H2O2-KI-FRCT反应体系荧光猝灭法测定小麦粉添加剂葡萄糖氧化酶活性

Determination of GOD activity in flour additive by fluorescence quenching method using H2O2-KI-FRCT reaction system

中文关键词:  荧光素  葡萄糖氧化酶  活性  过氧化氢  荧光猝灭法

英文关键词:Fluorescein  Glucose oxidase  Activity  Hydrogen peroxide  Fluorescence quenching method

中文摘要:

      为解决面粉添加剂葡萄糖氧化酶（GOD）活性检出限偏高、测试过程复杂等问题，特提出H2O2-KI-FRCT反应体系荧光猝灭法测定GOD活性新思路。荧光素（FRCT）于515nm处有特征荧光峰，GOD酶促反应生成的H2O2氧化KI生成碘单质，从而引起FRCT体系的荧光猝灭，荧光强度递减量（ΔF）与GOD活性有关联，据此构建GOD活性测定新方法。GOD活性测定分三步完成：建立H2O2标准曲线方程；完成GOD酶反应；记录ΔF，计算酶活性E（单位时间1分钟内H2O2浓度增量，μmol/(min·L)，U/L）。完成酶反应、KI氧化反应及FRCT荧光猝灭反应环境优化；在最佳测试条件下，测得新配GOD工作液及储备液真实活性，并进行方法精密度考察；以已知活性的GOD储备液为母本，配制GOD标准系列，逐个测其对应ΔF，得到GOD标准曲线方程，相关系数ｒ=0.9985，检测下限为0.0216 U/L，最低检测限达到2×10-4 U/L。新方法具有良好的重现性和准确性，用于市售面粉添加剂GOD活性测试，结果与双酶法基本一致。

英文摘要:

      To address the issues of high detection limit and complex testing process of glucose oxidase (GOD) activity in flour additives, A new approach for determining GOD activity using the H2O2- KI -FRCT reaction system fluorescence quenching method has been proposed.The fluorescent substance fluorescein (FRCT) has a characteristic fluorescence peak at 515nm. The H2O2 generated by GOD enzymatic reaction further oxidizes KI to form elemental iodine, which causes fluorescence quenching in the FRCT system. The decrease in fluorescence intensity (ΔF) is related to the activity of GOD and GOD activity determination was completed based on this.The GOD activity assay is completed in three steps: establishing the H2O2 standard curve equation; Complete GOD enzyme reaction; Record ΔF and calculate enzyme activity E (H2O2 concentration increment within 1 minute per unit time, μmol/(min·L),U/L）.The enzyme reaction, KI oxidation reaction, and FRCT fluorescence quenching reaction environments were optimized;Under the optimal testing conditions, The true activity of the newly prepared GOD working fluid and reserve fluid was determined, and the precision of the method was investigated; Finally, using the known active GOD reserve fluid as the parent material, a GOD standard series was prepared and its ΔF was measured one by one,GOD linear equation was obtained with a correlation coefficient of r=0.9985. The detection limit was 0.0216U/L, and the lowest detection limit was reached 2×10-4 U/L. This method has good reproducibility and accuracy, and is used for rapid detection and testing of GOD activity in flour additives,The results are basically consistent with the colorimetric method.