高效液相色谱法测定环二酰胺酶催化水解马来酰亚胺动力学参数

Determination of Kinetic Parameters of a Cyclic Imidase Hydrolyzing Maleimide by High Performance Liquid Chromatography

采用高效液相色谱法（HPLC）分析并测定了环二酰胺酶水解马来酰亚胺反应的动力学参数。以Symmetry C18柱（4．6mm×150mm，5μm）为色谱柱，甲醇：10mmol／L K2HPO4～KH2PO4缓冲液（pH6．5）体积比5：95为流动相，检测波长为255nm，流速1．0mL／min等色谱条件，马来酰亚胺与其水解产物马来酰胺酸得到较理想的分离效果。按照Lineveaver-Burk的双倒数作图法求得环二酰胺酶催化马来酰亚胺水解的动力学参数为Km=39．09mmol／L，Vmax=154．02μmol／（min·mg），Vmax／Km=3．94，kcat=28750．4min^-1，kcat／Km=735．5mmol／（L·min）。30min环二酰胺酶催化底物马来酰亚胺转化率达到95％。该方法快速、准确、重复性好。

Kinetic parameters of a cyclic imidase with maleimide as the substrate was assayed by HPLC method. The separation of the reaction mixture was conducted on a Symmetry C18 column with methanol： 10 mmol/L phosphate buffer, pH 6.5 （5 ： 95 v/v） as the mobile phase at a flow rate of 1. 0 mL/min, and the detection was carried out at 255 nm. Under the above conditions, maleimide and maleamic acid were perfectly separated with a good linear relationship between sample amounts and chromatograms. Under the optimal conditions of enzyme reaction, the yield of maleamic acid catalyzed by cyclic imidase reached 95% within 30 min. Kinetic parameters of the cyclic imidase for hydrolyzing substrate maleimide were calculated based on Lineveaver-Burk method e.g. Km = 39. 09 mmol/L, Vmax=154. 02μmol/（min · mg）, Vmax/Km = 3. 94, kcat= 28750.4min^-1, kcat/Km = 735. 5 mmol/（L · min）. The method is rapid, accurate and reproducible.