Magnetic Multifunctional Module Multiphasic Maneuver Mimicking Multienzyme Magic

Coimmobilizing multiple proteins onto a single nanosupport holds great promise for mimicking natural multiprotein complexes and creating efficient cascade biocatalytic systems. However, controlling the spatial arrangement and loading ratio of different proteins poses a significant challenge, and coimmobilization often requires purified proteins. In this study, we developed a plug-and-display, sequential, and tunable coimmobilization strategy by combining Catcher/Tag pairs with HaloTag technology for the first time. We designed a chloroalkane-functionalized magnetic multifunctional module multiphasic maneuver mimicking multienzyme magic platform that combines HaloTag, SpyCatcher, DogCatcher, etc., allowing for rapid and stable capture of enzymes functionalized with corresponding Tags. By varying the number and order of Catchers or switching the tags, the spatial arrangement and loading ratio of different enzymes can be precisely controlled. The immobilized enzymes demonstrated high protein loading capacity and retained their catalytic activity. Moreover, the magnetic beads preserved their responsiveness, facilitating easy enzyme separation and recovery. This system also efficiently captured target enzymes directly from cell lysates, streamlining the immobilization process while reducing the time and cost. These findings represent a significant improvement over current methods for diverse advanced, sustainable, and biomedical applications.