Cai Yixuan, Chen Junhua, Yao Dongsheng, Liu Daling
Institute of Microbial Biotechnology, College of Life Science and Technology, JinaP University, Guangzhou 510632, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Dec;25(12):1969-75.
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 degrees 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 degrees 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.
我们研究了通过“构象记忆”在共价固定化过程中防止蛋白质变性的可行性，“构象记忆”是通过在酶活性构象下冷冻干燥并在微水介质（MAM）中与载体交联来实现的。辣根过氧化物酶（HRP）和壳聚糖珠分别用作模型酶和载体。MAM由99%的二氧六环和1%的水组成。我们比较了MAM条件下固定化的HRP与传统水性溶剂条件下固定化的HRP，发现两者的最适温度均提高到了60℃，最适pH为6.5。然而，MAM固定化的HRP在使用过程中的活性损失较小，且活性比在水性溶剂中固定化的HRP高六倍。在70℃孵育30分钟后，MAM固定化的HRP仍保留75.42%的活性，而水性介质固定化的酶仅保留15.4%的活性。MAM固定化的HRP表现出更好的操作稳定性，重复操作5次后残留活性为77.69%，而水性介质固定化的酶仅为16.67%。此外，MAM固定化的HRP在用于去除苯酚时也表现出更多优势。我们构建了酶电极（CS-HRP-SWCNTs/Au）以进一步展示两种固定化HRP的不同特性。MAM固定化的HRP电极对H2O2的响应信号比水性介质中固定化的HRP电极强两倍，这表明MAM固定化的HRP具有更好的酶活性。我们的研究表明，以HRP为模型时，构象记忆在一定程度上确实有助于防止蛋白质变性，并且在微水介质下通过共价交联法固定化酶是可行的。
