Developing a Protein Scaffolding System for Rapid Enzyme Immobilization and Optimization of Enzyme Functions for...

Immobilization of enzymes is required for most biocatalytic processes, but chemistries used in enzyme immobilization are limited and can be challenging. Genetically encoded protein-based biomaterials could provide easy-to-use immobilization platforms for biocatalysts. We recently developed a self-assembling protein scaffold that covalently immobilized SpyTagged enzymes by engineering the bacterial microcompartment protein EutM from Salmonella enterica with a SpyCatcher domain. We also identified a range of EutM homologues as robust protein nanostructures with diverse architectures and electrostatic surface properties. In this work, we created a modular immobilization platform with tunable surface properties by developing a toolbox of self-assembling, robust EutM-SpyCatcher scaffolds. Using an alcohol dehydrogenase as model biocatalyst, we show that the scaffolds improve enzyme activity and stability. This work provides a modular, easy-to-use immobilization system that can be tailored for the optimal function of biocatalysts of interest.