金属螯合载体定向固定化木瓜蛋白酶的研究

Oriented Immobilization of Papain on Metal Chelating Carriers

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体(IDACu2+)与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu2+1.5×10-2molg载体、固定化时间4h、固定化pH7.0、给酶量30mgg载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4%,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。

Based on the technology of protein separation with immobilized metal ion affinity chromatography（IMAC）, a method for oriented immobilization of papain has been selected. Papain was successfully immobilized on magnetic agarose carriers. Cu^2＋ with iminodiacetate （IDA） was used as the chelating ligand to be correlated with the histidine on papain （His-81）. The optimum immobilization conditions of enzyme were as flows： Cu^2＋ 0.15× 10^-2 mol/g carriers, time was 4h, enzyme load was 30mg/g carrier , pH was 7.0 , respectively. The pH and temperature were 8.0 and 70℃ for immobilized enzyme. The recovery activity of immobilized enzyme was retained 68.4% . The carrier could be recovered from the spent immobilized enzyme, to be reused. After 5 times, the reimmobilization of papain on the regenerated matrix was 79.71% effective with the retention of maximum enzyme activity. The cost of carriers used for industrial applications is very important. The regenerability of carriers is therefore, relevant. The mild conditions used for immobilization, the high recovery of immobilized preparations, the stability and the regenerability of the matrix are the main features of the method reported here. All above indicate this method can be applicable and premising in enzyme immobilization field.