Purification and Characterization of Phloroglucinol Oxidase from Broccoli (Brassica oleracea L. Italica Group)

Abstract

　Phloroglucinol oxidase (PhO) was purified from broccoli. The purified enzyme appeared as a single band by SDS-PAGE. The enzyme was purified about 80-fold with a recovery rate of 26%. The estimated molecular weight of the enzyme was 63 kDa and 65 kDa, obtained by SDS PAGE and gel filtration, respectively. The purified enzyme oxidized phloroglucinol (1,3,5-trihydroxybenzene) with a K_m value of 11 mM. Peroxidase (POD) activity was also present in the enzyme preparation which was purified about 33-fold with a recovery rate of 11%. The PhO and POD showed highest activities at pH8.0 and pH7.0, respectively. The activities of PhO and POD were stable between pH5.0 and 10.0 at 5℃ for 20h. The optimum temperature was 60℃ for PhO and 20℃ for POD. The activities of PhO and POD were strongly inhibited by sodium diethyldithiocarbamate, KCN, L-ascorbic acid, chlorogenic acid, and hydroquinone at 1m M.