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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (15): 245-248.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

高效液相色谱法测定地黄蔗糖合成酶、蔗糖磷酸合成酶活性

刘燕 伊伟贞 焦连魁 王露 李先恩   

  • 收稿日期:2011-03-03 修回日期:2011-03-29 出版日期:2011-06-25 发布日期:2011-06-25
  • 基金资助:

    基金项目：国家科技重大专项课题“中药材种子种苗和种植（养殖）标准平台”

Determination of sucrose synthase and sucrose shosphate synthase activity in Rehmannia glutinosa by HPLC

  • Received:2011-03-03 Revised:2011-03-29 Online:2011-06-25 Published:2011-06-25

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摘要：

为了建立地黄蔗糖合成酶、蔗糖磷酸合成酶的高效液相色谱测定方法，利用高效液相法测定蔗糖合成酶、蔗糖磷酸合成酶反应前后蔗糖含量的变化，计算出酶活性。采用Waters XBridgeTM Amide 3.5 ?m （4.6×150 mm Column）色谱柱，流动相乙腈-水（70:30），流速0.5 mL/min，ELSD检测器：漂移管温度40℃，雾化气体30 psi，喷雾模式：冷却；地黄中蔗糖在0.128~3.2 μg的范围内，峰面积与含量呈良好线性关系（r=0.9999），且HPLC测定两种酶活性的RSD均小于5%。HPLC法稳定、可靠、精密度高、重复性好，可作为地黄蔗糖合成酶、蔗糖磷酸合成酶活性的测定方法。

关键词: 油茶枯饼, 油茶枯饼, 脱毒, 发酵, 培养基

Abstract:

To establish a HPLC method for the determination of sucrose synthase and sucrose phosphate synthase activity in Rehmannia glutinosa by HPLC, the activity of sucrose synthase and sucrose phosphate synthase was calculated through detecting the changes of sucrose content by HPLC. The column was Waters XBridgeTM Amide 3.5 ?m (4.6×150 mm Column)，with acetonitrile-water (70:30) as mobile phase, at a flow rate of 0.5 mL/min and a column temperature of 25℃. ELSD condition was optimized at the drift tube temperature of 40℃, the atomization gas of 30 psi and the spray mode of cooling. The regression line was linear over the concentration range of 0.128-3.2 μg (r=0.9999). The RSD by HPLC method was less than 5%. This method is stable and reliable, and has good precision and reproducibility for the determination of sucrose synthase and sucrose phosphate synthase Activity in Rehmannia glutinosa.