| Multi-enzyme cascade catalysis has an important application value in biomedicine,the use of multi-enzyme co-immobilization technology can improve its reusability,but the traditional co-immobilization method for multi-enzyme fixation randomness,resulting in low cascade efficiency.DNA directed immobilization（DDI）is a new technology that uses DNA self-assembly to realize co-immobilization of multiple enzymes.The principle of DDI is that enzymes are fixed to the surface of the carrier through DNA base complementation pairing,so as to improve the activity of multiple enzymes cascade.In this study,epoxy-modified polyvinyl acetate（PVAC）magnetic microspheres were used as carriers,and glucose oxidase（GOD）and horseradish peroxidase（HRP）were co-fixed to the surface of magnetic microspheres via DNA"star"scaffold by DDI technology.Magnetic GOD/HRP-DNA@PVAC multi-enzyme co-immobilized system was prepared.The DNA"star"scaffold can regulate the distance between two enzymes,so as to optimize the magnetic multi-enzyme co-immobilization system.The main content includes the following parts:Firstly,preparation of epoxide modified functional magnetic microspheres.Magnetic PVAC microspheres were prepared by polymerization with oleic acid coated Fe3O4magnetic particles as the core and vinyl acetate（VAC）as the monomer.The magnetic microspheres were characterized by microscopy,particle size distribution and thermogravimetry.The results showed that the magnetic response and redispersity of the prepared PVAC magnetic microspheres were good.The particle size distribution of PVAC magnetic microspheres was uniform before and after esterification.Then the prepared PVAC magnetic microspheres were modified with epoxide group and characterized by infrared spectroscopy.The content of epoxide group on PVAC magnetic microspheres was 1067μmol/g,which could be used as the carrier of multi-enzyme co-immobilization system.Secondly,the preparation and optimization of magnetic DNA"star"scaffold co-immobilized multiple enzymes.Firstly,epoxy-modified PVAC magnetic microspheres were used as carriers to design a set of DNA single-stranded Y1,Y2 and Y3（original group）with appropriate chain length.Y1@PVAC magnetic microspheres,GOD-Y2 complex and HRP-Y3 complex were prepared by coupling epoxy-modified PVAC magnetic microspheres,GOD and HRP,respectively.They were characterized by ir and polyacrylamide gel electrophoresis.Then,the magnetic GOD/HRP-DNA@PVAC multi-enzyme co-immobilized system was prepared by co-immobilizing GOD-Y2 complex and HRP-Y3 complex onto the surface of Y1@PVAC magnetic microspheres using DDI technology.The magnetic GOD/HRP-DNA@PVAC multi-enzyme co-immobilized system was characterized by infrared,thermogravimetry,fluorescence and fluorescence resonance energy transfer.After that,the solid load and the cascade activity were measured,and compared with the traditional random co-immobilization method（co-adsorption,co-coupling,etc.）,the results showed that the cascade activity had obvious advantages.The secondary structure of the immobilized enzyme was analyzed by infrared spectroscopy,and the results showed that DDI fixation had little effect on the secondary structure of the immobilized enzyme,and could keep its activity well.The experiment further designed two groups of DNA with different chain lengths（extended group and shortened group）to prepare magnetic GOD/HRP-DNA@PVAC multi-enzyme co-immobilized system respectively,and optimized the system by adjusting the DNA chain length.The results showed that the initial group of DNA(three DNA chains were30 bases in length,The magnetic GOD/HRP-DNA@PVAC co-immobilized system with enzyme spacing of 10.08 nm had the highest cascade activity.Thirdly,the performance and kinetic parameters of magnetic DNA"star"scaffold co-immobilized multiple enzymes were investigated.The optimum p H was 5.0,which was lower than that of free enzyme（7.0）,and the optimum temperature was 35℃,which was the same as that of free enzyme.The magnetic GOD/HRP-DNA@PVAC multi-enzyme co-immobilization system has a wider range of temperature and p H adaptation,better acid-base stability,thermal stability,placement stability,reversibility,reusability,and higher catalytic efficiency and kinetic properties.In this study,three single-ended modified DNA single strands were paired to form a"star"scaffold,and the multi-enzyme co-immobilized system was prepared.As a kind of biomaterial,DNA can be used as a scaffold for the preparation of multi-enzyme co-immobilization system,which can not only accurately regulate the position of the enzyme,but also have little impact on the spatial structure of the enzyme,so as to retain its activity to the maximum extent.At the same time,the DNA"star"scaffold has a certain rigidity,which can better maintain the enzyme spacing between double enzymes,and the enzyme spacing can be regulated by adjusting the length of DNA single strand,so as to realize the construction of dynamic multi-enzyme co-immobilization system with adjustable enzyme spacing.Compared with the DNA origami technique with complex structure design reported in the literature to regulate the spacing of enzymes,the DNA"star"shaped scaffold designed in this study only uses three DNA single strands,which is simple in structure and convenient in preparation,and greatly reduces the preparation cost. |
