Study On Co-Immobilization Of Glucose Oxidase And Catalase By Chitosan-Based Magnetic Microspheres

To alleviate the huge manpower needed for industrial production process,from oxidation of glucose to generate gluconate,this paper get the theoretical level of sodium gluconate,using efficient of enzymic catalytic reaction,using the immobilized enzyme solution to the free enzyme easy deactivation,and the problems of difficult to recycle,reduce the use of double enzyme system by-product hydrogen peroxide on the toxicity of glucose oxidase,Keep its efficient activity,the use of magnetic materials to ensure the subsequent enzyme immobilization,reuse,recycle use of chitosan on the surface of positive charge and ferroferric oxide surface negative charge,make whole material is more stable,and utilization of porous materials,large specific surface area characteristics improve the efficiency of enzyme quantity and load enzyme,finally from chitosan material modification and crosslinking methods improving immobilized system,The material with high enzyme load and stable system was obtained,and the efficient catalysis of enzyme was ensured.The immobilization process studied in this paper provides a theoretical basis for the production of sodium gluconate by double enzyme direct catalysis and lays a certain foundation for the application of immobilized enzyme in the future chemical industry and food industry.The main research contents and results are as follows:(1)Chitosan microspheres were prepared by emulsification method.The particle size distribution of chitosan microspheres was investigated from seven aspects,including the concentration of chitosan,the proportion of dispersant,the ratio of oil to water,the concentration of crosslinking agent,the crosslinking temperature,the crosslinking time and the rotational speed.The average particle size and the particle size distribution span were taken as the objects of investigation.When the concentration of chitosan was 1%,the proportion of dispersant was 6%,the ratio of oil to water was 9/1,the dosage of crosslinking agent was 0.5 m L,the crosslinking temperature was 50?,the crosslinking time was 2 h,and the rotation speed was 600 r/min,the chitosan microspheres with uniform particle size could be obtained.In order to improve the specific surface area of microspheres and explore the effect of pore size on the enzyme loading,different pore size of porous chitosan microspheres were prepared by adding different pore size of silica and using sodium hydroxide solution to corrode them.Taking the porosity,enzyme carrying capacity and enzyme activity of chitosan microspheres as the research object,the optimal parameters for silica corrosion were determined as 3 h immersion time of sodium hydroxide,70?immersion temperature,1.5 mol/L sodium hydroxide concentration and 500 nm size of porogenic silica.At the same time,the pore size,wall thickness,porosity and enzyme load of the microspheres were 790.15±250.91 nm,354.81±32.57 nm,58.23±0.86%,58.28±2.64 mg/g,and the enzyme activity of the microspheres reached 16.93±0.14 U.(2)The co-immobilization of CAT and GOD eliminated the harmful by-product H₂O₂ in time and effectively solved the problem of continuous deactivation of GOD during the reaction.When the mass ratio of PMCSM/GOD/CAT was 100/6.02/10.96(mg/mg/mg),the relative enzyme activity of GOD reached the highest,which was133.32±0.68%.The thermal stability and acid-base stability of the enzyme were greatly improved after co-immobilization.At 60?,the activity of the enzyme was57.27±3.04%for PMCSM@GOD@CAT and 28.76±4.10%for free GOD.The relative enzyme activity of PMCSM@GOD@CAT was above 63%when p H was 5?10,while the relative enzyme activity of free GOD was only 4.98±0.72%when p H was 10.Under the reaction temperature of 30?and p H of 6,the yield of sodium gluconate from glucose catalyzed by PMCSM@GOD@CAT can reach 96.80±1.36%.The relative enzyme activity of PMCSM@GOD@CAT remained 69.77±0.78%after 10 times of repeated use,and the initial activity of PMCSM@GOD@CAT was maintained76.52±1.41%after 30 days of storage.(3)By introducing carboxyl groups into the porous magnetic chitosan microspheres,PMCSM-COOH contained more GOD than PMCSM,and the stability of the immobilized enzyme was also enhanced.The results showed that when the mass ratio of PMCSM-COOH/GOD/CAT was 100/9.34/10.78(mg/mg/mg),the relative enzyme activity of GOD reached the highest value(131.91±0.58%),which was 1.46times higher than that of PMCSM@GOD.In addition,the temperature stability and p H stability of PMCSM-COOH@GOD@CAT are both improved compared with PMCSM.The results of infrared spectrum show that the?-helix content of PMCSM-COOH@GOD is higher than that of free GOD,that is,it has better rigid structure and stability.Similarly,through the analysis of the secondary structure of the free CAT and PMCSM-COOH@GOD@CAT,the content of?-helical and?-folding of the free CAT was higher than that of the free CAT,which improved the stability of the enzyme molecule,which was reflected in that the immobilized enzyme had better temperature stability and p H stability than the free enzyme.The immobilized enzyme system studied in this paper will lay a foundation for future chemical and food industry applications.