Controllable Preparation Of Enzyme/Metal-Organic Framework Composites For Cascade Reactions

The metal organic framework?MOF?has the characteristics of high pore size distribution,high specific surface area,structural designability and rich coordination of unsaturated metal sites,which makes it have higher potential in enzyme immobilization.At present,methods for immobilizing enzymes include in-situ coating,post-impregnation,and covalent modification.These methods have problems such as limited types of MOFs that can be loaded and inactivation of enzymes;and the current MOF is more used as a carrier if it is constructed.It is also an important issue to expand the scope of application of bio-chemical catalytic systems.On the other hand,these methods are difficult to effectively orient the position of the immobilized enzyme in the metal organic framework to avoid the problem of mutual interference of the enzymes.How to solve these problems becomes a challenge in the field of metal organic framework immobilized enzymes.In this paper,from the perspective of the opposite charge of MOF and enzyme surface and the biochemical mineralization of enzymes,the electrostatically induced preparation of enzyme-metal organic framework complex and step biochemical mineralization are proposed to realize the spatial segmentation of multi-enzyme system and apply it to the level of cascade reaction.?1?Preparation of glucose oxidase-metal organic framework complex?GOx/PCN-222?by electrostatic induction based on high stability biomimetic catalytic properties of zirconium-porphyrin-based metal organic framework PCN-222?Fe??positive charge?and enzyme surface?negative charge?.The metal-organic framework of the loaded enzyme maintains the original rod shape and crystallinity.Glucose oxidase was successfully modified on the surface of PCN-222?Fe?by means of FTIR,confocal microscopy,etc.GOx/PCN-222?Fe?shows a good cascade of catalyzed glucose oxidation and catalyzed oxidation of ABTS?or pyrogallol?by H₂O₂,and the enzyme loading 0.01 mg/mg,and.Kinetic studies indicate that the reaction follows the Michiaelis-Menten process mediated by conventional enzyme kinetics.It is indicated that the constructed chemical-biocatalyst still exhibits the characteristics of biomimetic biocatalysis when applied to cascade catalysis.Further stability experiments show that the acid stability is enhanced after loading on PCN-222?Fe?,which may be due to the strong interaction between GOx and PCN-222?Fe?that stabilizes the GOx second structure and stabilize its catalytic activity.GOx/PCN-222?Fe?has good stability,after repeated use for 5 times.And there is no obvious attenuation of catalytic performance,which further proveed that electrostatic interaction promotes strong interaction between enzyme and metal organic framework.?2?The lipase@ZIF-8/lipase@ZIF-8 core-shell structure was synthesized by biomineralization.The prepared sample can be separated by adding magnetic nanoparticles such as Fe₃O₄ to realize its industrial value.Characterization and catalytic performance were tested by XRD,BET,TGA,confocal microscopy,etc.These Characterizations proved that lipase can biomineralize the core-shell structure by ZIF-8,which can maintain its crystallinity,and gave a maximum total enzyme loading is 0.12 mg/mg.The catalytic reaction of 4-nitrophenyl palmitate proved that the synthesized core-shell structure maintained its good biocatalytic perform-ance.Moreover,lipase@ZIF-8/lipase@ZIF-8 has good stability.After repeated use for5 times,there is no obvious change of catalytic performance,which indicates that the lipase is more stabl after coprecipitation.Compared with the traditional ZIF-8bio-enzyme coprecipitation material,the core-shell structure is closer to the biological structure,and the obtained product shows good stability and durability,which lays a foundation for further simulating the organelle reaction.?3?The GOx@ZIF-8/HRP@ZIF-8,lipase@ZIF-8/tripsin@ZIF-8 core-shell struc-ture was synthesized by biomineralization.Characterization and catalytic performance were tested by SEM,XRD,BET,TGA,confocal microscopy,etc.These Characteri-zations proved that HRP,GOx,lipase and tripsin can biomineralize synthesis of the core-shell structure of ZIF-8,among which GOx@ZIF-8/HRP@ZIF-8 and lipase@ZIF-8/tripsin@ZIF-8 gave a maximum total enzyme loading of 0.11 mg/mg and 0.15 mg/mg,respectively.GOx@ZIF-8/HRP@ZIF-8 can perform multi-enzyme system cascade reaction on substrate,oxidize substrate glucose and produce hydrogen peroxide,which oxidize ABTS exhibit good catalytic performance.The window size of ZIF-8 is selective for different substrates.Lipase@ZIF-8/tripsin@ZIF-8 uses vinyl acetate and n-butanol as substrates,exhibiting a catalytic efficiency of over95%.Therefore,compared with the single-enzyme core-shell structure,the multi-enzyme core-shell structure is closer to the biological structure,and the organelle reaction can be further studied.