Nanoparticle Purification Of Immobilized Histidine-tagged Glucose Dehydrogenase

Glucose dehydrogenase(GDH),a tetramer short chain dehydrogenase,is widely used in clinical blood glucose detection,biofuel cells and cofactor regeneration.Due to the several drawbacks of free GDH that easy inactivation,difficult to separate from the reaction solution or product,difficult to recover and reuse,and needed to carry out a series of complex purification processes before application.The strategy of one-step purification and immobilization GDH can effectively reduce the cost,improve the work efficiency,enhance the stability of the enzyme,and achieve the rapid separation and reuse.In this thesis,two magnetic nanoparticles contained Ni²⁺were synthesized and used for purification and immobilization of His-tagged recombinant GDH in one step.In 2^sd chapter,the NiFe₂O₄ magnetic nanoparticles were prepared by a one-pot hydrothermal method,and the specific and affinity immobilization and purification of His-tagged GDH on NiFe₂O₄ was investigated.The immobilization conditions were investigated and optimized,and the highest activity recovery and protein bindings were71.39%and 38.50?g mg^-1 support,respectively.The optimum temperature of immobilized enzyme was 45?,5?lower than that of free enzyme(50?).The optimum pH of the immobilized enzyme was 9.0.The thermal stability and pH stability of the immobilized enzyme were 3.86 and 27 times higher than those of the free enzyme,respectively.After 10 cycles,the residue activity of NiFe₂O₄ immobilized GDH on NiFe₂O₄ magnetic nanoparticles retained more than 65%of the initial activity.In chapter 3,Fe₃O₄ nanoparticles were synthesized by co-precipitation method,and then SiO₂ was coated on Fe₃O₄ nucleus to form Fe₃O₄@SiO₂ nanoparticles by St(?)ber method.After that,Fe₃O₄@SiO₂-PDA-Ni²⁺magnetic nanoparticles were obtained by linking Ni²⁺on the surface of nanoparticles with dopamine.The prepared Fe₃O₄@SiO₂-PDA-Ni²⁺magnetic nanoparticles were used to purify and immobilize the His-tag recombinant GDH from the cell lysis in one step.The effect of temperature,immobilization time,pH,buffer concentration,and protein-support ratio on the immobilization were investigated,and the highest activity recovery and protein loading were 44.27%and 26.33?g mg^-1 support respectively.The thermal stability and pH stability of the immobilized GDH were tested,and the results showed that the immobilization could effectively improve the stability of the enzyme.The Fe₃O₄@SiO₂-PDA-Ni²⁺magnetic nanoparticles immobilized GDH were used to catalyze the conversion of glucose to gluconolactone.After 10 repeated uses,56.42%of the initial activity of the immobilized enzyme was retained.In chapter 4,the application of two magnetic nanomaterials containing Ni²⁺for purification of His-tagged recombinant GDH was studied and compared with three commercial Ni-NTA purified resin products from different manufacturers.The experimental results showed that the purity of His-tagged GDH purified by three commercial resin products was low,indicating that their specificity to His-tagged protein was poor,while the NiFe₂O₄ and Fe₃O₄@SiO₂-PDA-Ni²⁺magnetic nanoparticles prepared in this work showed high affinity and specificity to His-tagged GDH.The results showed that the SDS-PAGE bands of three commercial resin materials all contained a variety of miscellaneous proteins when purifying His-tagged GDH,However,when NiFe₂O₄ and Fe₃O₄@SiO₂-PDA-Ni²⁺magnetic nanomaterials prepared in this thesis were used to purify the His-tagged GDH.The results of SDS-PAGE indicated that single band was obtained,and GDH could not be eluted with the solution with a lower imidazole concentration range(10?200 m M).This phenomenon indicated that the two magnetic nanomaterials showed higher affinity and specificity to His-tagged GDH than three commercial Ni-NTA resin products.The binding force between the nanomaterials and His-tagged GDH was stronger than commercial products used in this work.Therefore,the study that one-step purification and immobilization of His-tagged recombinant GDH by magnetic nanomaterials in this thesis is helpful to promote the further application of GDH,and it also has important reference value for the purification and immobilization of other His-tagged recombinant proteins.