Magnetosomes From Burkholderia Sp.XM01 Possessing Enzyme Mimic Properties And Their Applications

In recent years,magnetic nanoparticles have attracted considerable attention because of their catalytic activity of enzyme mimic and magnetic properties.However,artificial magnetic nanoparticles have the disadvantages of poor dispersibility,low biocompatibility,and high environmental cost in the synthesis process,which limit their development and application to a certain extent.In contrast,the method of synthesizing nanoparticles by microorganisms can effectively reduce or eliminate the potential harm of toxic by-products in the synthesis process,which is generally considered as a new environmentally friendly synthesis strategy.It specifically refers to the use of microorganisms such as fungi,actinomycete,bacteria,etc.to synthesize magnetic nanoparticles at normal temperature and pressure through intracellular or extracellular mineralization process.The most representative of these is that magnetotactic bacteria can synthesize magnetosome independently,which is composed of magnetic crystal and thin membrane encapsulation outside.Aiming at the many shortcomings of artificial magnetic nanoparticles,this thesis carried out research on microbial synthesis of magnetosomes,enzyme-like activity and applications.A strain of magnetotactic bacteria was isolated from muddy water samples.The culture conditions affecting the growth of this bacteria strain and the synthesis of magnetosomes were studied.Magnetosomes were purified from the collected bacterial cells,and their morphological composition.magnetic properties,dispersibility and recyclability in the aqueous phase were investigated.In addition,the enzyme-like avtivity of magnetosomes and the kinetics of enzymatic reactions were studied.Magnetosomes were applied to the analysis detection of cholesterol,the degradation of organic pollutant and the construction of electrochemical sensor.A novel magnetotactic bacteria strain producing magnetosomes was successfully isolated from the bottom sediment of Wuqi Reservoir in Yanshou County.It was named as Burkholderia sp.XM01 by 16S rDNA,physiological and biochemical experiments.There existed two or three magnetic nanoparticles in single cell,which were arranged along the long axis of the cell.The optimal culture condition for the growth of strain XM01 and the proportion of magnetic cells were obtained:pH 6.5,10%oxygen concentration,succinic acid as optimized carbon source at a concentration of 400 mg/L,sodium nitrate as optimized nitrogen source at concentration of 150 mg/L,iron quinate as an effective iron source at a concentration of 25?30 ?mol/L,the culture temperature was set at 30?,and the stationary culture time was 6 days.After the operation of ultrasonic disruption,high-speed centrifugation,repeated washing and magnet adsorption,the morphologically complete magnetosomes were successfully extracted from the strain XM01 cells with a yield of 3.16 mg/Lgrowth medium.Transmission electron microscope(TEM),X-ray diffraction(XRD),and X-ray photoelectron spectroscopy(XPS)characterization results showed that magnetosomes produced by strain XM01 have an average particle size of about 80 nm,and single particle was consisted of internal cubic Fe3O4 crystal and external phospholipid membrane.This is the first report that intact magnetosomes have been purified from Burkholderia sp.XM01 cells.The particle size distribution of magnetosomes in pure water was relatively uniform,and the overall distribution showed a right-skewing tendency.The hysteresis loop measurement result showed a curve with wide waist characteristic,indicating that magnetosomes exhibited ferromagnetic characteristics at room temperature,with a saturation magnetization of 61.2 emu/g,a saturation remanence of 21.7 emu/g,and a magnetic coercive force of 142.6 Oe.Magnetosomes can be stably suspended in pure water,PBS,HEPES buffer.When an external magnetic field was applied to the suspension system,magnetosomes can be almost completely separated from the three aqueous phase systems after 120 s.Magnetosomes produced by strain XM01 possess intrinsic peroxidase-like activity under acidic condition.The optimal conditions for the oxidation of the substrate tetramethylbenzidine(TMB)catalyzed by magnetosomes were pH at 4 and temperature of 40?.The range of pH and temperature tolerance