Biomimetic Construction Of Spatially Isolated Multienzyme Systems And Performance Intensification

Multienzymatic catalysis is considered to be the next generation of biocatalysis.As a central part, rational design and construction of multeinzyme systems haveattracted great attention and become a hot research topic nowadays. When designingan efficient multienzyme system, three crucial issues should be taken intoconsideration:1) synergistic catalysis among multienzymes;2) efficient mass transferof substrates and products; and3) easy recyclability of multienyzmes. Inspired by theexistence form and synergistic catalytic behavior of multienzymes in organisms, weput forward to the strategy of constructing spatially isolated multienzyme systems. Inthe present study, a series of spatially isolated multienzyme systems （containingformate dehydrogenase and formaldehyde dehydrogenase） enabled bynanospheres/microcapsules are constructed by combining biomimetic mineralizationwith other biomimetic platform techniques （e.g., biomimetic adhesion, Pickeringemulsion, surface segregation and layer-by-layer （LbL） self-assembly）. When utilizedfor the conversion of CO₂, these systems exhibit desirable catalytic performance andeasy recyclability.The details of this study were summarized as follows:Firstly, a series of size-controlled active titania nanospheres were synthesizedthrough the synergy between biomimetic mineralization and biomimetic adhesion.Biomimetic mineralization was in charge of the formation and growth of nanospheres,while the addition of biomimetic adhesive （oligodopa） was responsible forterminating the mineralization process and functionalizing the nanospheres surface.By adjusting the species/concentration of inducer as well as the addition time ofoligodopa, the chemical composition and size of nanospheres could be facilelytailored. Besides, the biomimetic adhesive layer of oligodopa can anchor amino-orsulfhydryl-containing molecules through Michael addition reaction or Schiff’sreaction, rendering the nanospheres surface multifunctionality.Secondly, a spatially isolated multienzyme system enabled by active nanosphereswas successfully constructed through the synergy between biomimetic mineralizationand biomimetic adhesion, achieving the efficient process intensification. The first enzyme （formate dehydrogenase） was entrapped accompanying the formation ofnanospheres through biomimetic titanification. After in situ surface functionalizationof nanospheres with oligodopa, the second enzyme （formaldehyde dehydrogenase）was immobilized on the surface of active nanospheres through amine-catechol adductreaction. When utilized for the conversion of CO₂, the as-constructed multienzymesystem exhibited a formaldehyde yield of higher than60.0%. Besides, after evaluatingthe performance of different-sized multienzyme systems, it was found that smallersized multienzyme system possessed higher enzymatic activity but lowerrecyclability.Thirdly, a spatially isolated multienzyme system enabled by nanosphere-stabilizedcapsules （NSSCs） was constructed through the synergy of biomimetic mineralizationand Pickering emulsion, achieving the efficient process intensification. The firstenzyme-containing nanospheres were monodispersely spread on the surface of oildroplets through Pickeirng emulsion, maintaining their structural characteristics andintegrity. Then, the gel titania, that was produced through sol-gel process of tetrabutyltitanate, was in charge of crosslinking the nanospheres, forming a stable andcontinuous capsule wall. The second enzyme was immobilized on the surface ofcapsule wall through amine-catechol adduct reaction between oligodopa and enzyme.During the construction process, the released butanol as a result of the sol-gel processof butyl titanate could be adsorbed on the surface of nanospheres, further inducing theself-assembly of the nanospheres at the oil/water interface. This process played keyrole on the formation of Pickering emulsions. When utilized for the conversion ofCO₂, the as-constructed multienzyme system acquired a formaldehyde yield of higherthan50.0%. Meanwhile, the lower density of the oil core than that of water endowedthe multienzyme system with excellent recyclability, especially the formaldehydeyield kept almost unaltered after10times recycling.Fourthly, a spatially isolated multienzyme system enabled by mesoporous hybridmicrocapsules was constructed through the synergy of biomimetic mineralization andsurface segregation, achieving the efficient process intensification. The first enzymewas entrapped accompanying the formation of the PAH-segregated CaCO₃template.Then, the PAH polymeric network was formed through crosslinking between GA andPAH. Afterwards, this polymeric network was utilized for implementing biomimeticmineralization with in situ entrapping the second enzyme. After removing thetemplate, a spatially isolated multienzyme system was acquired. During the construction process, the CaCO₃microsphere served as the dual templates forformation of both capsule lumen and mesopores on the capsule wall, whereas PAHplayed the following two crucial roles:（1） forming the bulk polymer network torender confined space for biomimetic mineralization and （2） triggering themineralization of Ti-BALDH to produce titania nanoparticles. When utilized for theconversion of CO₂, the as-constructed multienzyme system exhibited a formaldehydeyield of higher than70.0%. Meanwhile, the reaction rate was nearly two times higherthan the average level. And no catalytic activity was lost after10times recycling.Besides, the mesoporous hybrid microcapsules can be utilized in various otherapplications and exhibited desired perforamnce.Fifth, a spatially isolated multienzyme system enabled by mitochondria-likemicrocapsules was constructed through the synergy of biomimetic mineralization andLbL self-assembly, achieving the efficient process intensification. Inspired by thedouble membrane structure of mitochondria, we proposed to utilize process andstructure biomimicism strategy instead of just process biomimicism strategy forconstructing multienzyme systems. The first enzyme was entrapped accompanyingthe formation of the PSS-doped CaCO₃template. The organic inner membrane wasacquired via LbL self-assembly of oppositely charged polymers/proteins on thetemplate. The silica template layer was then formed onto the inner membrane throughbiomimetic silicification with in situ entrapping the second enzyme, while theorganic-inorganic hybrid outer membrane was acquired via biomimetic titanificationsubsequently. After removing the CaCO₃template and the silica template, a spatiallyisolated multienzyme system was estabilished. When utilized for the conversion ofCO₂, the as-constructed multienzyme system exhibited a formaldehyde yield of higherthan82.0%, with a formaldehyde selectivity of90.0%. Meanwhile,70.0%of theinitial activity was retained after10times recycling.