by Ni+, Mn+ and Ba+2, and slightly stimulated by K+ and Ca+2.
authors.affiliation: Department of Biotechnology, Dongguk University, Gyeongju 780-714, Korea.
authors.forename: Kyung Mi
authors.initials: KM
authors.lastname: Park
authors.forename: Sang-Shin
authors.initials: SS
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D042845:Laccase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011124:Polyporaceae / Q000737:chemistry / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D042845:Laccase