鲜切荸荠类黄酮3’-羟化酶分离纯化及其动力学性质

Isolation,Purification and Characterization of Flavonoid 3'-Hydroxylase from Fresh-Cut Chinese Wate Chestnut

以鲜切荸荠为材料,通过p H 7.5 Tris-HCl缓冲液浸提得到类黄酮3’-羟化酶(flavonoid 3′-hydroxylase,F3’H)粗酶液,然后采用硫酸铵分级沉淀、透析、二乙氨乙基(dicthylaminoethyl,DEAE)-纤维素离子交换层析及Sephadex G-100凝胶过滤层析进行分离纯化,最终所得纯化酶的纯化倍数达到14.01,比活力提高到478.49 U/mg,回收率为6.38%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)进行纯度鉴定,已达到电泳纯,测定得到其分子质量约为53.09 k D。纯化酶F3′H的最适温度为30℃,最适p H值为7.5;以柚皮素作为底物,测得该酶的米氏常数(K_m)为1.08 mmol/L,最大反应速率(v_(max))为416.67 U/(min·m L);对于酶活性,高浓度的Na+表现出微弱的抑制作用,Ca^(2+)和柠檬酸具有强烈的抑制作用,而Fe^(2+)、Mg^(2+)、NADPH和VC却表现出强烈的激活作用。

Crude flavonoid3'-hydroxylase(F3'H)from fresh-cut Chinese water chestnut was extracted by Tris-HCl buffer(pH7.5),and then isolated and purified successively by ammonium sulfate precipitation,dialysis,DEAE-cellulose ionexchange column chromatography and Sephardi G-100gel filtration chromatography.After purification,the enzyme was finally purified14.01folds with a specific activity of478.49U/mg and a protein yield of6.38%.The purified enzyme showed a single protein band with a molecular mass of about53.09kD as estimated via sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Further enzymatic characterization showed that the purified enzyme activity attained its maximal value at30℃and pH7.5,and had a Km and vmax of1.08mmol/L and416.67U/(min·mL),respectively,using naringein as the substrate.The flavonoid3′-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.