磁性壳聚糖微球固定化乳糖酶及其酶学性质  

Immobilization of lactase on magnetic chitosan microspheres and its effect on enzymatic properties

作　　者：李黎[1] 马力[2] Li Li;Ma Li(Chengdu Agricultural College,Chengdu 611130,Sichuan Province,China;Xihua University,Chengdu 610039,Sichuan Province,China)

机构地区：[1]成都农业科技职业学院,四川省成都市611130 [2]西华大学,四川省成都市610039

出　　处：《中国组织工程研究》2021年第4期576-581,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:游离酶在使用过程中存在稳定性差、不能重复使用等问题,可利用磁性高分子微球作为结合酶的载体制备固定化酶,使其既保持酶的天然活性并可以重复使用,又为自动化生产管理提供便利的条件。目的:采用磁性壳聚糖微球作为结合酶的载体制备出便于回收、可重复使用、酶活及稳定性较高的固定化乳糖酶。方法:将一定量磁性壳聚糖微球加入磷酸缓冲液中溶胀2 h,用磁铁收集溶胀后的磁性壳聚糖微球,加入到一定浓度的乳糖酶磷酸缓冲液中,置于恒温震荡器中震荡1 h,于4℃冰箱中静置,然后用磁铁将微球沉淀,倒出上清液。用缓冲液充分洗涤后得到固定化乳糖酶。并就磁性微球本身的性质(制作微球时戊二醛用量2,4,6,8,10,12,14 mL)、加酶量(0.5,1,1.5,2 g/L)、缓冲液的pH值(6.4,6.8,7.0,7.2)、固定化时间(1,2,5,10,15,20 h)进行实验,以确定最佳固定化条件。结果与结论:①实验发现用磁性壳聚糖微球固定化乳糖酶的最佳条件为:选择戊二醛用量为10 mL制备的磁性壳聚糖微作为乳糖酶的固定化载体,加酶量取0.3 g/L,pH=7.0,固定化时间为5 h;②与游离酶相比,固定化酶的最适作用温度范围、pH值范围更宽;③乳糖酶在固定化后其结合底物的能力有所增强;④固定化酶反复使用5次后,酶活仍保持了65%;⑤乳糖酶被固定化后,其贮存稳定性也有所提高。BACKGROUND:The free enzyme has the problems of poor stability and inability to be reused during application.Using magnetic polymer microspheres as the carrier of the binding enzyme to prepare immobilized enzyme can maintain the natural activity of the enzyme,and can be reused,but also provide convenient conditions for automatic production management.OBJECTIVE:Magnetic chitosan microspheres as carrier of binding enzyme were used to prepare the immobilized lactase,which is easy to recycle,can be reused,and has high enzyme activity and stability.METHODS:By inputting a certain amount of magnetic chitosan microspheres into the phosphoric acid buffer for swelling for 2 hours,the swelling magnetic chitosan microspheres were collected with a magnet and added to a certain concentration of lactase phosphate buffer.They were shocked in a constant temperature shaker for 1 hour and preserved in refrigerator at 4°C.The microspheres were precipitated with a magnet to pour out the supernatant.After full washing with buffer solution,the immobilized lactase was obtained.The properties of the magnetic microspheres(the amount of glutaraldehyde used in the preparation of microspheres was 2,4,6,8,10,12,14 mL),the amount of enzymes(0.5,1,1.5,2 g/L)added,the pH(6.4,6.8,7.0,7.2)of the buffer,and the immobilization time(1,2,5,10,15,20 hours)were tested to determine the optimal immobilization conditions.RESULTS AND CONCLUSION:(1)The optimum conditions for immobilized lactose with magnetic chitosan microspheres were as follows:magnetic chitosan microspheres prepared with 10 mL of glutaraldehyde were selected as the immobilized carrier of lactase.The amount of enzyme added was 0.3 g/L,pH 7.0 and the immobilization time was 5 hours.(2)Compared with the free enzyme,immobilized lactase showed a wider range of reaction temperature and pH value.(3)The ability of immobilized enzyme binding substrate was enhanced.(4)After repeated use of the immobilized enzyme five times,the enzyme activity remained 65%.(5)The storage stability of lactase was also

关 键 词：材料 磁性微球 高分子微球 生物材料 壳聚糖 固定化酶 磁响应 乳糖酶 

分 类 号：R459.9[医药卫生—治疗学] R-331[医药卫生—临床医学]