“构象记忆”的辣根过氧化物酶的微水相共价固定化

Investigation of micro-aqueous covalent immobilization of horseradish peroxidase by “conformation memory”

摘要:

本研究利用酶在微水溶剂中的“构象记忆”特性, 以壳聚糖微球为载体, 以辣根过氧化物酶(Horseradish peroxidase, HRP)为研究对象, 将HRP于活性构象下冻干“固定”后, 在二氧六环:水=99:1(V/V)微水介质中与载体进行共价交联, 同时与传统水介质中共价交联固定化的HRP进行比较。结果发现, 两种介质中固定化HRP的最适温度都提高到60°C, 最适pH均为6.5, 而微水相中固定的酶活力损失较低, 酶活比传统水相中固定的酶高6倍以上; 70°C保温30 min后, 微水相中固定的酶保留75.42%的活力, 而水相中固定的HRP仅存15.4%的活力; 微水相中固定的HRP具有更好的操作稳定性和热稳定性, 60°C下连续操作5次之后, 微水相固定的HRP保留77.69%的酶活, 而水相固定的HRP仅存16.67%的酶活; 微水相中固定的HRP在苯酚的去除中表现得更具优势; 微水相中共价交联制备的CS-HRP-SWCNTs/Au酶修饰电极对H2O2的响应信号比水相中共价固定的酶电极强2.5倍, 灵敏度更高。本研究表明利用酶的“构象记忆”在微水介质中进行共价交联是固定化酶的一种可行方法, 所制备的固定化酶具有更优良的性质。

Abstract:

We has studied the feasibility of preventing protein from denature during covalent immobilization by “conformation memory”, which was achieved by freeze-drying under enzyme active conformation and cross-linked with carrier under micro-aqueous media (MAM). Horseradish peroxidase (HRP) and chitosan beads have been used as the model enzyme and carrier. The MAM consisted of 99% dioxane and 1% water. We compared the immobilized HRP under MAM with that under traditional aqueous solvent, found that the optimum temperature of both was raised to 60°C, and the optimum pH was 6.5. However, the MAM-immobilized HRP had shown less activity loss during usage and six times higher activity than that immobilized under aqueous solvent. After 30 min incubation at 70°C, the MAM-immobilized HRP remained 75.42% activity while the aqueous-media-immobilized enzyme only 15.4%. The MAM-immobilized HRP has shown a better operation stability with 77.69% residue activity after 5 times of repeat operation while the aqueous-media-immobilized enzyme only 16.67%. In addition, the MAM-immobilized HRP had also shown more advantages when used in phenol removal. We constructed enzyme electrodes (CS-HRP-SWCNTs/Au) to further display the different properties of the two immobilized HRP. MAM-immobilized HRP-electrode has shown two times stronger response signal to H2O2 than that immobilized under aqueous media, which indicated a better enzyme activity of MAM-immobilized HRP. Our research demonstrated that the conformation memory, to some extent, did contribute to preventing protein from denaturing when use HRP as a model, and it is feasible to immobilize enzyme by covalent cross-linking method under micro-aqueous media.