新型温敏网络半互穿多孔水凝胶的制备及其固定化酶的研究

以甲基丙烯酸羟乙酯(Hydroxyethyl methacrylate,HEMA)、N,N-二乙基丙烯酰胺(N,N-diethyl-Acrylamide,DEAAm)为原材料,通过自由基聚合反应合成一种温敏型多孔水凝胶,经接枝反应引入乙烯基,制得新型温敏网络半互穿多孔水凝胶。利用红外光谱(IR)、核磁共振碳谱(13 C NMR),结合扫描电镜(SEM)表征中间产物和最终产物。采用称重法研究最终产物的溶胀、去溶胀动力学,分析其溶胀性能和温度响应性。运用点击反应原理,以新型温敏网络半互穿多孔水凝胶固定肌酸激酶。釆用BCA(Bicinchoninic Acid)蛋白试剂盒法测定酶的固载量,pH法测定酶活。结果表明,新型温敏水凝胶合成成功,肌酸激酶的平均固载量为24.77mg·g~(-1),肌酸激酶的平均酶活为9.67U·g~(-1)。该新型温敏水凝胶对肌酸激酶具有良好的选择性和温度响应性,对实现肌酸激酶的高效利用具有很好的借鉴意义。

Preparation of the Novel Temperature-sensitive Semi-interpenetrating Porous Hydrogel and the Use in Terms of Immobilized Enzyme

The novel temperature-sensitive porous hydrogel was synthesized by using free radical polymerization method ,the raw materials of hydroxyethyl methacrylate (HEMA) and N ,N-diethyl-acrylamide (DEAAm) were use as reactants .Then propyl was introduced by grafting reaction .The intermediate product and the final product were characterized by infrared spectroscopy (IR) ,nuclear magnetic resonance spectroscopy (NMR) ,scanning electron microscope (SEM) .The swelling and desorption kinetics of the final product were studied by weighing method ,and the swelling properties and temperature response were also analyzed .Fol-lowing the principle of 'click reaction' ,the creatine kinase was fixed on the matrix material of novel temperature-sensitive semi-in-terpenetrating porous hydrogel .The enzyme loading was measured by BCA (bicinchoninic acid) protein kit method ,and the enzyme activity was measured by pH method .The experimental results showed that the novel temperature-sensitive hydrogels was success-fully synthesized and the average loading of creatine kinase was 24.77 mg · g -1 ,the average activity of creatine kinase was 9.67 U · g-1 . The novel temperature-sensitive hydrogel has good selectivity and temperature response to creatine kinase ,which is of great reference for the effective utilization of creatine kinase .