Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis.
The white-rot fungus Phellinus ribis produced a single form of laccase, which was purified to apparent electrophoretic homogeneity from cultures induced with 2,5-xylidine. This protein was a dimer, consisting of two subunits of 76 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that the enzyme contained about 28% carbohydrate content. The laccase appeared to be different from other known laccases by the UV-visible absorption spectrum analysis. One enzyme molecule contained one copper, one manganese, and two zinc atoms. The laccase showed optimal activity at pH 4.0-6.0, 5.0, and 6.0 with 2,6-dimethoxyphenol, ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], and syringaldazine, respectively. The enzyme preferably oxidized dimethoxyphenol and aromatic amine compounds. The stability of the laccase was low at acidic pH, whereas it showed high stability at neutral pH and mild temperature. The N-terminal amino acid sequence revealed a very low homology with other microbial laccases. With some substrates, the addition of manganese and H2O2 resulted in a remarkable increase in the oxidation rate. Without an appropriate phenolic substrate, the enzyme could not oxidize Mn(II) in the presence of H2O2 or pyrophosphate.
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Sir Mortimer B. Davis-Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, PQ, Canada. klmin@sympatico.ca
Kim
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Kim
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Jung
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Hah
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D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D052160:Benzothiazoles; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry*; D006835:Hydrazones / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D042845:Laccase; D037102:Lectins / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D011748:Pyrogallol / Q000031:analogs & derivatives / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013451:Sulfonic Acids / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D052160:Benzothiazoles; D002241:Carbohydrates; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D006835:Hydrazones; D037102:Lectins; D013451:Sulfonic Acids; D011748:Pyrogallol; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C010120:pyrogallol 1,3-dimethyl ether; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D042845:Laccase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010100:Oxygen