Compartmentalization Of Bi-enzymes Within Hydrogel-emulsion Composite Particles For Tandem Catalysis

In nature,a single cell often contains several adjacent compartments separated from each other,where different enzymes are loaded to catalyze tandem reactions,an important part of the ecosystem’s material and energy cycle.Inspired by this,many researchers have adopted the strategy of compartmentalization,where different catalysts are loaded in separate compartments to mimic the tandem enzymatic reactions in a cell,resulting in the efficient synthesis of target products.The water-in-water emulsion is a system consisting of droplets of an aqueous solution dispersed in a continuous aqueous solution.The dispersed phase and the continuous phase contain different water-soluble molecules respectively.The solvent in water-in-water emulsion only contains water,so it has non-toxic,has excellent biocompatibility,green environmental protection and other significant advantages,and has a wide range of applications in the packaging and delivery of bioactive substances.However,due to its low surface tension and large interface thickness,the stability of this emulsion is relatively low.Sodium alginate can form gels quickly under mild conditions.Meanwhile,it has excellent biocompatibility and can sequester and prevent the inactivation of active substances such as enzymes,cells,proteins and sensitive drugs.In this paper,glucose oxidase（GOX）modified with polyethylene glycol derivatives was prepared into granules and used as an emulsifier to stabilize the water-in-water emulsion.Then,the continuous phase was gelated to form hydrogel-emulsion composite particles.During the gelation process,another enzyme,horseradish peroxidase（HRP）,was separated to make the hydrogel-emulsion composite particles contain two enzymes,and on this basis,a tandem reaction was carried out.Specific research results are as follows:1.Preparation of hydrogel-emulsion composite particles and study of their stability.Methoxy-polyethylene glycol propanal（m PEG-p ALD）and glucose oxidase（GOX）react with the Schiff base to form m PEG-GOX granules.Due to mild reaction conditions,glucose oxidase（GOX）remains active after modification.And that provides the basis for what we’re going to do in tandem.The synthesized m PEG-GOX particles were used as an emulsifier to stabilize polyethylene glycol/dextran（PEG/Dex）water-in-water emulsion.The stability of the water-in-water emulsion was adjusted by varying the concentration of m PEG-GOX particle emulsifier,the concentration of polyethylene glycol or glucan,and the volume ratio between the two aqueous solutions.Subsequently,sodium alginate solution was added to the water-in-water emulsion,mixed well and then added dropwise to the calcium chloride solution to form hydrogel-emulsion composite particles with a particle size of 3 to 4 mm.The results showed that the catalytic activity of the m PEG-GOX particles loaded in the hydrogel-emulsion composite particles was higher than that of the free enzyme under the same conditions when the particles were placed in different organic solvents,at different p H and temperature.Thus,the prepared hydrogel-emulsion composite particles are well resistant to environmental interference.At the same time,the encapsulation efficiency of the prepared hydrogel-emulsion composite particles is good,even if soaked in a water solution for 24 h,the encapsulation rate is still as high as 70%.It is proved that it has good stability.2.Preparation of hydrogel-emulsion composite particles with separated solid-loaded dual enzymes and their tandem catalytic application.In this system,the prepared m PEG-GOX particles act as an emulsifier at the interface between the two phases.At the same time,the external phase aqueous solution used contains horseradish peroxidase（HRP）,which is thus anchored in the external phase gel by gelation,resulting in hydrogel-emulsion composite particles that are separated and anchored with two different enzymes.A hydrogel-emulsion composite particle with a solid double enzyme is added to an aqueous solution containing a mixture of glucose,3,3’5,5’-tetramethylbenzidine（TMB）and other reaction substrates in a tandem reaction.The first step of the tandem reaction is the generation of H₂O₂by glucose under the action of m PEG-GOX particles located at the interface,and the second step is the conversion of TMB into ox TMB by HRP located in the external phase with the participation of the intermediate product H₂O₂.The results show that H₂O₂content is an important factor affecting the effect of tandem reactions.As the intermediate product of a tandem reaction,the catalytic reaction rate of H₂O₂increases with the increase of its content.However,when the content of H₂O₂reaches 130μmo L/m L,the reaction rate decreases gradually.At the same time,by comparing the reaction rates between different systems,it was found that the tandem reaction efficiency of the system was 7 times higher than that of the system formed by using horseradish peroxidase（HRP）in the inner phase to form a tandem reaction.Moreover,the catalytic reaction efficiency of the system did not change much after recycling the hydrogel-emulsion composite particles 6 times,indicating that it had good cycling performance.