Natural macromolecular scaffolds have also been widely employed where nucleic acids and proteins are used as scaffold materials. Glucose oxidase and horseradish peroxidaseīiosensor and biotechnological application Pharmaceutical and nanotechnological application Human carbonic anhydrase, Retro-aldolase, and Kemp eliminase Glucose-6-phosphate dehydrogenase and Hexokinase 2 Peroxygenase CYP BM321B3 and Glucose oxidaseīiotransformation of endocrine disruptor compounds To date, numerous versatile nanomaterials such as nanotubes, nanowires, nanoparticles, nanosponges, nanoflowers, metal-organic frameworks, nanocages, and nanocomposites have been reported to be utilized for the construction of artificial scaffolds for enzyme cascades.Ĭlarification and stabilization of fruit juicesĭetection of organophosphorus and carbamate pesticideīacteriophages P22 virus-like particles (VLPs) This strategy provides better conversion yield and improved catalytic activity. In the context of synthetic scaffolds, diverse nanomaterials with a high aspect ratio (ratio of surface area to volume) have been explored to improve enzyme loading and thereby reducing the diffusional barrier. Scaffolding material can be classified into two categories: synthetic scaffolds and natural macromolecular scaffolds ( Table 1). These improvements compensate for the high cost of enzyme biocatalyst in biotechnological industries. Several types of scaffold materials and technologies have been investigated to improve the overall processivity, stability, and substrate accessibility of enzymes along with the aim of increasing the enzyme loading capacity of scaffold materials. Tremendous efforts have been made in the field of biocatalyst engineering and biomimetics to design such multi-enzyme nanostructures resembling the naturally occurring scaffolded multi-enzyme complexes which would catalyze industrially relevant biochemical reactions with an enhanced rate of productivity. In recent decades, biocatalysis has been gaining considerable attention owing to its sustainable and environmentally friendly nature that in turn provides a greener alternative to traditional chemical synthesis. Thus, the scaffolded clusters play an immense role in carrying out multi-step biochemical reactions. Such multi-enzyme clusters are often formed with the aid of scaffolds wherein enzymes assemble on desired docking sites on the scaffold biomolecule. Indeed, in its highly ordered form, enzymes work more efficiently with better substrate channeling which subsequently enhances the overall productivity. Nature has always witnessed the highly organized enzymatic complexes that drive various metabolic reactions with a high degree of specificity. Such mega-enzyme complexes promise wider applications in the field of biotechnology and bioengineering. Various analytical and characterization tools that have enabled the development of these scaffolding strategies are also reviewed. Moreover, different conjugation strategies viz dockerin-cohesin interaction, SpyTag-Sp圜atcher system, peptide linker-based ligation, affibody, and sortase-mediated ligation are discussed in detail. 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. The scaffolding improves the catalytic performance, enzyme stability and provides an optimal micro-environment for biochemical reactions. With this respect, scaffolding proteins play an immense role in bringing different enzymes together in a specific manner. However, operating different enzymes together in a single vessel limits their operational performance which needs to be addressed. 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.
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