Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni2.

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and NC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.

Study on the properties of a dual-system-based protein scaffold for orthogonal self-assembly.

Protein scaffolds possessing the ability to efficiently organize enzymes to improve the catalytic performance, enzyme stability and provide an optimal micro-environment for biocatalysis. Here, SpyCatcher fused to the C-terminus of Treptavidin (a variant of streptavidin) to construct a chimeric tetramers protein scaffold (Tr-SC) with dual orthogonal conjugation moieties. The results showed that the expressed Tr-SC scaffold was an active tetramer with good stability under 80 °C and pH 6.5-8.5, which could bind 4 SpyTag-mCherry and 4 Biotin-EGFP. Tr-SC scaffold can bind 1-4 ligands alone under different conditions. The order in which protein scaffolds bind to proteins has little effect on the final complex structure. It is more difficult for SpyTag-mCherry than Biotin-EGFP to bind to Tr-SC, so incomplete conjugates of a hexameric complex composed of 2 SpyTag-mCherry and 4 Biotin-EGFP form when the molar ratio of scaffold and two ligands is 1:4:4. Therefore, it was suggest that the Tr-SC can first bind to excess SpyTag-protein and mixed with Biotin-protein to promote the formation of higher multimers. The results can be important reference for more extensive use of Tr-SC to construct heterologous protein polymers and assembly of heterologous enzyme molecular machine in vitro to carry on efficient cascade reaction in the future.

Performance of microenvironment-induced lipase immobilization on diversify surface of magnetic particle.

The orientation of the enzyme molecular on the interface of the carrier affects its activity. Therefore, it is very important to controllably induce the orientation of the enzyme on the surface to improve the performance of the immobilized enzyme. Magnetic nanoparticles were used to construct microenvironments with the different surface hydrophobicity and charge characteristics by controlled modification, and those particles with various microenvironments were further used to study their interaction with the lipase. The amount and activity of immobilized enzyme on different magnetic nanoparticles surfaces were studied by physical adsorption and covalent binding. Through the enzyme surface and particle surface characteristics analysis, the possible preferred orientation of enzyme and enzyme conformation on different surfaces were inferred, which well explained the effect of surface induction on enzyme loading and activity. The methods of surface microenvironment regulation and the strategy of controllable induction of enzyme orientation adopted in this study are enlightening for the rational design of immobilized enzyme methods.