Study On Preparation And Application Of Magnetic Nano-starch Immobilized Enzyme

Enzymes are biocatalysts with high efficiency and high specificity,but they have the defects of structural instability,expensive but difficult to be recovered,which can be effectively solved by immobilization.Meanwhile,magnetic Fe₃O₄nanoparticles(Fe₃O₄)have gradually become a research hotspot in the field of immobilized enzyme supports,but the immobilization effect is limited due to the lack of reactive groups on their surface.Therefore,it is of great significance to develop a Fe₃O₄with surface activity and use it as supports to prepare immobilized enzyme catalysts with high catalytic activity and high stability.Based on this,the research of this paper are as follows:1.Preparation of magnetic starch(Fe₃O₄@Starch)nanoparticlesMagnetic Fe₃O₄nanoparticles were prepared by co-precipitation method,and their particle sizes were optimized by single-factor and response surface experiments.On this basis,the magnetic Fe₃O₄nanoparticles were surface modified by starch pasting to obtain Fe₃O₄@Starch.The results showed that the optimal time,temperature,molar ratio,ammonia addition,ammonia drop acceleration and rotational speed for the preparation of magnetic Fe₃O₄nanoparticles were55 min,60°C,0.5:0.3,5 m L,140 m L/min,and 760 r/min.The optimal additions of immobilized carrier starch for trypsin(TRY),Thermomyces lanuginosus lipase(TLL)and Horseradish peroxidase(HRP)were 30%,25%and 30%(wt%).2.Preparation and characterization of Fe₃O₄@Starch immobilized enzymesTRY,TLL and HRP were immobilized by glutaraldehyde cross-linking method to study the effect of each factor on the immobilized enzymes and to characterize the structural and enzymatic properties of the immobilized enzymes.The results showed that the optimal cross-linking concentrations for the three enzymes immobilizations were 2.0%,1.5%and 1.0%,the optimal cross-linking times were 60 min,60 min and 120 min,the optimal enzyme addition amounts were 2.5 m L,4.0 m L and 5.0 m L,the optimal immobilization temperatures were 40°C,45°C and 35°C,and the optimal 2.0 h,5.0 h,and 3.5 h,and the optimal immobilization p H values were 6.5,7.5,and 7.5.The structural characterization demonstrated that the enzymes were successfully immobilized on Fe₃O₄@Starch.The particle sizes of Fe₃O₄,Fe₃O₄@Starch,Fe₃O₄@Starch@TRY,Fe₃O₄@Starch@TLL and Fe₃O₄@Starch@HRP were about 7.3±2.1 nm,12.5±3.7 nm,16.6±3.2 nm,18.3±4.1 nm and 14.5±2.7 nm.The enzymatic properties demonstrated that the affinity and catalytic rate of the enzymes for the substrate were reduced after immobilization,but the optimum temperature,p H,thermal stability,p H stability,storage stability,organic solvent tolerance and operational stability of the enzymes were significantly improved compared with that of the free enzyme.3.Verification of catalytic activity of Fe₃O₄@Starch immobilized enzymesThe enzymatic reaction mediated by magnetic nanoimmobilized enzymes showed that Fe₃O₄@Starch@TRY could efficiently hydrolyze casein up to 85.19%after 2.0 h reaction.Fe₃O₄@Starch@TLL can efficiently catalyze the acylation reaction of gastroside and polygonin with vinyl octanoate,with 97.04%and 98.17%conversions for both.The Fe₃O₄@Starch@HRP catalyzed reaction for 2.0 h showed a high removal rate of 93.17%for phenol.It shows the good potential of the above immobilized enzyme for application.This study not only enriches the theoretical study of nano-immobilized enzymes technology,but also provides a reference for the application of nano-magnetic starch in the field of immobilized enzymes,which has important theoretical research and industrial application significance.