Isolation and Purification of enzymes from ligninolytic complex of the basidial fungus Trametes pubescens (Schumach.) Pilát and study of their properties.
A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.
Nikitina
O V
OV
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071, Russia.
Shleev
S V
SV
Gorshina
E S
ES
Rusinova
T V
TV
Serezhenkov
V A
VA
Burbaev
D Sh
DSh
Belovolova
L V
LV
Yaropolov
A I
AI
D001487:Basidiomycota / Q000201:enzymology*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D042845:Laccase / Q000302:isolation & purification* / Q000378:metabolism; D008031:Lignin / Q000378:metabolism*; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008031:Lignin; D042845:Laccase; D010544:Peroxidases; C042858:lignin peroxidase