In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria

In situ cell separation and immobilization of bacterial cells for biodesulfurization were developed by using superparamagnetic Fe₃O₄ nanoparticles (NPs). The Fe₃O₄ NPs were synthesized by coprecipitation followed by modification with ammonium oleate. The surface-modified NPs were monodispersed and the particle size was about 13 nm with 50.8 emu/g saturation magnetization. After adding the magnetic fluids to the culture broth, Rhodococcus erythropolis LSSE8-1 cells were immobilized by adsorption and then separated with an externally magnetic field. The maximum amount of cell mass adsorbed was about 530 g dry cell weight/g particles to LSSE8-1 cells. Analysis showed that the nanoparticles were strongly absorbed to the surface and coated the cells. Compared to free cells, the coated cells not only had the same desulfurizing activity but could also be easily separated from fermentation broth by magnetic force. Based on the adsorption isotherms and Zeta potential analysis, it was believed that oleate-modified Fe₃O₄ NPs adsorbed bacterial cells mainly because of the nano-size effect and hydrophobic interaction.     

Biodesulfurization of dibenzothiophene by microbial cells coated with magnetite nanoparticles

Microbial cells of Pseudomonas delafieldii were coated with magnetic Fe3O4 nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe3O4 nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe3O4 nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe3O4 nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (delta(s)) was 8.39 emu.g(-1). The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe3O4 nanoparticles had greater desulfurizing activity and operational stability.     

Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe₃O₄ nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g⁻¹ saturation magnetization. When the mixture of gellan gel and the Fe₃O₄ nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe₃O₄ nanoparticles was 9 mg ml⁻¹ and the saturation magnetization of magnetically immobilized cells was 11.08 emu g⁻¹. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe₃O₄–chitosan (Fe₃O₄–CS) nanoparticles as support. The magnetic Fe₃O₄–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe₃O₄ nanoparticles made through co-precipitation of Fe²⁺ and Fe³⁺. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe₃O₄–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.     

Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and Its Potential Application in Bisphenol A Degradation

In the present study, Rhus vernicifera laccase (RvLac) was immobilized through covalent methods on the magnetic nanoparticles. Fe₂O₃ and Fe₃O₄ nanoparticles activated by 3-aminopropyltriethoxysilane followed with glutaraldehyde showed maximum immobilization yields and relative activity up to 81.4 and 84.3% at optimum incubation and pH of 18 h and 5.8, respectively. The maximum RvLac loading of 156 mg/g of support was recorded on Fe₂O₃ nanoparticles. A higher optimum pH and temperature of 4.0 and 45 °C were noted for immobilized enzyme compared to values of 3.5 and 40 °C for free form, respectively. Immobilized RvLac exhibited better relative activity profiles at various pH and temperature ranges. The immobilized enzyme showed up to 16-fold improvement in the thermal stability, when incubated at 60 °C, and retained up to 82.9% of residual activity after ten cycles of reuses. Immobilized RvLac exhibited up to 1.9-fold higher bisphenol A degradation efficiency potential over free enzyme. Previous reports have demonstrated the immobilization of RvLac on non-magnetic supports. This study has demonstrated that immobilization of RvLac on magnetic nanoparticles is very efficient especially for achieving high loading, better pH and temperature profiles, and thermal- and solvents-stability, high reusability, and higher degradation of bisphenol A.     

Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.     

Conjugation of α-chymotrypsin on a polymeric hydrophilic nanolayer covering magnetic nanoparticles

Magnetic nanoparticles, covered by a polymeric hydrophilic nanolayer containing reactive amino groups, were obtained via Hoffman degradation of the polyacrylamide-coated Fe₃O₄ nanoparticles synthesized by photochemical in situ polymerization, and then conjugated the model enzyme––α-chymotrypsin (CT) by use of EDC· HCl and NHS at room temperatures. The mechanism of photochemical in situ polymerization was briefly proposed in this paper. Superparamagnetic properties were retained for Fe₃O₄ after enzyme immobilization while slightly reducing the value of saturation magnetization. Crystalline structure of Fe₃O₄ after CT immobilization was consistent with that of the freshly prepared Fe₃O₄ by X-ray diffraction (XRD) analysis. The binding capacity was 69 and 61 mg enzyme/g nanogel determined by thermogravimetric (TG) analysis and by standard BCA protein assay, respectively. Specific activity of the immobilized CT was 0.93 U/(mg min), only 59.3% as that of free CT. Thermal stability of CT was improved after being bound to the amine-functionalized magnetic nanogel.     

