Co-immobilization of multiple enzymes by self-assembly and chemical crosslinking for cofactor regeneration and robust biocatalysis

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 KgBDH-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 KgBDH-SC-ST-FDH-SiO2 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, KgBDH-SC-ST-FDH-SiO2 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. •KgBDH-SC and FDH-ST can spontaneously link together by self-assembly.•KgBDH-SC-ST-FDH-SiO2 can asymmetrically prepare (R)-PED with cofactor regeneration.•KgBDH-SC-ST-FDH-SiO2 showed a higher catalytic activity than FDH-KgBDH-SC-SiO2.•KgBDH-SC-ST-FDH-SiO2 showed excellent pH stability and organic solvents stability.•KgBDH-SC-ST-FDH-SiO2 remained 52.5% of catalytic activity after 8 cycles.