The Preparation Of Novel Magnetic Composites And Application For Cellulase/Horseradish Peroxidase Immobilization

As a natural catalyst,enzymes have characteristics of high catalytic efficiency,mild reaction condition and high specificity in the catalytic process,but they also have characteristics of easy deactivation and difficult recovery,which inhibit their industrial application.In order to using the excellent catalytic performance of enzyme,overcoming the weakness,immobilized enzyme became the focus field for experts and scholars.And the performance of the immobilized enzyme mainly depends on the immobilization methods and carrier materials.In this work,we prepared three kinds of composites as carriers and immobilized cellulase or horseradish peroxidase in covalent bonding.The specific works are listed as follows:(1)The magnetic composite microspheres Fe3O4@SiO2@P(GMA-co-NIPAM)was synthesized and characterized.The microspheres exhibited a core-shell-shell structure with a diameter of about 350 nm and were superparamagnetic.They were applied to the cellulase immobilization process,and the cellulase loading amount was about 233.00 mg g-1 under the optimum immobilization conditions.The enzymatic properties of the immobilized enzyme were determined.Compared with the free enzyme,the catalytic temperature range of the immobilized enzyme was widened,and the catalytic pH value was not greatly affected.The thermal stability and and storage stability were improved.After repeated hydrolysis of carboxymethyl cellulose for 6 times,the relative activity was 65.6%.In addition,immobilized cellulase has thermal denaturation and refolding ability.(2)The magnetic multi-arm composite microspheres Fe3O4 @ PAA @6-arm-PEG-NH2 were designed and synthetized,and their properties and morphology were characterized.The glutaraldehyde was used to connect the horseradish peroxidase and the amino groups on the surface of the material to obtain covalently immobilized horseradish peroxidase.Secondly,the effects of immobilization process(glutaraldehyde concentration,amount of material,time and temperature)on the immobilization results were discussed.The enzyme loading amount was about 139.82 mg g-1,which was much higher than that of other scholars.The enzymatic properties of immobilized horseradish peroxidase have been improved.The relative activity of immobilized horseradish peroxidase was 71.05%after storage for 60 days.The catalytic activity was 61.06%after 8 times of catalytic reaction.Finally,the ability of the immobilized horseradish peroxidase degrading phenol in aqueous solution was discussed.The degradation process,such as phenol concentration,hydrogen peroxide content,immobilized enzyme dosage and reaction time,were optimized.On the basis of this,the degradation rate of the immobilized enzyme was significantly higher than that of the free enzyme and the carrier material.(3)The magnetic graphene composites GO-Fe3O4-6-arm-PEG-NH2 were designed and synthesized,and its morphological structure was characterized.The material exhibits a nearly transparent film,with superparamagnetism and a saturation magnetization of 30.80 emu g-1.The optimal enzyme loading amount was about 186.34 mg g-1 by using GO-Fe3O4-6-arm-PEG-NH2 immobilized horseradish peroxidase.Compared to free enzymes,immobilized horseradish peroxidase has slightly enhanced tolerance to the catalytic environment.Thermal stability,storage stability and operational stability have been improved.The efficiency of immobilized horseradish peroxidase on the removal of phenol was much higher than that of free enzyme and carrier material,because of the enzyme-like catalytic performance of the graphene material.