Biocatalytic 3D binary crystals formed through the self-assembly of enzyme-embedded ferritin

Abstract

Protein crystals are traditionally used to aid structural analysis but have recently gained attention as functional materials due to their intrinsic order, defined porosity, and high chemical programmability. While enzymes have been incorporated into protein crystals, most existing systems rely on post-crystallization loading or nonspecific adsorption, offering limited control over the spatial distribution of catalytic components. Furthermore, there are few examples of catalytically active crystals formed through the ordered assembly of protein nanocages. These modular and uniform building blocks provide precise size, surface chemistry, and cargo loading, making them ideal candidates for constructing functional crystalline materials. In this study, we report a binary protein crystal formed by electrostatic co-crystallization of oppositely charged ferritin nanocages, with one component encapsulating the peroxidase-mimicking enzyme enhanced ascorbate peroxidase 2 (APEX2). The resulting material exhibits long-range order, retains enzymatic activity, and can be reused in multiple catalytic cycles. This platform provides a framework for building multienzyme crystalline assemblies and studying spatially programmed biocatalysis.