Using Magnetic Nanoparticles To Covalently Immobilize Recombinant Escherichia Coli

Escherichia coli is a typical gram-negative bacterium.As an important biocatalyst,it has been widely used in biocatalysis,drug production,clinical diagnosis,environment protection and so on.However,free E.coli cells are unstable in the reaction solution.When cells are used for catalysis,it is difficult to repeat using them and separate them from subsequent products.Compared with the free cells,the immobilized cells are reusable,which could simplify the separation and purification process and reduce the cost of reaction.Immobilization has been proved to enhance the properties of cells.In the present work,covalent immobilization of recombinant E.coli was performed by the click chemical reaction of azido-alkynyl group on the surface of magnetic nanocarrier with alkynyl group.Fe₃O₄ magnetic nanoparticles were prepared by co-precipitation method.Then the nanoparticles were coated using tetraethyl silicate to obtain Fe₃O₄@SiO₂ magnetic nanoparticles.And the surface amine group modification of Fe₃O₄@SiO₂ magnetic nanoparticles was performed using silylation reagent APTES.Finally,the reaction of amine and carboxyl groups was used to transfer Fe₃O₄@SiO₂-NH₂ magnetic nanoparticlestoalkynylfunctionalizedmagneticnanoparticles Fe₃O₄@SiO₂-NH₂-alkyne.FTIR,TEM,SEM,XRD and DSC results indicated that the alkynyl modified magnetic nanoparticles Fe₃O₄@SiO₂-NH₂-alkyne were successfully prepared.D-arabinose was treated with p-toluenesulfonyl chloride to protect hydroxyl group,then acetic anhydride was added to activate its C-5.After activation,NaN₃ was added to obtain 5-azido-1,2,3-tri-O-acetyl-D-arabinofuranose,fowlling treated with sodium methoxide to obtain 5-azido-5-deoxy-D-arabinofuranose.KDO-N₃ was synthesized by condensation of 5-azido-5-deoxy-D-arabinofuranose with sodium oxalacetate,and its ammonium salt KDO-N₃·NH₃ was obtained after decarboxylation under weak acid conditions.And the structure of product was analyzed by FTIR and NMR,which indicated that KDO-N₃ was successfully prepared.The prepared KDO-N₃ was inserted into the surface of recombinant E.coli through metabolic integration,and then covalently immobilized cells were obtained by linking the KDO-N₃ and the above-prepared magnetic nanoparticles Fe₃O₄@SiO₂-NH₂-alkyne.Several affecting immobilization factors?cell load,immobilization time,immobilization temperature,pH and concentration of divalent copper ions?were optimized respectively.The optimum conditions for immobilization were obtained as following:cell load 0.67 mg/mg,immobilization temperature 45 ^oC,immobilization time 10 minutes,immobilization pH 6.0,copper ions 20 mM.Under the optimum immobilization conditions,characterization of the immobilized cells and free cells showed that immobilized cells had better pH stabilities.After 10 cycle reuses,the immobilized cells retained more than 50%of their initial activity.Under the optimum conditions,the activity of immobilized cells was 5 times and 2.5 times higher than that of the free and KDO-N₃ modified cells,respectively.The catalytic efficiency of the immobilized cells was also calculated by monitoring the conversion of glycerol to 1,3-dihydroxyacetone.After 12 hours of reaction,the DHA production of immobilized cells was 1.4 times than that of the free cells.In summary,the immobilization of whole cells with click chemistry could be a potential immobilization method for industrial production.