The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Enzymological Characterization of EpoA, a Laccase-Like Phenol Oxidase Produced by Streptomyces griseus
Kohki EndoYutaka HayashiTakahiro HibiKuniaki HosonoTeruhiko BeppuKenji Ueda
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2003 Volume 133 Issue 5 Pages 671-677

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Abstract

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N, N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40°C, respectively. The enzyme retained 40% activity after preincubation at 70°C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

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