Preparation Of Multi-enzyme-loaded Colloidosomes Based On Pickering Emulsion Template And Its Catalytic Performance

Immobilized enzymes as biocatalysts have great potential both industrially and scientifically because of their technological and economic importance.However,in many reactions,a single enzyme cannot meet the needs of industrial production,and multiple enzymes are often required to work together.Since in the traditional step-by-step catalytic production,each intermediate product has to be separated and purified,the loss during the operation will be large and the yield will be low.Therefore,if the catalytic process is reasonably designed to realize multi-enzyme cascade catalysis in a certain space,the initial input of reaction steps and reaction equipment can be effectively reduced,and the loss of intermediate products in the separation process can be reduced.In this paper,GOx@ZIF-8 and HRP@ZIF-8 were prepared by encapsulating glucose oxidase（GOx）and horseradish peroxidase（HRP）in Zeolitic Imidazolate Framework-8（ZIF-8）by biomimetic mineralization method.On this basis,the colloidosomes with a core-shell hollow structure were prepared using enzyme-loaded ZIF-8 as particle stabilizer via Pickering emulsion template method.By directionally assembling GOx and HRP on the wall and cavity of the colloidosome,two kinds of enzyme-loaded colloidosomes with different enzyme distribution positions were obtained.The catalytic mechanism of the colloidosome loaded with dual enzymes was systematically analyzed,and the regulatory relationship between the positioning and assembly of dual enzymes and the catalytic process was revealed.The main research is as follows:（1）GOx@ZIF-8 and HRP@ZIF-8 were prepared by biomimetic mineralization method,and the relationship between the amount of enzyme added in the preparation process and the amount of enzyme loading and the recovery rate of enzyme activity was investigated.SEM,FTIR,XRD and CLSM were adopted to characterize the ZIF-8,GOx@ZIF-8 and HRP@ZIF-8.The enzymatic properties of free and immobilized enzymes were systematically studied.The optimum temperature of free and immobilized GOx were both 45℃,the optimal p H of free and immobilized GOx were both 6.The optimum temperature of free and immobilized HRP were both 35℃,the optimal p H of free and immobilized HRP were both 7.Moreover,the stability（thermal stability,p H stability,solvent tolerance,storage stability）of the immobilized enzymes were better than that of the free enzymes.After repeated use of 8 times,GOx@ZIF-8 and HRP@ZIF-8 remained 76.71%and 77.78%relative enzyme activity recovery.（2）The effect of oil-water ratio,the addition amount of enzyme-loaded ZIF-8nanoparticles and Span 80 on the morphology of Pickering emulsion was systematically investigated.The best conditions for preparing the emulsion are:oil-water ratio=3:1,enzyme-loaded ZIF-8 nanoparticles addition amount is 30 mg,Span 80 addition amount is 30 mg.The colloidosomes with hollow structure were prepared by Pickering emulsion template method and the positioning and assembly of GOx and HRP on the wall and cavity of the colloidosome were realized.CLSM verified the separated immobilization of GOx and HRP in the colloidosome.The morphology of the colloidosomes was observed by SEM,and the hollow structure of the colloidosomes was verified.Besides,the small particle size ZIF-8 coating that grew on the outside of the emulsion droplet during the curing process of the emulsion provided additional structural stability for the emulsion.The enzymatic properties of free and immobilized double enzymes were studied.The optimum temperature of free and immobilized double enzymes were both 35℃,the optimal p H of free and immobilized double enzymes were both 6.At the same time,it was proved that the temperature stability,p H stability,storage stability and solvent tolerance of S_GOxL_HRP（GOx is located on the wall of the colloidosome and HRP is located in the cavity of the colloidosome）and S_HRPL_GOx（HRP is located on the wall of the colloidosome and GOx is located in the cavity of the colloidosome）were significantly stronger than that of the free double enzymes.（3）Two kinds of microreactors with double enzymes were constructed and the glucose-o-phenylenediamine cascade reaction was used as a model.By analyzing the difference in the utilization degree of intermediate product H₂O₂in the two bioreactors,the relationship between double enzymes assembly and reaction efficiency was established.The comparison of S_GOxL_HRP,S_HRPL_GOx and other independent immobilized enzyme systems highlights the advantages of co-immobilization over independent immobilization.