Study On The Effect Of Crosslinker On Cross-linked Encapsulated And Immobilizated Enzyme

Enzyme immobilization should not only improve the stability and reusability of the enzyme,but also retain or even increase the activity of the enzyme to the greatest extent.In order to improve the performance of the immobilized enzyme,we use epoxy crosslinker to crosslink and encapsulate the immobilized enzyme in the"molecular cage"on the surface of the carrier,so that the enzyme molecule could be firmly fixed and its natural conformation could be well maintained.Firstly,the magnetic microspheres of polymethyl methacrylate（PMMA）were prepared,and then grafted with polyvinyl alcohol（PVA）and modified with thiol groups.Then,the magnetic microspheres were used as carriers to adsorb Candida rugosa lipase（CRL）.Finally,the enzyme was encapsulated and fixed on the surface of the magnetic microspheres by PVA chain crosslinking.The main content includes the following parts:Firstly,PMMA magnetic microspheres were prepared with Fe₃O₄magnetic particles as the core and PMMA as the shell,and the surface was carboxyl grouped by esterification and acidification.The carboxyl group content was measured to be 0.40mmol/g.PMMA magnetic microspheres were measured by scanning electron microscopy（SEM）and particle size distribution.The results showed that the microspheres have regular sphericity,good monodispersity,and an average particle size of 22.74μm.Then,PVA was grafted onto the surface of the microspheres,and the measured hydroxyl content was 5.6 mmol/g.The thiol modification of3-mercaptopropyltriethoxysilane（MPTES）was used to obtain MPTES-PVA@PMMA magnetic microspheres.The thiol modification density was 7.12μmol/g.The experiment used MPTES-PVA@PMMA magnetic microspheres as a carrier to adsorb CRL lipase,and then add a double epoxy crosslinker to crosslink the PVA chain,thereby encapsulating and fixing the enzyme on the surface of the magnetic microspheres.Fluorescence characterization and desorption experiment analysis show that CRL was successfully encapsulated on the surface of magnetic microspheres.Secondly,The effects of the chain length and hydrophobicity of crosslinker on the performance of the cross-linked encapsulated immobilized enzyme were investigated.The experimental results show that the chain length of the crosslinker mainly affects the loading capacity of the encapsulated immobilized CRL,and at the same time,the chain length of the crosslinker also has a certain effect on the enzyme activity.As the chain length of the crosslinker increased from 2.93 nm to 4.70 nm,the loading capacity of the enzyme increased by 37.9%,and continued increase in the chain length led to the leakage of the enzyme.Another interesting phenomenon is that the hydrophobicity of the crosslinker has an important effect on the activity of the cross-linked encapsulated immobilized CRL.The activity of immobilized CRL enzyme encapsulated by crosslinker with higher hydrophobicity was higher,indicating that the construction of hydrophobic molecular cage is conducive to enzyme activity.The study also used Autodock software to simulate the interaction between crosslinker 1,4-cyclohexane dimethanol diglycidyl ether（CHDMDGE）,diethylene glycol diglycidyl ether（DEGDGE）and CRL enzyme molecules,and the results showed that the highly hydrophobic crosslinker CHDDGE could better maintain the open state of the lid in the enzyme conformation,which made the enzyme more easily combine with the substrate,and thus played a positive role in the enzyme activity.The experiment also explored the optimal conditions for the immobilization of crosslinking encapsulation,and found that polyethylene glycol diglycidyl ether（PEGDGE,Mn 400）is the best crosslinker,when the amount of thiol and epoxy material of 1:1 is the best dosage of crosslinker,p H 7.0 is the best crosslinking p H,under the optimal crosslinking conditions,the loading capacity of the encapsulated immobilized CRL is 21.64 mg/g,and the enzyme activity is 63.55 U/mg,and free the CRL enzyme activity（50.67 U/mg）compared with improved obviously.Thirdly,the experiment further studied the catalytic performance of the cross-linked encapsulated immobilized CRL enzyme,and investigated the optimal temperature,p H,stability（including thermal stability,storage stability and resistance to denaturants）and reusability of the immobilized CRL.The experiment measured that the optimal p H of the cross-linked encapsulated immobilized enzyme was 7.5,and the optimal temperature was 40°C,which was higher than the optimal temperature of the free enzyme（37°C）.Moreover,the cross-linked encapsulated immobilized enzyme has a wider temperature and p H adaptation range than the free enzyme,as well as higher thermal stability,better storage stability and better denaturant tolerance.After repeated use for four times,the relative enzyme activity of the cross-linked encapsulated immobilized enzyme was still about 70%,while that of the physical adsorption CRL was only about 40%,indicating that the cross-linked encapsulated immobilized enzyme had better reusability than that of the physical adsorption enzyme.