Sequential co-immobilization of multienzyme nanodevices based on SpyCatcher and SpyTag for robust biocatalysis

Sequential immobilization of multiple enzymes can not merely improve the enzymatic activity and enzyme stability but also facilitate the recovery for reuse, well imitating the natural enzyme complexes. Here, we developed a novel approach of preparing sequential multienzyme nanomachineries based on chemical crosslinking and protein assembly, and then characterized the nanodevice. In the preparation process, KgBDH-SC (SpyCatcher-fused (2R,3R)-2,3-butanediol hydrogenase) was firstly immobilized onto the functionalized silicon dioxide nanoparticles with glutaraldehyde, and then FDH-ST (SpyTag-fused formate hydrogenase) was immobilized by the spontaneous protein reaction between SpyTag and SpyCatcher to achieve the sequential co-immobilized enzymes FDH-ST-SC-Kg[email protected]2, which can asymmetrically reduce 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol by cofactor regeneration. In comparison to the mixture of free enzymes, the sequential co-immobilized enzymes created under the design conditions performed around 50% activity recovery. Furthermore, FDH-ST-SC-Kg[email protected]2 presented wider pH tolerance and greater organic solvents tolerance than the mixture of free enzymes as well as retained 51.7% of overall activity after being recycled eight times. Finally, the sequential co-immobilized enzyme enabled the biosynthesis of (R)-1-phenyl-1,2-ethanediol from 80 mM 2-hydroxyacetophenone in the phosphate buffer reaction system, with 87.4% yield and over 99% optical purity.