Isolation, Purification and Characterization of Flavonoid 3’-hydroxylase in Fresh-cut Chinese Water-chestnut

Abstract

Abstract: The crude enzyme solution of flavonoid 3’-hydroxylase (F3’H) in fresh-cut Chinese water-chestnut was extracted by Tris-HCl buffer (pH7.5), and then isolated and purified by ammonium sulfate precipitation, dialysis, and ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-100. After purification procedure, the enzyme exhibited final purification fold of 14.01, specific activity of 478.49 U/mg and protein yield of 6.38%, respectively. The purified enzyme showed a single protein band and the molecular mass was about 53.09 kDa via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Further enzymatic characterization assays showed that the purified enzyme attained maximal activity 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, strongly inhibited by Na+, however, Fe2+, Mg2+, NADPH and ascorbic acid exhibited strongly inhibition activated on flavonoid 3’-hydroxylase activity.

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

CLC Number:

References

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