Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus.
Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, DEAE Sepharose CL-6B, Sephacryl S-200 HR, Hitrap SP, and Mono S chromatography. The purification was 14.5-fold with an overall yield of 32.3%. The enzyme is a monomeric glycoprotein with 11% carbohydrate content, an isoelectric point of 7.4, and a molecular mass of 73 kDa. The N-terminal amino acid sequence showed low homology to those of the laccases of other white-rot basidiomycetes. Spectroscopic analyses revealed a typical laccase active site in the C. hirsutus enzyme, as all three Cu centers were identified. The absorption spectrum showed a type 1 signal at around 600 nm and a type 3 signal near 330 nm. Type 3 Cu showed fluorescence emission near 418 nm and an excitation maximum at 332 nm. The EPR spectrum yielded parameters for the type 1 and type 2 Cu of gII = 2.191 and AII = 0.0097 cm(-1), and gII = 2.222 and AII = 0.0198 cm(-1), respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for the enzyme was reached at 45 degrees C, and the pH optima of the enzyme varied and was substrate dependent in the range of 2.5 to 4.0. The enzyme oxidized a variety of the usual laccase substrates, including lignin-related phenols and had highest affinity toward guaiacol. Under standard assay conditions, the apparent Km value of the enzyme toward guaiacol was 10.9 microM. The enzyme catalyzed single electron transfer via the phenoxy radical as an intermediate and was completely inhibited by L-cysteine and sodium azide but not by EDTA.
Shin
K S
KS
Department of Microbiology, College of Sciences, Taejon University, Republic of Korea. shinks@dragon.taejon.ac.kr
Lee
Y J
YJ
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D042845:Laccase; D017705:Lignans / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D017705:Lignans; D010088:Oxidoreductases; D042845:Laccase