HPLC法测定猪尿酸酶活性

Determination of Pig-Uricase Enzyme Activity by HPLC

建立了一种测定重组猪尿酸酶活性的高效液相色谱方法。将重组猪尿酸酶基因工程菌的粗酶液与尿酸溶液反应5min,加入高氯酸溶液终止,经超滤后以体系中尿酸浓度的降低值为指标,采用HPLC法[色谱条件:AgilentZorbax 300SB-C18色谱柱、流动相为磷酸-甲醇-双蒸水(3∶25∶472,体积比)、流速1.0mL·min-1、波长292nm]测定粗酶液活性。结果表明,尿酸在10～500μmol·L-1范围内线性关系良好,样品回收率在99%～104%之间。该方法不受杂质干扰、稳定、灵敏度高,适用于尿酸酶粗酶液活性的检测。

A HPLC method for determining the activity of fermentation liquor of recombinant E. coli ex- pressing pig-uricase was established. The fermentation liquor was reacted with uric acid for 5 min,then the reac- tion was terminated by adding perchloric acid. The analysis was conducted by HPLC after ultrafiltration using Agilent Zorbax 300SB-C,s column with a mobile phase consisting of phosphoric acid-methanol-water（3 ：25 ： 472,volume ratio）. The flow rate was 1.0 mL. min^-1 and the wavelength was 292 nm. The enzyme activity of fermentation liquor was calculated indirectly by lowering the concentration of uric acid. The calibration curve of uric acid concentration displayed highly relative linearity over the range of 10-500μmol. L^-1. The recovery was 99-104%. The method is stable, sensitive, and is not disturbed by impurities. It＇s suitable for determi- ning activity of crude enzyme.