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 (N-C) 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 N-C 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 N-C. 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 multienzyme complexes in vitro from the crude cell lysis.