Cross-linked Encapsulation For Enzyme Immobilization And Catalytic Performance On Magnetic Microspheres

At present,the development of high efficiency emenzy immobilization technology is still a challenge.In order to securely fix the emenzy and maintain its natural conformation,we designed a structure similar to a"birdcage"on the surface of the carrier to wrap the emenzy.The poly?methyl methacrylate??PMMA?magnetic microspheres were first prepared and polyethyleneamine?PEI?was grafted onto the microspheres.Then the proteins were adsorbed into these chains on the magnetic microsphere.Finally,the“birdcage”were constructed by cross-linking the amino groups on PEI chains using glutaraldehyde?GA?as crosslinker,and the adsorbed protein was thus encapsulated in t the structure.The immobilization method used in the process was called cross-linked encapsulation method.The main contents of this paper are listed as below:First,the Fe₃O₄ magnetic nanoparticles were prepared by chemical precipitation,and the PMMA magnetic microspheres were prepared by a modified suspension polymerization with methyl methacrylate?MMA?as monomer.Then the surface of microspheres was functionally modified,and the carboxyl content of microspheres after Esterification was 0.85 mmol/g by chemical titration,and the amino content on the surface of microspheres after grafted polyethyleneamine 1800?PEI 1800?was 0.275 mmol/g.Secondly,Bovine serum albumin?BSA?as protein template was immobilized by cross-linked encapsulation method using PEI 1800-PMMA magnetic microspheres as carrier.And the immobilization principle,encapsulation process,effect of this method,and the change of protein structure were studied.The results showed that the crosslinked encapsulation method conforms to our original design expectations,which was to encapsulate BSA on the surface of the carrier by using the Schiff alkali structure that was generated because of PEI and GA reaction.Crosslinked encapsulation method could not only strengthen the bonding force between BSA and carrier,but also maintain the natural structure of BSA.Thirdly,Candida rugosa lipase?CRL?as protein template was immobilized by cross-linked encapsulation method using PEI 1800-PMMA magnetic microspheres as carrier.It is proved by the desorption experiment that the bonding force between CRL lipase and carrier is significantly enhanced,and the stability of immobilized CRL is improved.SDS-PAGE experiments show that the Schiff alkali structure realizes the encapsulation of the CRL,and the two-stage structure of infrared spectroscopy shows that the immobilized lipase of crosslinking package maintains its conformation structure well.The enzyme activity analysis showed that the lipase activity of crosslinked encapsulation fixation was significantly higher than that of covalent immobilized enzyme,which was close to the activity of adsorption immobilized enzyme.In addition,the effects of various parameters on CRL encapsulation were investigated,and at 35°C,the concentration of glutaraldehyde 0.75‰and crosslinking time 1 h,CRL loading and enzyme ratio on PEI 1800-PMMA microspheres respectively reached 38.6 mg/g,2.10 U/mg,respectively.Fourthly,compared with the immobilized CRL by adsorption method,the experiments investigated the immobilized CRL crosslinking temperature ranges packaging method,the scope of pH,thermal stability and pH stability.The results showed that the range of crosslinked CRL package immobilized lipase temperature and pH suitable for expansion,and the thermal stability and pH stability were improved significantly.In addition,the Km value?5.65 mg/ml?of the immobilized CRL by the Crosslinked encapsulation was higher than that of the free CRL?4.65 mg/ml?,while the Vmax value was lower than free enzyme.