Characterization of an extracellular azo dye-oxidizing peroxidase from Flavobacterium sp. ATCC 39723

Abstract

A peroxidase from Flavobacterium sp. ATCC 39723 was selected from nine peroxidase-producing bacterial strains for its highest azo dye-oxidizing capacity among the peroxidases examined. Comparison of azo dye No. 1 (3,5-dimethyl-4-hydroxyazobenzene-4′-sulfonic acid) oxidation with native and heat-inactivated enzyme preparations, and with and without H 2O 2, confirmed the involvement of this peroxidatic enzyme in azo dye decolorization. The peroxidase activity in this gram-negative Flavobacterium sp. was predominantly extracellular. The enzyme was actively expressed in a minimal medium with sodium glutamate as sole carbon source. The addition of either pentachlorophenol or azo dye No. I into culture media did not enhance enzyme production. The Flavobacterium peroxidase was concentrated by ultrafiltration and partially purified by Q-Sepharose column chromatography. The optimal conditions for azo dye oxidation by the Flavobacterium peroxidase were pH 7–8, and 5 mM H 2O 2 concentration, when the azo dye concentration was held constant at 50 μM. Cobalt chloride, mercury chloride, and manganese sulfate had significant inhibitory effects on dye oxidation by the peroxidase. Although guaiacol was a strong inhibitor of dye oxidation by this enzyme, veratryl alcohol was not, even at I mM concentration. A heme protein inhibitor, potassium cyanide, showed considerable inhibition. The substrate specificity of the peroxidase toward 22 azo dyes was also examined. Specificity was compared to that of another peroxidase from the Streptomyces sp. YCED 105. The presence of syringyl or guaiacyl groups in the dye structures resulted in enhanced oxidation by both peroxidases. These data showed that azo dyes can be made more readily oxidizable by bacterial peroxidases by appropriately modifying the substitution pattern of their aromatic rings.