Nature Inspired Multienzyme Immobilization: Strategies and Concepts

Abstract

In a biological system, the spatiotemporal arrangement of enzymes in a dense cellular milieu, subcellular compartments, membrane-associated enzyme complexes on cell surfaces, scaffold-organized proteins, protein clusters, and modular enzymes have presented many paradigms for possible multienzyme immobilization designs that were adapted artificially. In metabolic channeling, the catalytic sites of participating enzymes are close enough to channelize the transient compound, creating a high local concentration of the metabolite and minimizing the interference of a competing pathway for the same precursor. Over the years, these phenomena had motivated researchers to make their immobilization approach naturally realistic by generating multienzyme fusion, cluster formation via affinity domain–ligand binding, cross-linking, conjugation on/in the biomolecular scaffold of the protein and nucleic acids, and self-assembly of amphiphilic molecules. This review begins with the discussion of substrate channeling strategies and recent empirical efforts to build it synthetically. After that, an elaborate discussion covering prevalent concepts related to the enhancement of immobilized enzymes’ catalytic performance is presented. Further, the central part of the review summarizes the progress in nature motivated multienzyme assembly over the past decade. In this section, special attention has been rendered by classifying the nature-inspired strategies into three main categories: (i) multienzyme/domain complex mimic (scaffold-free), (ii) immobilization on the biomolecular scaffold, and (iii) compartmentalization. In particular, a detailed overview is correlated to the natural counterpart with advances made in the field. We have then discussed the beneficial account of coassembly of multienzymes and provided a synopsis of the essential parameters in the rational coimmobilization design.

Combining a Genetically Engineered Oxidase with Hydrogen‐Bonded Organic Frameworks (HOFs) for Highly Efficient Biocomposites

“…Although this approach resulted in high enzyme loading, protein surface functionalization protocols require multistep procedures, [41,43,44] depend on the specific sequence of amino acids and their accessibility, [45] and can influence the native bioactivity [46] . A strategy that has been overlooked to increase protein‐framework interactions is protein engineering, where progress in both molecular biotechnology and DNA manipulation has enabled a straightforward and cost‐effective expression of fused protein systems [47–49] . For example, enzymes can be expressed with arginine‐rich mini‐proteins (modules) connected by a polypeptide chain (Z basic2 made of 58 amino acid; 7 kDa) to improve non‐covalent immobilization on inorganic substrates [50] .…”

DNA as a Recyclable Natural Polymer

“…We looked for a multi-enzymes bio-catalysis as an efficient approach. [12,13] In the biosystem, the phosphorylation of nucleotides is catalyzed by intracellular phosphotransferase and kinase. [14,15] Therefore, cell lysate is an excellent catalyst and has been applied for this nucleotide phosphorylation reaction, [16,17] but the reported phosphorylation yields are relatively low (especially for dTTP).…”

Kombination einer genetisch engineerten Oxidase mit wasserstoffbrückengebundenen organischen Gerüsten (HOFs) für hocheffiziente Biokomposite

“…[42] A strategy that has been overlooked to increase protein-framework interactions is protein engineering, where progress in both molecular biotechnology and DNA manipulation has enabled a straightforward and cost-effective expression of fused protein systems. [43][44][45] For example, enzymes can be expressed with arginine-rich mini-proteins (modules) connected by a polypeptide chain (Zbasic2 made of 58 amino acid; 7 kDa) to improve noncovalent immobilization on inorganic substrates. [46] We hypothesize that such positive surface charge enrichment via Zbasic2 modules would enhance the protein immobilization in HOFs (Figure 1a).…”