Protein-based scaffolds for enzyme immobilization.

Biocatalysis is emerging as an alternative approach to chemical synthesis of industrially relevant complex molecules. To obtain suitable yields of compounds in a cost-effective manner, biocatalytic reaction cascades must be efficient, robust, and self-sufficient. One approach is to immobilize biocatalysts on a solid support, stabilizing the enzymes and providing optimal microenvironments for reaction sequences. Protein-based scaffolds can be designed as immobilization platforms for biocatalysts, enabling the genetically encoded spatial organization of single enzymes and multistep enzyme cascades. Additionally, protein scaffolds are versatile, are easily adapted, and remain robust under different reaction conditions. In this chapter, we describe methods for the design and production of a self-assembling protein scaffold system for in vitro coimmobilization of biocatalytic cascade enzymes. We provide detailed methods for the characterization of the protein scaffolds, as well as approaches to load biocatalytic cargo enzymes and test activity of immobilized cascades. In addition, we also discuss methods for the development of a scaffold building block toolbox with different surface properties, which could be adapted for a diversity of biocatalysts requiring alternative microenvironments for function.

Building a toolbox of protein scaffolds for future immobilization of biocatalysts.

Biological materials that are genetically encoded and can self-assemble offer great potential as immobilization platforms in industrial biocatalysis. Protein-based scaffolds can be used for the spatial organization of enzymes, to stabilize the catalysts and provide optimal microenvironments for reaction sequences. In our previous work, we created a protein scaffold for enzyme localization by engineering the bacterial microcompartment shell protein EutM from Salmonella enterica. Here, we sought to expand this work by developing a toolbox of EutM proteins with different properties, with the potential to be used for future immobilization of enzymes. We describe the bioinformatic identification of hundreds of homologs of EutM from diverse microorganisms. We specifically select 13 EutM homologs from extremophiles for characterization, based on phylogenetic analyses. We synthesize genes encoding the novel proteins, clone and express them in E. coli, and purify the proteins. In vitro characterization shows that the proteins self-assemble into robust nano- and micron-scale architectures including protein nanotubes, filaments, and scaffolds. We explore the self-assembly characteristics from a sequence-based approach and create a synthetic biology platform for the coexpression of different EutM homologs as hybrid scaffolds with integrated enzyme attachment points. This work represents a step towards our goal of generating a modular toolbox for the rapid production of self-assembling protein-based materials for enzyme immobilization.