Using Magnetic Nanomaterials To Specifically Immobilize Recombinant E. Coli

As an important biocatalyst,bacteria have been widely used in drug production,pharmaceutical intermediates,food preparation,sewage,environmental remediation,chemical industry and light industrial products.However,the free cells have poor stability and reusability in the culture medium or the reaction solution which have high requirements for the reaction environment.The immobilized cells can realize the repeated use of cells,and extremely high concentration of bacteria can be obtained in the bioreactor.Based on the specific recognition between protein and sugar,the immobilization of recombinant Escherichia coli on the magnetic nanoparticles was studied in this paper.In the second chapter,a magnetic nanocarrier grafted with concanavalin A was prepared.Concanavalin A?conA?is a protein extracted from the concanavalin,which has specific recognition through mannose components on bacterial cell surfaces.Firstly,magnetic Fe₃O₄ nanoparticles were prepared by co-precipitation method,the oleic acid was coated to obtain Fe₃O₄@OA nanoparticles for preventing the Fe₃O₄nanoparticles from oxidation,and the Fe₃O₄@OA@DP nanoparticles were simply prepared by dipping the Fe₃O₄@OA magnetic nanoparticles in dopamine solution.After modification,a nanocarrier Fe₃O₄@OA@DP with a large number of quinone groups onitssurfacewasobtained.Theimmobilizednanocarrier Fe₃O₄@OA@DP-conA was prepared by covalently bonding between the quinone groups of Fe₃O₄@OA@DP and the amino group of conA.Finally,immobilization of recombinant E.coli harboring glycerol dehydrogenase was achieved by the specific recognition between glycoconjugates and glycoprotein.The effects of various factors on the immobilization including temperature,pH,cell concentration and immobilization time were studied.The highest immobilization yield of 91%was obtained under the conditions:enzyme/carrier 1.28 mg/mg,pH 8.0,immobilization time 2 h,temperature 4 ^oC.After optimization,immobilized cells exhibit higher thermal stability than free cells.After ten cycles,the immobilized cells can still maintain an initial activity of 62%.These results indicate that immobilization cell on Fe₃O₄@OA@DP-conA nanocarrier by specific recognition of glycosyl and glycoproteins is a potential method for the production of stable immobilized cells.In the third chapter,the mannose functionalized magnetic nanocarriers?Fe₃O₄@OA@DP-mannose?were prepared.The Fe₃O₄@OA@DP-mannose nanocarrier was successfully synthesized between the quinone groups of Fe₃O₄@OA@DP nanoparticles and amino groups of mannosamine by using Michael addition and Schiff base formation reaction.As a gram-negative bacterium,E.coli has the FimH protein on the tip of pilus which has a specific binding capacity with mannose,and this interaction is used for immobilization of E.coli.The effects of cell concentration,immobilization time,pH,temperature,and salt concentration on the immobilization were studied.The highest immobilization yields of 83%and 82%of residual activity were obtained under the conditions:cell concentration 13.5 mg/mL,immobilization time 2 h,pH 8.0,temperature 4 ^oC,phosphate buffer concentration 50mM.After incubation at 37 ^oC for 3 h,the thermal stability of immobilized cells was1.2 times higher than that of free cells.After ten cycles,the initial activity of the immobilized cells remained above 50%.Furthermore,the DHA concentration of immobilized cells was 2 times higher than that of free cells after 12 h reaction time by catalyzing the glycerol to 1,3-dihydroxyacetone.The specific binding of sugar and protein provides a novel method for efficient immobilization of whole cells.Characerization of the immobilized whole cells showed that the immobilized cells had better stability and reusability than that of free cells.Moreover,the immobilized cells had better catalytic efficiency in the biocatalytic reaction.The research in this work provides a new idea for further research on the application of immobilization of whole cells.