HPLC法测定固定化青霉素酰化酶合成头孢羟氨苄的活力

Measurement of the activity of the synthesized cefadroxil from immobilized penicillin acylase by HPLC

目的建立一种HPLC法测定固定化青霉素酰化酶合成头孢羟氨苄活力的方法。方法酶与其底物7-ADCA及D-对羟基苯甘氨酸甲酯,在20℃的溶液中,搅拌转速为250r/min条件下反应10min,通过测定反应液中头孢羟氨苄的含量,从而换算出合成酶活力的数值。采用十八烷基硅烷键合硅胶为填充剂;流动相:0.02mol/L磷酸二氢钾溶液(用lmol/L氢氧化钾溶液调节pH值至5.0)-甲醇(98:2);流速:1.0mL/min;柱温:25℃;检测波长:230nm。结果头孢羟氨苄在0.24～0.39mg/mL范围内线性关系良好(r=1.0000),回收率均在99%～101%范围内,RSD为0.40%。结论该测定方法专属性强、准确可靠、重现性好,适用于青霉素酰化酶合成酶活力的检测。

Objective To establish a method of HPLC to measure the activity of the synthesized cefadroxil from immobilized penicillin acylase.Methods The concentration of cefadroxil in the sample solution was determined, which reacted 10min after adding the enzyme and 7-ADCA and D-p-hydroxyphenylglycine methyl ester as its reaction substrates (reaction conditions:20℃ in solution,stirring speed 250r/min),in order to calculate the value of synthetic enzyme activity.The column was used with the octadecylsilane bonded silica gel as the filler;with 0.02mol/L potassium dihydrogen phosphate solution (adjusted to pH5.0 with lmol/L potassium hydroxide solution)-methanol (98:2)as the mobile phase with a flow rate at 1.0mL/min.The detection wavelength was 230nm,and the temperature of the column was 25℃.Results The calibration curve for cefadroxil was linear in the range of 0.24 to 0.39mg/mL (r=1.000),the recoveries were in the range of 99% to 101%,and the RSD was 0.40%.Conclusion The method is highly specific, accurate,reliable,and reproducible.It is suitable for the detection of penicillin acylase synthetase activity.