The simple and quick method for co-immobilization of multiple enzymes with clear spatial distribution has presented great challenges for decades. Herein, Zr⁴⁺ and 2-methylimidazole (2MIm) coordination polymers (CPs) were used to synthetize co-immobilized nanoreactor by a simple two-step procedure in aqueous environment. The CPs was first self-assembled in situ encapsulating glucose-6-phosphate dehydrogenase (G6PD) and then utilized coordination unsaturated metal sites on the surface of CPs to selectively adsorb hexahistidine-tagged α, β-unsaturated ketoreductase (his-tagged KRED). The obtained multi-enzymes system (G6PD@Zr-2MIm/KRED) was employed as an enzymatic reactor involving coenzymes regeneration. G6PD@Zr-2MIm/KRED still exhibited good repeatability and storage stability. The bi-enzymatic reactor could achieve more than 95% chalcone conversion ratio after 15 min and good tolerance at high temperature and different pH, retained about 70% and 80% of its initial activity after storage for 4 days and after 4 cycles, respectively. This step-wise enzyme immobilization method is easy to operate and can be used to prepare multi-enzyme systems with clear spatial distribution of the biocatalysts and allowing the coenzymes regeneration.

数十年来，简单，快速的固定多种酶并固定空间分布的方法提出了巨大的挑战。本文中，Zr ⁴⁺和2-甲基咪唑（2MIm）配位聚合物（CPs）用于在水性环境中通过简单的两步程序来合成共固定的纳米反应器。CP最初是在现场自行组装的封装葡萄糖6-磷酸脱氢酶（G6PD），然后利用CP表面上的配位不饱和金属位点选择性吸附六组氨酸标签的α，β-不饱和酮还原酶（他标签的KRED）。将获得的多酶系统（G6PD @ Zr-2MIm / KRED）用作涉及辅酶再生的酶反应器。G6PD @ Zr-2MIm / KRED仍然表现出良好的重复性和存储稳定性。该双酶反应器在15分钟后可以达到95％以上的查尔酮转化率，并且在高温和不同pH下具有良好的耐受性，分别在储存4天和4个循环后保留其初始活性的70％和80％。