Studies Of The Immobilization Technology For Co-immobilized Glucose Oxidase And Catalase

Immobilized enzyme technology has been of interest for the excellent properties than those of free enzymes.As an important part of enzyme engineering,immobilized enzymes could address the disadvantages of high-cost,low stability,and non-recyclable nature of free enzymes.In the complicated industrial process,Multienzyme combination is a common method to meet the requirement of the production.In is work,the process of glucose acid production by co-immobilization of glucose oxidase?GOD?and catalase?CAT?on porous materials was studied.And the preparation and optimization of porous materials,the modification of functional groups of carrier materials,the optimization of immobilization process,and the properties of immobilized enzyme were tested.The main contents were as following:Poly Glycidyl methacrylate?PGMA?as the porous materials carrier,were prepared by suspension polymerization with glycidyl methacrylate?GMA?as monomer.A series of porous carriers with varied particle size and pore size were obtained by adjusting the preparation speed and porogen,which were used for protein adsorption and enzyme activity determination.The PGMA spheres with moderate size have uniform pores on its surface.The average particle size of the spheres was between 0.5 to 1 mm,and the average pore diameter was in the range of 100?250 nm.The immobilized enzyme has reached the highest protein adsorption capacity and relative enzyme activity when the preparation speed was 500 rpm,the pore forming agent formula was toluene/nano-CaCO₃?1:1?.Four types of immobilized enzyme with carrier surface modification were compared,including PGMA were treated with the plasma system;PGMA were treated with the ethylene diamine solution;PGMA were modified by plasma and then treated with ethylene diamine solution;PGMA were immersed in the ethylene diamine solution followed by plasma treatment.The results show that plasma treatment followed by ethylenediamine treatment has displayed the highest relative activity,and the optimal plasma processing power and time was 80 W and 60 s.The optimal concentration of ethylenediamine was 1%.Compared with the four modified immobilized enzymes,the immobilized enzyme treated with plasma followed by ethylenediamine showed the highest activity.The relative activity was approximately 70%after storage for 5 days and82%after three consecutive cycles.GOD and CAT has been co-immobilization on the modified PGMA by covalent crosslinking.The optimal activity ratio of GOD and CAT was 1:1.25,glutaraldehyde concentration was 1%.Moreover,the optimized temperature and pH were 35?and 7.0.Scanning Electron Microscopy and Fourier Infrared Spectroscopy were used to characterize the immobilization process.The results of electron microscopy showed that the surface roughness of the PGMA changed significantly before and after immobilization.The results of infrared spectroscopy showed that,the content of epoxy group in the surface on PGMA decreased after modification.And the amino group increased due to the corresponding increases of amide bond on PGMA.The highest enzyme activity and protein adsorption capacity of immobilized enzyme were 25.98 U/g and 6.07 mg/g,respectively.Immobilized enzyme has stronger substrate affinity than free enzyme through kinetic analysis.Compared with free enzyme,immobilized enzyme has a wider range of temperature of 25?40?and pH 5.5?7.5.