聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶

Immobilization of Lipase Through Cross-linking of Polyethylene Glycol Diglycidyl Ether with Amino Carrier LX-1000EA

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EAPEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA-PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。

Polyethylene glycol diglycidyl ether(PEGDGE),a double-functional epoxy reagent,was used to covalently immobilize marine lipase through cross-linking amino carrier LX-1000EA.The treated carrier behaved good effect in the covalent immobilization of lipase.After single factor screening and orthogonal experiment,the best conditions of crosslinking and immobilized is 0.75%concentration of crosslinking agent,crosslinking temperature 35℃,crosslinking time 3 h,carrier quantity 1.25 g,pH 9.0 and immobilization temperature 55℃,immobilization time of 1 h.The enzymatic properties of LX-1000EA-PEGDGE immobilized enzyme were compared with that of free enzyme and glutaraldehyde crosslinked immobilized enzyme LX-1000HA-GA.Compared with free enzymes,the optimal reaction temperature of LX-1000EA-PEGDGE immobilized enzyme did not change;and similar with LX-1000HA-GA,the optimal reaction p H increased from 7.0 to 8.0.Under the optimal condition,the enzyme activity of LX-1000EA-PEGDGE reached 78.84 U/g.Immobilization changed the acid-base tolerance of lipase,and the thermal stability and the operation stability were improved compared with free enzyme and LX-1000HA-GA immobilized enzyme.The immobilized enzyme exhibited excellent thermal stability,retaining 90%initial enzyme activity after 3 h incubation at 60℃;the residual enzyme activity retained 50%of its original activity after 5 times of operation;the residual enzyme activity retained 60%of its original activity after preserved for 30 days at 4℃.The new crosslinking agent PEGDGE and organic amino carrier were combined for the first time to immobilize lipase,which provided technical support for more effective immobilization methods.It was also found that the cross-linking agent had a great impact on the properties of the immobilized enzyme.