食品科学 ›› 2017, Vol. 38 ›› Issue (8): 17-23.doi: 10.7506/spkx1002-6630-201708004

• 生物工程 • 上一篇    下一篇

鲜切荸荠类黄酮3’-羟化酶分离纯化及其动力学性质

何凤平,潘永贵,张伟敏   

  1. 海南大学食品学院,海南 海口 570228
  • 出版日期:2017-04-25 发布日期:2017-04-24
  • 基金资助:
    国家自然科学基金地区科学基金项目(31360414)

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

HE Fengping, PAN Yonggui, ZHANG Weimin   

  1. College of Food Science and Technology, Hainan University, Haikou 570228, China
  • Online:2017-04-25 Published:2017-04-24

摘要: 以鲜切荸荠为材料,通过pH 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-polyacrylamidegel 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 kD。纯化酶F3′H的最适温度为30 ℃,最适pH值为7.5;以柚皮素作为底物,测得该酶的米氏常数(Km)为1.08 mmol/L,最大反应速率(vmax)为416.67 U/(min·mL);对于酶活性,高浓度的Na+表现出微弱的抑制作用,Ca2+和柠檬酸具有强烈的抑制作用,而Fe2+、Mg2+、NADPH和VC却表现出强烈的激活作用。

关键词: 鲜切荸荠, 类黄酮3′-羟化酶, 分离纯化, 动力学性质

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

中图分类号: