Magnetic Nanoparticles Immobilized Of Lipase CRL

As a green biocatalyst,lipase(EC 3.11)is widely used in biopharmaceutical,food industry,chemical analysis and other fields.Immobilization strategy was an effective method for the reusability and stability of lipase.Magnetic nanoparticles can be used as immobilized carrier materials to provide convenient and rapid recovery.In this paper,three kinds of magnetic nanocomposite carriers were prepared,including magnetic graphene oxide nanocomposites(MGO)prepared by co-precipitation,surfactant modified magnetic graphene oxide(SMGO)and surfactant modified Fe3O4 magnetic nanoparticles(Span/Tween Fe3O4).These three magnetic composite materials were applied with immobilized of lipase(CRL),the immobilization performance of magnetic composites and the effect of Span/Tween surfactant-modified carrier on the catalytic properties of lipase were investigatedThe preparation process of MGO was optimized by orthogonal experiment,the optimal preparation process was obtained by taking enzyme activity and specific activity as indicators.FT-IR and SEM characterization indicated that MGO was successfully prepared.During the preparation process,the carboxyl functional group of GO layer surface was partially reduced under strong alkali.Under the optimal immobilization conditions,the immobilized enzyme activity recovery and protein recovery were 92.41%and 88.2%,respectively.The immobilized enzyme relative enzyme activity decreased to about 40%reused for a second time,it is speculated that most of the lipase immobilized on the MGO carrier was physically adsorbed,and very few were covalently immobilized.MGO was prepared by chemical co-precipitation method,span series surfactant was directly added to the reaction system to prepare surfactant-modified magnetic graphene oxide(SMGO)through one pot process.The results of X-ray diffraction(XRD),scanning electron microscopy(SEM)and Fourier transform infrared spectroscopy(FT-IR)indicate that SMGO is successfully prepared and performs well in magnetic separation.With GA_as cross-link agent and Candida rugose lipase(CRL)as model enzyme,CRL was immobilized covalently on the SMGO materials.The activity recovery of Span40 MGO immobilized enzyme was 116.5%±1.7,which was 6 times that of MGO immobilized enzyme;the specific activity was up to 32.5 U/mg,1.6 times that of free enzyme;Kcat/Km was about 0.5 times higher than that of free enzyme.The storage stability and thermal stability were improved,and 73.6%of the relative enzyme activity was retained after 6 cycles of hydrolysis reaction.Preliminary analysis indicated that the surface of MGO was changed from hydrophilic to strong hydrophobic after modification,which simultaneously caused the activation of hydrophobic interface during the process of covalent immobilization,it is one of the reasons that increase the enzyme activity.The modification strategy reported in the article can provide new ideas for the modification of similar carrier.For the Span/Tween Fe3O4 carrier,the activity recovery and protein recovery of Span Fe3O4 immobilized enzyme were higher than that of Tween Fe3O4,which is believe to be due to the difference in surface hydrophobicity of Tween/Span surfactant modified carrier.Kinetic parameter measurements showed that the Span Fe3O4 immobilized enzyme had higher catalytic efficiency.The Span40 Fe3O4 immobilized enzyme Kcat/Km was 33.38 s-1·rmM-1,which was 1.7 times that of the free enzyme and 6.9 times that of the Fe3O4 immobilized enzyme.It indicates that the activity center of the lipase CRL is more exposed to the substrate when immobilized on the surface of the hydrophobic carrier,and the enzymatic performance is activated.Under the optimal immobilization conditions,the immobilized enzyme activity recovered to 112.8%,the enzyme activity was 19.2 U,and the immobilized enzyme was reused for 6 times still retaining 72.7%of relative enzyme activity,immobilized enzyme storage stability and thermal stability get improvement.The preparation method of Span40 Fe3O4 is simple and cheap,and can realize the interfacial activation of lipase while covalently immobilizing,thereby improving the catalytic performance of immobilized enzyme,providing a feasible immobilization strategy for industrial application of immobilized enzyme.