Song Jiayi, Shen Hao, Yang Ye, Zhou Zixin, Su Ping, Yang Yi
Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, College of Science, Beijing University of Chemical Technology, Beijing 100029, P. R. China.
J Mater Chem B. 2018 Sep 28;6(36):5718-5728. doi: 10.1039/c8tb01842a. Epub 2018 Aug 29.
The overall stability and activity of immobilized enzyme systems have suffered from non-specific adsorption immobilization of enzymes at the surface of carriers through electrostatic and hydrophobic interactions. Elimination of these non-specific adsorptions of enzymes on the surface of carriers is critical for enzyme reactions. Herein, for the first time, we have prepared zwitterion-functionalized magnetic particles with amino, phosphonate, and thiol functional groups to coimmobilize glucose oxidase and horseradish peroxidase by DNA directed immobilization. The zwitterionic surface of the multifunctional magnetic particles could efficiently suppress non-specific adsorption of different kinds of enzymes, and the immobilized multienzyme catalyst without non-specifically adsorbed enzymes exhibited excellent enzymatic activity, stability, and reusability compared with those of free and non-specifically adsorbed enzymes. The immobilized multienzymes maintained 87% of their enzymatic activity after two weeks of storage at 4 °C and 58% of their enzymatic activity after 90 min of thermal incubation at 60 °C. Furthermore, the immobilized multienzymes exhibited more than 87% enzymatic activity after reuse for 10 cycles. The apparent K and catalytic efficiency (k/K) values of the immobilized multienzymes were 12.6 mM and 4.03 s mM, respectively, which were 0.2- and 11.8-fold better than those of the free enzymes, indicating effective cascade efficiency and substrate affinity. The optimal ratio of GOx and HRP was 1 : 5, and the prepared immobilized enzymes could detect low concentrations of glucose (0.5 μM) with excellent selectivity. Therefore, we believe that the strategy developed in this study can be widely applied in biotechnology, industrial catalysis, and biomedical engineering.
固定化酶系统的整体稳定性和活性一直受到酶通过静电和疏水相互作用在载体表面非特异性吸附固定的影响。消除酶在载体表面的这些非特异性吸附对于酶反应至关重要。在此，我们首次制备了具有氨基、膦酸酯和硫醇官能团的两性离子功能化磁性颗粒，通过DNA定向固定共固定葡萄糖氧化酶和辣根过氧化物酶。多功能磁性颗粒的两性离子表面能够有效抑制不同种类酶的非特异性吸附，与游离酶和非特异性吸附酶相比，没有非特异性吸附酶的固定化多酶催化剂表现出优异的酶活性、稳定性和可重复使用性。固定化多酶在4℃储存两周后保持其酶活性的87%，在60℃热孵育90分钟后保持其酶活性的58%。此外，固定化多酶在重复使用10个循环后仍表现出超过87%的酶活性。固定化多酶的表观K值和催化效率（k/K）分别为12.6 mM和4.03 s mM，分别比游离酶好0.2倍和11.8倍，表明有效的级联效率和底物亲和力。葡萄糖氧化酶和辣根过氧化物酶的最佳比例为1:5，制备的固定化酶能够以优异的选择性检测低浓度葡萄糖（0.5μM）。因此，我们认为本研究中开发的策略可广泛应用于生物技术、工业催化和生物医学工程。
