In this study, using sorbitan monooleate (Span-80), we developed a new strategy to achieve the positional assembly of a horse radish peroxidase/glucose oxidase bienzyme system within the zeolitic imidazolate framework-8 (ZIF-8)-based colloidosomes. Span-80 with self-adsorption property, was used as an auxiliary stabilizer to endow the enzyme-loaded ZIF-8 (enzyme@ZIF-8) with proper wettability in the formation of water–in–oil Pickering emulsion templates. Benefited from this, simultaneous immobilization of the bienzymes in the lumen and shell of the colloidosomes could be achieved. Meanwhile, Span-80 was found to act as a mediator to induce and control the overgrowth of the ZIF-8 crystallites on the enzyme@ZIF-8 layers, by which a “bricklaying” solidification protocol was established for the shell preparation. The resulting colloidosomes enabled the flexible positional change of the bienzymes, its shell exhibited the monolayer structure with increased compactness and homogenized micropores. Furthermore, the multi-enzyme cascade reaction was exemplified with the oxidization of o-phenylenediamine. An in-depth investigation showed that the assembly strategies of the bienzymes significantly affected their biocatalytic efficiency by influencing the utilization efficiency of the H₂O₂ intermediate between the step reactions. This study provides an insight towards a facile synthetic route for constructing multi-enzyme biocatalyst.

在这项研究中，我们使用失水山梨糖醇单油酸酯 (Span-80)，开发了一种新的策略来实现辣根过氧化物酶/葡萄糖氧化酶双酶系统在沸石咪唑酯框架 8 (ZIF-8) 基胶体中的位置组装。具有自吸附性能的 Span-80 作为辅助稳定剂赋予载酶 ZIF-8 (enzyme@ZIF-8) 以形成油包水 Pickering 乳液模板的适当润湿性。受益于此，可以实现双酶同时固定在胶体的腔和壳中。同时，发现 Span-80 作为介质诱导和控制酶@ZIF-8 层上 ZIF-8 微晶的过度生长，从而为壳制备建立了“砌砖”固化协议。由此产生的胶体使双酶灵活的位置变化成为可能，其外壳呈现出单层结构，具有更高的致密性和均质的微孔。此外，多酶级联反应以氧化邻苯二胺。深入研究表明，双酶的组装策略通过影响步骤反应之间的H ₂ O ₂中间体的利用效率来显着影响其生物催化效率。该研究为构建多酶生物催化剂的简便合成路线提供了见解。