Protein scaffolds: A tool for multi-enzyme assembly

The synthesis of complex molecules using multiple enzymes simultaneously in one reaction vessel has rapidly emerged as a new frontier in the field of bioprocess technology. However, operating different enzymes together in a single vessel limits their operational performance which needs to be addressed. With this respect, scaffolding proteins play an immense role in bringing different enzymes together in a specific manner. The scaffolding improves the catalytic performance, enzyme stability and provides an optimal micro-environment for biochemical reactions. This review describes the components of protein scaffolds, different ways of constructing a protein scaffold-based multi-enzyme complex, and their effects on enzyme kinetics. Moreover, different conjugation strategies viz; dockerin-cohesin interaction, SpyTag-SpyCatcher system, peptide linker-based ligation, affibody, and sortase-mediated ligation are discussed in detail. Various analytical and characterization tools that have enabled the development of these scaffolding strategies are also reviewed. Such mega-enzyme complexes promise wider applications in the field of biotechnology and bioengineering.