Abstract: Crude flavonoid 3’-hydroxylase (F3’H) from fresh-cut Chinese water chestnut was extracted by Tris-HCl 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.

Key words: fresh-cut Chinese water chestnut, flavonoid 3′-hydroxylase, isolation and purification, enzymatic characterization