高效液相色谱测定D-阿洛酮糖3-差向异构酶酶活力

A High Performance Liquid Chromatography Method for the Determination of D-psicose 3-epimerase Activity

D-阿洛酮糖3-差向异构酶可以将D-果糖异构化生成D-阿洛酮糖。为建立D-阿洛酮糖3-差向异构酶酶活力测定方法,首先建立了高效液相色谱法测定产物D-阿洛酮糖含量的方法,进而测得D-阿洛酮糖3-差向异构酶酶活力。采用50 mg/L EDTA钙溶液作为流动相等度洗脱Waters Sugar-Pak I色谱柱(柱温90℃,流速0.3 m L/min,进样量10μL),采用示差检测器测定D-阿洛酮糖含量。结果表明,该方法测定酶反应产物D-阿洛酮糖含量得检测限为4.12 mg/L,定量限为10.3 mg/L,在0.5~5.0 g/L范围内线性度良好,R~2=1,重复性(RSD)小于1.0%,加标收回率为98.92%。在此基础上进一步确定D-阿洛酮糖3-差向异构酶酶活力测定时的酶反应最适温度45℃,最适pH值7.0,D-果糖底物质量分数24%,反应时间20 min。经检验,该酶活力检测方法的精密度RSD小于5.0%,达到酶活力检测要求。

D-psicose 3-epimerase can isomerize D-fructose to form D-Allulose.In order to establish a method for the determination of D-psicose 3-epimerase activity,a HPLC method was established firstly to determine the content of the product D-Allulose,and then the activity of D-psicose 3-epimerase was determined.A Waters Sugar-Pak I column was eluted isocratically with 50 mg/L EDTA calcium solution,the column temperature was 90℃,the flow rate was 0.3 m L/min,the injection volume was 10μL,and the content of D-Allulose was determined by differential detector.The results showed that the detection limit of the method for the content determination of the enzyme reaction product D-Allulose was 4.12 mg/L,the limit of quantification was 10.3 mg/L,the linearity was good in the range of 0.5 g/L to 5 g/L,R~2=1,the repeatability(RSD)was less than 1.0%,and the recovery rate of the spiked standard was 98.92%.On this basis,it was further determined that for the determination of D-psicose 3-epimerase activity,the optimal reaction temperature was 45℃,the optimum pH was 7.0,the concentration of the substrate D-fructose was 24%,and the reaction time was 20 min.By verification test,the precision(RSD)of the method for the determination of the enzyme activity is less than 5.0%,which meets the requirements of the enzyme activity detection.