Magnetic Clustering Effect during the Association of Biofunctionalized Magnetic Nanoparticles with Biomarkers

We report herein an investigation into dynamic magnetic clustering that occurs during immunoassays as biofunctionalized magnetic nanoparticles (BMNs) become associated with biotargets. We measure the dynamic effective relaxation time τ _eff(t) and use scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to investigate the C-reactive protein (CRP) as it associates with the BMN Fe₃O₄-antiCRP to form the magnetic cluster Fe₃O₄-antiCRP-CRP. The results indicate that τ _eff(t) increases with increasing association time. In addition, the ration Δτ _eff/τ ₀ as a function of CRP concentration follows a characteristic logistic function, which provides a basis for estimating the quantity of biomolecules with a detection sensitivity close to 0.1 ppm. After the association, SEM and TEM images show that CRP and Fe₃O₄-antiCRP conjugate to form Fe₃O₄-antiCRP-CRP clusters hundreds of nanometers in size. The SEM and TEM images provide direct evidence of the formation of magnetic clustering.     

DNA binding and cytotoxicity studies of magnetic nanofluid containing antiviral drug oseltamivir

In this work, the possibility of preparing a nanoparticle with improved treatment properties was investigated. In this regard, synthesis, characterization, in vitro cytotoxicity and DNA binding of Fe₃O₄@oleate/oseltamivir magnetic nanoparticles (MNPs) were investigated. Fe₃O₄ nanoparticles were synthesized via chemical co-precipitation and coated by oleate bilayers. Then, Fe₃O₄@OA MNPs were functionalized with an antiviral drug (oseltamivir), for better biological applications. The MNPs were subsequently characterized by zeta sizer and Zeta potential measurements, Fourier transform infrared (FT-IR) spectroscopy, vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) analyses. The TEM image demonstrated that average sizes of Fe₃O₄@OA/oseltamivir MNPs were about 8?nm. The in vitro cytotoxicity of Fe₃O₄@OA/oseltamivir MNPs was studied against cancer cell lines (MCF-7 and MDA-MB-231) and compared with oseltamivir drug. The results illustrated that Fe₃O₄@OA/oseltamivir magnetic nanoparticles have better antiproliferative effects on the mentioned cell lines as compared with oseltamivir. Also, in vitro DNA binding studies were done by UV–Vis, circular dichroism, and Fluorescence spectroscopy. The results indicated that Fe₃O₄@OA/oseltamivir MNPs bound to DNA via groove binding. Moreover, this magnetic nanofluid has potential for magnetic hyperthermia therapy due to magnetic core of its nanoparticles.

Communicated by Ramaswamy H. Sarma     

In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution

A new and simple method has been proposed to prepare magnetic Fe₃O₄-chitosan (CS) nanoparticles by cross-linking with sodium tripolyphosphate (TPP), precipitation with NaOH and oxidation with O₂ in hydrochloric acid aqueous phase containing CS and Fe(OH)₂, and these magnetic CS nanoparticles were used to immobilize lipase. The effects on the sequence of adding NaOH and TPP, the reaction temperature, and the ratio of CS/Fe(OH)₂ were studied. TEM showed that the diameter of composite nanoparticles was about 80 nm, and that the magnetic Fe₃O₄ nanoparticles with a diameter of 20 nm were evenly dispersed in the CS materials. Magnetic measurement revealed that the saturated magnetisation of the Fe₃O₄-CS nanoparticles could reach 35.54 emu/g. The adsorption capacity of lipase onto nanoparticles could reach 129 mg/g; and the maximal enzyme activity was 20.02 μmol min⁻¹ mg⁻¹ (protein), and activity retention was as high as 55.6% at a certain loading amount.     

