“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"

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30min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。

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℃, 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℃, the MAM-irnmobilized 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.