【作者】 应国清； 王玉姣；

【Author】 Ying Guo-Qing, Wang Yu-jiao (College of Pharmaceutical Science ,Zhe Jiang University of Technology,Hang Zhou 310014, China)

【机构】 浙江工业大学药学院；

【摘要】 为确定一条采用亲和层析技术分离纯化酵母中磷酸激酶的较佳工艺路线,采用分步盐析初步纯化己糖激酶(HK),考察硫酸铵饱争度对盐析效果的影响;以ATP为配基,SESA活化纸纤堆素制备亲和层析剂,确定最佳偶联条件和最佳静态吸附pH值后,将粗酶液直接上亲和层析柱,看分离纯化的效果;在载体和配基之问接上1．6-己二胺为间隔臂,比较接臂前后对吸附选择性的影响．粗酶液经分步盐析后纯化倍数达3．5倍;粗酶液直接上亲和层析柱,一步纯化后纯化倍数达3．7倍;并确定了接间隔臂可以提高载体对HK的选择性．实验证明:以ATP为配基的亲和层析法纯化酵母细胞中的磷酸激酶是可行的．更多还原

【Abstract】 To search a suitable process route of purification of kinase in yeast by affinity chromatography, hexokinase(HK) was preparatorily purified by salt preciptation. After confirming the optimized coupling condition and absorbing pH, the crude enzyme was purified by affinity chromatographic medium combined with ATP and with SESA as surfactant The space arm 1,6-Hexanediamine was coupled between the matrix and the ligand to compare the absorbtion effect without the space arm. The crude enzyme was purified 3.5 fold by fractional salt preciptation, 3.7fold by one-step affinity chramatography, and the selectivity was better with coupling of the space arm . It is feasible to purify the kinase in yeast by affinity chromatography with ATP as ligand.更多还原