Characterization of direct cellulase immobilization with superparamagnetic nanoparticles

Abstract

Methods of cellulase immobilization on magnetic particles via glutaraldehyde binding were studied. The binding was confirmed by transmission electronic microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM). Samples analyzed by TEM and XRD showed that the magnetic particles with or without bound cellulase were all nanosized particles with a mean diameter of 11.5 nm, and the binding process did not cause significant changes in particle size and structure. Analysis by FTIR showed that the binding of cellulase to the magnetic nanoparticles might be via covalent bonding between residual amine groups on Fe₃O₄ nanoparticles and amine groups of the cellulase. The VSM analysis showed that magnetic nanoparticles with or without bound cellulase were all superparamagnetic. The immobilized cellulase had a wider pH and temperature range and improved storage stability compared with the free enzyme. Determination of the Michaelis constants revealed that the immobilized cellulase had a greater affinity for the cellulosic substrate than the free enzyme. The immobilized cellulase showed better performance on hydrolysis of steam-exploded corn stalks than of bleached sulfite bagasse pulp.     

Preparation,Characterization and Application of Magnetic Fe3O4-CS for the Adsorption of Orange I from Aqueous Solutions

Fe₃O₄ (Fe₃O₄-CS) coated with magnetic chitosan was prepared as an adsorbent for the removal of Orange I from aqueous solutions and characterized by FTIR, XRD, SEM, TEM and TGA measurements. The effects of pH, initial concentration and contact time on the adsorption of Orange I from aqueous solutions were investigated. The decoloration rate was higher than 94% in the initial concentration range of 50–150 mg L⁻¹ at pH 2.0. The maximum adsorption amount was 183.2 mg g⁻¹ and was obtained at an initial concentration of 400 mg L⁻¹ at pH 2.0. The adsorption equilibrium was reached in 30 minutes, demonstrating that the obtained adsorbent has the potential for practical application. The equilibrium adsorption isotherm was analyzed by the Freundlich and Langmuir models, and the adsorption kinetics were analyzed by the pseudo-first-order and pseudo-second-order kinetic models. The higher linear correlation coefficients showed that the Langmuir model (R² = 0.9995) and pseudo-second-order model (R² = 0.9561) offered the better fits.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

Desulfurization with <Emphasis Type="Italic">Thialkalivibrio versutus</Emphasis> immobilized on magnetic nanoparticles modified with 3-aminopropyltriethoxysilane

Objective

Thialkalivibrio versutus D301 cells were immobilized on Fe₃O₄ nanoparticles (NPs) synthesized by an improved chemical coprecipitation method and modified with 3-aminopropyltriethoxysilane (APTES), then the immobilized cells were used in sulfur oxidation.

Results

The prepared Fe₃O₄–APTES NPs had a narrow size distribution (10 ± 2 nm) and were superparamagnetic, with a saturation magnetization of 60.69 emu/g. Immobilized cells had a saturation magnetization of 34.95 emu/g and retained superparamagnetism. The optimum conditions for cell immobilization were obtained at pH 9.5 and 1 M Na⁺. The immobilization capacity of Fe₃O₄–APTES NPs was 7.15 g DCW/g-NPs that was 2.3-fold higher than that of Fe₃O₄ NPs. The desulfurization efficiency of the immobilized cells was close to 100%, having the same sulfur oxidation capacity as free cells. Further, the immobilized cells could be reused at least eight times, retaining more than 85% of their desulfurization efficiency.

Conclusion

Immobilization of cells with the modified magnetic NPs efficiently increased cell controllability, have no effect on their desulfurization activity and could be effectively used in large-scale industrial applications.

     

Efficient phenol degradation by laccase immobilized on functional magnetic nanoparticles in fixed bed reactor under high‐gradient magnetic field

Enzymatic degradation of emerging contaminants has gained great interest for the past few years. However, free enzyme often incurs high costs in practice. The immobilized laccase on the polyethylenimine (PEI)‐functionalized magnetic nanoparticles (Fe₃O₄–NH₂–PEI–laccase) was fabricated to efficiently degrade phenolic compounds continuously in a newly fixed bed reactor under a high‐gradient magnetic field. The degradation rate of continuous treatment in the bed after 18 h was 2.38 times as high as that of batch treatment after six successive operations with the same treatment duration. Under the optimal conditions of volume fraction of nickel wires mesh, flow rate of phenol solution, phenol concentration, and Fe₃O₄–NH₂–PEI–laccase amount, the degradation rate of phenol kept over 70.30% in 48 h continuous treatment. The fixed bed reactor filled with Fe₃O₄–NH₂–PEI–laccase provided a promising avenue for the continuous biodegradation of phenolic compounds for industrial wastewater in practice.     

