Single electron transfer by an extracellular laccase from the white-rot fungus Pleurotus ostreatus.
Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.
Youn
H D
HD
Department of Microbiology, College of Natural Sciences, Seoul National University, Republic of Korea.
Kim
K J
KJ
Maeng
J S
JS
Han
Y H
YH
Jeong
I B
IB
Jeong
G
G
Kang
S O
SO
Hah
Y C
YC
D000595:Amino Acid Sequence; D000946:Antigens, Fungal; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D042845:Laccase; D008667:Metalloproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011124:Polyporaceae / Q000201:enzymology* / Q000276:immunology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000946:Antigens, Fungal; D008667:Metalloproteins; D003300:Copper; D010088:Oxidoreductases; D042845:Laccase