乙酰胆碱酯酶壳聚糖膜的制备研究

Research on Acetylcholinesterase and Chitosan Membrane

关键词:  乙酰胆碱酯酶  壳聚糖  固定化酶

Key words:  Acetylcholinesterase  chitosan  immobilization

摘要:

以新鲜母鸡血为材料,30%~70%(V/V)乙醇分步沉淀制备粗酶制剂,再经DEAE-Cellulose与Sephadex G-75柱层析,部分纯化鸡红细胞乙酰胆碱酯酶(AChE),再以壳聚糖制备成膜作为载体,采用吸附-交联法固定AChE制备壳聚糖酶膜,分别考察制备壳聚糖酶膜的壳聚糖浓度(0、0.05、0.1、0.15、0.2g/ml),壳聚糖酶膜的吸附时间(2、4、6、8、10、12h)和吸附温度(0、4、25、30、37℃),以及戊二醛质量浓度(0.2%、0.4%、0.6%、0.8%、1.0%),戊二醛交联时间(40、80、120、160、1200min),戊二醛交联温度(0、4、25、37℃)和给酶量(0~2000 U/ml)对壳聚糖酶膜固定化效果的影响。结果显示,鸡红细胞AChE粗酶制剂的部分纯化倍数为12倍时,活力回收34.1%。优化的固定化条件是以0.1g/ml壳聚糖溶液制备载体膜,采用950 U/ml的酶液(蛋白质含量为0.43 mg/ml),在4℃下吸附10 h,再以质量浓度为0.6%的戊二醛溶液4℃下交联2h。此条件下制备的酶膜活力达11.5 U/cm²,活力回收达30%。

Abstract:

By 30%~70% ethanol fractional precipitation,DEAE-Cellulose and Sephadex G-75 chromatography,the Acetylcholinesterase is purified from hen reythrocyte,then the Acetylcholinesterase Membrane is immobilized by absorbing and crosslinking with carrier of Chitosan.In order to prepare the best Acetylcholinesterase Membrane,so difference activation mass of Acetylcholinesterase and difference conditions were studied such as the concentration of Chitosan for 0g/ml,0.05g/ml,0.1g/ml,0.15g/ml and 0.2g/ml,time for absorbing 2h,4h,6h,8h,10h and 12h,temperature for absorbing 0℃,4℃,25℃,30℃ and 37℃,concentration of glutaraldehyde for 0.2%,0.4%,0.6%,0.8% and 1.0%,time for crosslinking with glutaraldehyde for 40min,80min,120mon,160min and 1200min,temperature for crosslinking with glutaraldehyde for 0℃,4℃,25℃ and 37℃.The results show the Acetylcholinesterase purification from hen reythrocyte has reached 12-folds with a yield of 34.1%.By using as a bifunctional crosslinking reagent,the optimum conditions for membrane of immobilization are as follows:the concentration of chitosan is 0.1g/ml;the concentration of the free AChE is 950U/ml;absorbing time and temperature for 10h at 4℃;crosslinking temperature at 4℃ with the concentration of glutaraldehyde is 0.6% for 2h.The activity and recovery of immobilized AChE are 11.5 U/cm² and 30%,respectively.