基于无机载体的先交联后吸附固定化脂肪酶的方法

Immobilization of lipase by first crosslinking and then adsorption based on inorganic carrier

目的:使用双功能环氧交联剂新戊二醇二缩水甘油醚和硅藻土载体固定化海洋脂肪酶,并探究固定化酶的酶学性质.方法:采用先交联后吸附的方法固定化海洋脂肪酶,交联和吸附分别进行单因素与正交实验结合的实验方案,并测定固定化酶的酶活力.结果:确定最佳交联条件:pH5.5,温度35℃,交联剂质量分数0.5%,交联时间2h;最佳吸附条件:温度40℃,载体质量1.5g,吸附时间5h.在此工艺条件下,所制备的固定化脂肪酶活力为1.89μkat,酶活力回收率72.89%.与游离的脂肪酶相比,所制备的固定化脂肪酶具有更好的温度、pH、储存及操作稳定性.结论:基于环氧交联剂新戊二醇二缩水甘油醚和硅藻土载体,进行先交联后吸附固定化海洋脂肪酶的技术具有良好的工业应用前景.

Objective:The purpose of this research is to immobilize marine lipase with bifunctional epoxy cross-linking neopentyl glycol diglycidyl ether and kieselguhr,and to explore the enzymatic properties of immobilized enzyme.Methods:The marine lipase was immobilized by pre-crosslinking and adsorption,and the experimental scheme combining single factors and orthogonal experiment were carried out,and then the enzyme activity of the immobilized enzyme was determind.Results:The optimum crosslinking conditions were determined as follows:pH 5.5,temperature 35℃,crosslinker concentration 0.5%,crosslinking time 2 h;optimal adsorption conditions were:temperature 40℃,carrier quantity 1.5 g,adsorption time 5 h.Under the optimal process conditions,the prepared immobilized lipase activity reached 1.89μkat,and the enzyme activity recovery rate reached 72.89%.Compared with free lipase,the immobilized lipase exhibited better thermo-stability and pH-stability;meanwhile,it also showed good storage stability,with 87%of its original activity left after being stored at 4℃for 60 days.The immobilized lipase maintains more than 60%of its original activity after 5 consecutive operations.Conclusion:Based on the epoxy cross-linking agent neopentyl glycol diglycidyl ether and kieselguhr carrier,marine lipase adsorbed and immobilized after cross-linking has good industrial application prospects.