Biochemical characterization of a thermostable cobalt- or copper-dependent polyphenol oxidase with dye decolorizing ability from Geobacillus sp. JS12.

A putative laccase-like gene, GPPO, encoding a protein of 17.2 kDa and belonging to the multicopper oxidase family, was cloned and overexpressed in Escherichia coli cells. The purified recombinant protein GPPO is homodecameric protein with a molecular weight of 171.6 kDa. GPPO was not detected the ultraviolet-visible spectroscopy (UV/Vis) spectrum of typical laccases. Co2+ or Cu2+ was essential for substrate oxidation of GPPO, and the enzyme contained 1 mol of Co or Cu per mole of protein. The optimum pH required for the oxidation of 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) was 4.5 and 5.5, respectively, and the optimum temperature was 75 °C. The half-life of heat inactivation was about 8 min at 80 °C and 90 min at 90 °C, in the presence of Cu2+ and Co2+, respectively. The catalytic efficiency (kcat/Km) of GPPO containing Co2+ was 68 times higher than that of GPPO containing Cu2+. The enzyme reaction was inhibited by conventional inhibitors of laccase like metal chelators and thiol compounds. GPPO incubated with Cu2+ or Co2+ for 48 h decolorizes 45% or 47% of Nile blue, respectively. This is the first report of a novel thermostable polyphenol oxidase that shows the cobalt-dependent laccase activity and dye decolorization ability.