Versatile Enzyme Immobilization through Genetically Encoded Biomolecular Click Modules on the Spore Surface

Here, we report engineered bacterial spores for versatile covalent enzyme immobilization through a biomolecular click reaction (Click-TIED). This approach decouples crust protein folding and assembly during sporulation from the production of target enzymes, enabling a versatile immobilization platform. We identified a truncated CgeA protein as an effective fusion partner for successful covalent bond formation on the Bacillus subtilis spore surface. The resulting immobilized enzymes remained stable on the spore surface and showed robust, improved activity for long-term reuse for a week. This platform also enables enzyme co-immobilization for tandem catalysis and the direct harvesting of target enzymes from cell lysates. This work presents a straightforward method for covalently immobilizing enzymes on the bacterial spore, a recyclable and renewable biological bead, advancing the development of a deployable biocatalysis platform.