School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, PR China.
School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, PR China; Tianjin Key Laboratory of Chemical Process Safety, Hebei University of Technology, Tianjin 300130, PR China.
Enzyme Microb Technol. 2022 May;156:110001. doi: 10.1016/j.enzmictec.2022.110001. Epub 2022 Feb 2.
In this study, a novel kind of Ni-NTA modified monodispersed SiO nanoflowers (Ni-NTA@SiO nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)- 3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO nanoflowers was 40.17 mg/g and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of CC bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.
在这项研究中，成功合成了一种新型的 Ni-NTA 修饰的单分散 SiO2 纳米花（Ni-NTA@SiO2 纳米花）。所得到的 Ni-NTA@SiO2 纳米花被用于特异性吸附和纯化 His 标记的老黄色酶（OYE1）和葡萄糖脱氢酶（GDH），这使得通过不对称氢化反应获得光学纯的（3S）-3-甲基-环己酮，并形成辅因子再生系统。Ni-NTA@SiO2 纳米花上的蛋白质负载量为 40.17mg/g，OYE1 和 GDH 的活性回收率分别为 81.53%和 79.68%。研究了 pH 值和温度对游离酶和共固定化酶活性的影响，并测量了稳定性和可重复使用性。与游离酶相比，共固定化酶表现出更高的热稳定性和储存稳定性。共固定化酶被应用于 CC 键的不对称还原反应，用于合成手性中心，具有优异的对映选择性（ee > 99%），经过 7 次循环后转化率为 46.02%。这项工作介绍了一种一锅多酶纯化和共固定化策略，用于构建具有高活性和稳定性的高效辅因子再生系统。