A surfactant-coated lipase immobilized in magnetic nanoparticles for multicycle ethyl isovalerate enzymatic production

Gum arabic coated magnetic Fe₃O₄ nanoparticles (GAMNP) were prepared by chemical co-precipitation method and their surface morphology, particle size and presence of polymer-coating was confirmed by various measurements, including transmission electron microscopy (TEM), X-ray diffraction (XRD), thermo gravimetric analysis (TGA), and Fourier transform infra red (FTIR) analysis. Magnetic particles were employed for their potential application as a support material for lipase immobilization. Glutaraldehyde was used as a coupling agent for efficient binding of lipase onto the magnetic carrier. For this purpose, the surface of a Candida rugosa lipase was initially coated with various surfactants, to stabilize enzyme in its open form, and then immobilized on to the support. This immobilized system was used as a biocatalyst for ethyl isovalerate, a flavor ester, production. The influence of various factors such as type of surfactant, optimum temperature and pH requirement, organic solvent used, amount of surfactant in coating lipase and effect of enzyme loadings on the esterification reaction were systematically studied. Different surfactants were used amongst which non-ionic surfactant performed better, showing about 80% esterification yield in 48 h as compared to cationic/anionic surfactants. Enhanced activity due to interfacial activation was observed for immobilized non-ionic surfactant–lipase complex. The immobilized surfactant coated lipase activity was retained after reusing seven times.     

In vitro spectroscopic investigation of groove binding interaction of Fe3O4@CaAl-LDH@L-Dopa with calf thymus DNA

Abstract

The principal goal of this study is to evaluate the interaction of Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles with calf thymus DNA. The magnetic nanoparticles were previously prepared by a chemical co-precipitation method, and the surface of the Fe₃O₄ nanoparticles was coated with CaAl layered double hydroxides. The antiparkinsonian drug “L-Dopa” was carried by this core–shell nanostructure to achieve the drug delivery system with suitable properties for biological applications. Also, the interaction of Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles with CT-DNA was studied using, UV–Visible spectroscopy, viscosity, circular dichroism (CD), and fluorescence spectroscopy techniques. The results of investigations demonstrated that Fe₃O₄@CaAl-LDH@L-Dopa and Fe₃O₄@CaAl-LDH nanoparticles have interacted via minor groove binding and intercalated to CT-DNA, respectively.     

Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles

Magnetic Fe₃O₄-chitosan nanoparticles are prepared by the coagulation of an aqueous solution of chitosan with Fe₃O₄ nanoparticles. The characterization of Fe₃O₄-chitosan is analyzed by FTIR, FESEM, and SQUID magnetometry. The Fe₃O₄-chitosan nanoparticles are used for the covalent immobilization of lipase from Candida rugosa using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The response surface methodology (RSM) was employed to search the optimal immobilization conditions and understand the significance of the factors affecting the immobilized lipase activity. Based on the ridge max analysis, the optimum immobilization conditions were immobilization time 2.14 h, pH 6.37, and enzyme/support ratio 0.73 (w/w); the highest activity obtained was 20 U/g Fe₃O₄-chitosan. After twenty repeated uses, the immobilized lipase retains over 83% of its original activity. The immobilized lipase shows better operational stability, including wider thermal and pH ranges, and remains stable after 13 days of storage at 25 °C.     

A Magnetic Nanoparticle-Based Multiple-Gene Delivery System for Transfection of Porcine Kidney Cells

Superparamagnetic nanoparticles are promising candidates for gene delivery into mammalian somatic cells and may be useful for reproductive cloning using the somatic cell nuclear transfer technique. However, limited investigations of their potential applications in animal genetics and breeding, particularly multiple-gene delivery by magnetofection, have been performed. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using magnetic Fe₃O₄ nanoparticles as gene carriers. After surface modification by polyethylenimine, the spherical magnetic Fe₃O₄ nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNA_GFP) or red (DNA_DsRed) fluorescent protein. At weight ratios of DNA_GFP or DNA_DsRed to magnetic nanoparticles lower than or equal to 10∶1 or 5∶1, respectively, the DNA molecules were completely bound by the magnetic nanoparticles. Atomic force microscopy analyses confirmed binding of the spherical magnetic nanoparticles to stretched DNA strands up to several hundred nanometers in length. As a result, stable and efficient co-expression of GFP and DsRed in porcine kidney PK-15 cells was achieved by magnetofection. The results presented here demonstrate the potential application of magnetic nanoparticles as an attractive delivery system for animal genetics and breeding studies.