固定化乙酰胆碱酯酶的制备及其特性研究

Preparation and Characteristic Research of Immobilized Acetylcholinesterase

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.

A new method for preparation of a reusable immobilized acetylcholinesterase （AchE） was de- veloped in order to lay a foundation for screening AchE inhibitors from natural products. AchE was immolized on aminated silica gel microspheres with glutaraldehyde as cross-linking reagent. The optimum conditions and properties of immobilized enzyme were investigated. The optimal immobilization process was as follows： 0.05 g of aminated silica gel microspheres was activated by glutaraldehyde solution for 6 h, and mixed with AChE solution with the activity of 5 U. Then, the mixture was shook for 16 h at 28 ℃. The immobilized enzyme produced by this method had the highest enzyme activity, and it could remain better activity with a wide range of pH （pH 6-10） at room temperature （20-40 ℃）. The immobilized AChE showed higher storage stability and could be used repeatedly.