Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce Kg BDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming Kg BDH-SC-ST-FDH-SiO 2 with the capability of reducing 2-hydroxyacetophenone to ( R )-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, Kg BDH-SC-ST-FDH-SiO 2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of ( R )-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.
Natural proteins display organized hierarchical structures and tailored functionalities that cannot be achieved by synthetic approaches, highlighting the increased interest in developing protein-based materials. Protein self-assembly allows fabricating sophisticated supramolecular structures from relatively simple building blocks, a strategy naturally employed by amyloid proteins and intrinsically disordered proteins. However, the design of self-assembled bioinspired materials with multi functionalities is still challenging. Inspired by the natural self-assembly proteins (such as mussel foot proteins and amyloid proteins), a temperature-inducible engineering programable hydrogel-like amyloid nanostructure is developed by using a genetically modular fusion approach. The resulting hydrogel-like assemblies display outstanding adhesive capacity, high stability, and broad substrate universality. The employed SpyCatcher/SpyTag system allows modifying the hydrogel-like assemblies with any functional proteins of interest. Owing to their strong adhesive capacity and functional flexibility, such amyloid fibril-based hydrogel shows advantages in the immobilization of diverse enzymes for highly efficient biocatalysis, fabrication of multi-layered functional coatings, and construction of functionalized 3D scaffold for cell culture. Overall, a modular and straightforward approach is established to obtain a genetically programable nanostructure platform. The novel hydrogel-like assemblies described here may be potentially applied to but not limited to synthetic biology, surface/interface engineering, and tissue engineering.
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