Isolation, Purification and Characterization of Flavonoid 3'-Hydroxylase from Fresh-Cut Chinese Wate Chestnut
Crude flavonoid 3'-hydroxylase (F3'H) from fresh-cut Chinese water chestnut was extracted by Tris-HC1 buffer (pH 7.5),and then isolated and purified successively by ammonium sulfate precipitation,dialysis,DEAE-cellulose ionexchange column chromatography and Sephardi G-100 gel filtration chromatography.After purification,the enzyme was finally purified 14.01 folds with a specific activity of 478.49 U/mg and a protein yield of 6.38％.The purified enzyme showed a single protein band with a molecular mass of about 53.09 kD as estimated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Further enzymatic characterization showed that the purified enzyme activity attained its maximal value at 30 ℃ and pH 7.5,and had a Km and Vmax of 1.08 mmol/L and 416.67 U/(min·mL),respectively,using naringein as the substrate.The flavonoid 3'-hydroxylase activity was slightly inhibited by Ca2+ and citric acid and strongly inhibited by Na+.However,Fe2+,Mg2+,NADPH and ascorbic acid strongly activated its activity.
fresh-cut Chinese water chestnutflavonoid 3'-hydroxylaseisolation and purificationenzymatic characterization
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