of magnetosomes as peroxidase mimics significantly exceeded that of horseradish peroxidase(HRP).Magnetosomes catalytic kinetics was in accordance with regular Michaelis Menten equation.The dependence of magnetosomes on the substrate H2O2 was one order of magnitude higher than that of HRP,and the affinity to the substrate TMB was greater than that of HRP.The catalytic efficiency of magnetosomes was slightly higher than that of HRP.The peroxidase-like activity was related to the hydroxyl radicals produced by the iron ions on the surface of Fe3O4 nanocrystals.Furthermore,magnetosomes were applied to the determination of cholesterol.The linear range of detection was 2?150 ?mol/L with a detection limit of 0.58?mol/L.The sensitivity of the proposed system for cholesterol determination was higher than that of the similar colorimetric detection systems constructed by some artificial nanoenzymes.This method exhibited excellent selectivity for the determination of cholesterol in human serum.At the end of the reaction,magnetosomes can be recycled multiple times by magnet adsorption.The relative difference between the results was less than 5%after 7 consecutive cycles.The relative difference between the measurement results of proposed system and the automatic serum analyzer for 15 random serum samples was less than 3.18%.This is the first report that magnetosomes are used as efficient heterogeneous Fenton catalyst for the degradation of methyl orange.The optimized condition was obtained:magnetosomes with a concentration of 200?g/mL,H2O2 with a concentration of 1 50 mmol/L,pH 3.5 and a temperature of 60?.The magnetosomes/H2O2 heterogeneous Fenton system can completely degrade methyl orange with a concentration of 120 mg/L after a reaction time of 150 min.The effective concentration of the proposed system for catalyzing the degradation of methyl orange greatly exceeded the concentration that can be degraded by reported similar Fenton systems.The magnetosomes/H2O2 system catalyzed the degradation of methyl orange in accordance with the first-order kinetics equation,with an apparent activation energy of 12.15 kJ/mol.The rapid consumption of H2O2 in the catalytic system was accompanied by a large amount of OH formation.The dominant oxidation source of methyl orange was OH produced by magnetosomes catalyzing the decomposition of H2O2.Although magnetosomes and natural enzyme HRP have the ability to catalyze the degradation of methyl orange under ideal solution conditions,the high concentration of salts and heavy metal ions in the simulated dye wastewater can greatly reduce the degradation rate of methyl orange in the system containing HRP,but that has a slight effect on the catalytic activity of the system containing magnetosomes,which indicated that the application potential of magnetosomes in the treatment of methyl orange dye wastewater.After the magnetosome was reused seven times,the degradation rate of methyl orange in the system remained about 81%.This is the first report that magnetosomes possess intrinsic catalase-like activity under alkaline condition.The catalytic activity of magnetosomes was linear with the concentration of nano enzyme,with the optimum pH of 9 and temperature of 60?.The range of pH and temperature tolerance of magnetosomes as catalase mimics significantly exceeded that of catalase.After magnetosomes and catalase were treated with NaN3(100?mol/L)and SDS(5 mmol/L)respectively,the catalytic activity of catalase has been basically lost,while the activity of magnetosomes remained above 90%.The activation energy of magnetosomes catalyzing the decomposition of H2O2 was basically close to that of catalase.The catalytic kinetics of magnetosomes conformed to the Michaelis Menten equation.The affinity and catalytic efficiency of magnetosomes for H2O2 substrate were superior to catalase and artificial Co3O4 nanoenzyme.Based on the ability to electrochemically reduce H2O2,magnetosomes were applied to the construction of electrochemical sensor to detect H2O2.The linear range of detection was 0.02?2 mmol/L,and the detection limit was 0.14 ?mol/L.The method showed excellent selectivity and stability for the determination of H2O2.Compared with some reported enzymatic and non-enzymatic sensors,magnetosomes/GCE sensor possessed higher sensitivity and shorter response time,which can effectively determine trace amounts of H2O2.