Preparation Of MPTES-PVA-PMMA Magnetic Microspheres And Its Surface Cross-linked Encapsulating Immobilized Enzyme

The superiority of immobilized enzymes is well known,and how to obtain more efficient immobilized enzymes is still a topic of great research significance.In order to better retain or even improve the enzyme activity,we use cross-linking and encapsulation to immobilize the enzyme.The purpose is to build a nano-dimensional space for the enzyme,which can better protect the spatial conformation of the enzyme,thereby obtaining a firm and efficient immobilization.Enzyme.First prepare polymethyl methacrylate(PMMA)magnetic microspheres and graft polyvinyl alcohol(PVA)on the magnetic microspheres,then use 3-mercaptopropyltriethoxysilane(MPTES)to modify the mercapto group on the PVA,Finally,the Candida lipase(CRL)was adsorbed and cross-linked to encapsulate the CRL on the surface of the microspheres.This article can be divided into the following two parts:First,ferric oxide magnetic nanoparticles coated with oleic acid were prepared by chemical co-precipitation.Then,using methyl methacrylate(MMA)as a monomer,PMMA magnetic microspheres were prepared by suspension polymerization.Furthermore,the PMMA magnetic microspheres were esterified and acidified to obtain magnetic microspheres rich in carboxyl groups on the surface.The content of carboxyl groups on the surface of the microspheres was 0.51 mmol/g measured by chemical titration.Thereafter,PVA-PMMA magnetic microspheres were prepared by grafting PVA(Mw 10000)on the microspheres.The modification density of hydroxyl groups on the surface of the microspheres was determined by the acetic anhydride method to be 3.8 mmol/g.Furthermore,MPTES modified thiol groups on the surface of PVA-PMMA magnetic microspheres to obtain MPTES-PVA-PMMA magnetic microspheres.The 2-nitrobenzoic acid(DTNB)method measured the thiol content of 5.56 ?mol/g.Then,using MPTES-PVA-PMMA magnetic microspheres as the carrier and polyethylene glycol diglycidyl ether(PEGDGE)as the crosslinking agent,the CRL was fixed on the magnetic microspheres by crosslinking encapsulation under the protection of nitrogen.Fluorescence imaging experiments show that CRL is immobilized on the surface of magnetic microspheres,and desorption experiments show that cross-linking encapsulation method can firmly fix CRL to microspheres.Furthermore,the enzyme load and enzyme specific activity of the immobilized enzyme were measured experimentally.The experimental results meet the design expectations.Compared with the free enzyme specific activity of 50 U/mg,the specific activity of the cross-linked encapsulated immobilized enzyme prepared in this experiment reached 58 U/mg,which not only firmly immobilized the enzyme on the microspheres The surface,and well protected the spatial conformation of the enzyme.Second,combined with the previous experimental results,in order to better explain the reason why the cross-linked encapsulated immobilized enzyme has higher specific activity than free enzyme,we used Autodock molecular simulation software to compare the CRL enzyme molecule with PVA,MPTES-PVA,PEGDGE-MPTES-The interaction between PVA was studied separately.The results show that the grafting molecules,modifiers and cross-linking agents we selected are more suitable,do not produce obvious interaction with the enzyme molecules,and do not damage the active conformation of the enzyme.In the interaction between PEGDGE-MPTES-PVA and the enzyme molecule,its interaction with the CRL cap and the active center played a positive role in the activity of the immobilized enzyme we prepared.