| During enzyme immobilization,covalent coupling of enzyme molecules with the carrier causes enzyme deactivation to maintain the active conformation of the enzyme molecule while making the enzyme fixation more firm,we designed a disulfideencapsulated “birdcage” structure on the surface of the magnetic microsphere carrier for enzyme immobilization studies.We first prepared polyethyleneimine(PEI)-grafted polymethyl methacrylate(PMMA)magnetic microspheres,and then modified the grafted PEI with 3-(2-pyridine dimmercapto)propionic acid N-hydroxysuccinimide ester(SPDP).Then,the adsorbed lipase was trapped by charge gravity.finally,the crosslinked SPDP was fixed with 2,2-(1,2-ethylenedioxy)diethylenethiol(DMDO).The main elements include the following:First,the synthesis,graft modification,crosslinking encapsulation and characterization of magnetic microspheres.Firstly,the oleic acid modified Fe3O4 magnetic particles were prepared by chemical coprecipitation method.The PMMA magnetic microspheres were synthesized by suspension polymerization with it as the magnetic core,and the microspheres were esterified to carry the carboxyl group.The titration results showed that the surface carboxyl group content of the microspheres was 0.49 mmol/g.Then PEI was grafted on the surface of the microspheres to prepare the PEI@PMMA magnetic microspheres.The analysis showed that the content of primary amino groups on the surface of the microspheres was 53.3μmol/g.Furthermore,the SPDP-PEI@PMMA magnetic microspheres were prepared with SPDP modified PEI.UV analysis showed that the modified amount of SPDP was 9μmol/g.Finally,the DMDO crosslinked SPDP was used to complete the package.The UV analysis results showed that the content of diethylthiol after crosslinking was 4μmol/g.Second,the SPDP-PEI@PMMA magnetic microspheres were crosslinked to encapsulate the immobilized lipase.Firstly,using SPDP-PEI@PMMA magnetic microspheres as support,the Candida rugosa lipase(CRL)was trapped and adsorbed on the microspheres by charge gravity.The enzyme was then encapsulated internally by crosslinking the SPDP with DMDO.Fluorescence microscopy experiments showed that the CRL lipase was immobilized on the surface of the microsphere carrier;the results of high salt elution experiments showed that the binding force between the cross-linked encapsulated CRL lipase and the carrier was significantly enhanced compared with the adsorbed immobilized enzyme,indicating that the CRL enzyme was successfully cross-linked encapsulated on the surface of the microsphere carrier;the second-order structure of the cross-linked encapsulated immobilized enzyme showed that the activity of the cross-linked encapsulated enzyme was increased by 14.2% compared with the activity of the free crl enzyme(50 u/mg).In addition,the crosslinking time,crosslinking temperature of the cross-linked encapsulated immobilized enzyme were investigated.The results showed that the crosslinking temperature was 25℃,and the immobilized enzyme was the best prepared at 1 h of crosslinking reaction.The immobilization amount and enzyme activity were 28.8 mg/g microspheres and 57.1 u/mg,respectively.Third,the properties of magnetic microspheres cross-linked encapsulated immobilized enzymes.The optimum temperature,optimum PH,stability and reusability of immobilized CRL lipase were investigated.The results showed that the optimum temperature of crosslinking encapsulated immobilized enzyme was 37°C and the optimum PH was 7.5,which was the same as that of free enzyme.However,the temperature and pH range of the cross-linked encapsulated immobilized enzyme were significantly expanded compared with that of free enzyme,and its thermal stability and tolerance to denaturant were also significantly improved.The results of the reusability experiment showed that the cross-linked encapsulated immobilized enzyme still retained more than 55% after four consecutive use,while the activity of the adsorbed immobilized enzyme decreased significantly after four use,retaining only 20% of the initial activity. |
