Nano-sized enzyme aggregates, which preserve their catalytic activity are of great interest for flow processes, as these catalytic species show minimal diffusional issues, and are still sizeable enough to be effectively separated from the formed product. The realization of such catalysts is however far from trivial. The stable formation of a micro-to millimeter-sized enzyme aggregate is feasible via the formation of a cross-linked enzyme aggregate (CLEA); however, such a process leads to a rather broad size distribution, which is not always compatible with microflow conditions. Here, we present the design of a compartmentalized templated CLEA (c-CLEnA), inside the nano-cavity of bowl-shaped polymer vesicles, coined stomatocytes. Due to the enzyme preorganization and concentration in the cavity, cross-linking could be performed with substantially lower amount of cross-linking agents, which was highly beneficial for the residual enzyme activity. Our methodology is generally applicable, as demonstrated by using two different cross-linkers (glutaraldehyde and genipin). Moreover, c-CLEnA nanoreactors were designed with Candida antarctica Lipase B (CalB) and Porcine Liver Esterase (PLE), as well as a mixture of glucose oxidase (GOx) and horseradish peroxidase (HRP). Interestingly, when genipin was used as cross-linker, all enzymes preserved their initial activity. Furthermore, as proof of principle, we demonstrated the successful implementation of different c-CLEnAs in a flow reactor in which the c-CLEnA nanoreactors retained their full catalytic function even after ten runs. Such a c-CLEnA nanoreactor represents a significant step forward in the area of in-flow biocatalysis.
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