Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli

The application of nanoscale materials and structures, usually ranging from 1 to 100 nanometers (nm), is an emerging area of nanoscience and nanotechnology. Nanomaterials may provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, and water-treatment. The development of techniques for the controlled synthesis of nanoparticles of well-defined size, shape and composition, to be used in the biomedical field and areas such as optics and electronics, has become a big challenge. Development of reliable and eco-friendly processes for synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. One of the options to achieve this objective is to use ‘natural factories’ such as biological systems. This study reports the optimal conditions for maximum synthesis of silver nanoparticles (AgNPs) through reduction of Ag⁺ ions by the culture supernatant of Escherichia coli. The synthesized silver nanoparticles were purified by using sucrose density gradient centrifugation. The purified sample was further characterized by UV–vis spectra, fluorescence spectroscopy and TEM. The purified solution yielded the maximum absorbance peak at 420 nm and the TEM characterization showed a uniform distribution of nanoparticles, with an average size of 50 nm. X-ray diffraction (XRD) spectrum of the silver nanoparticles exhibited 2θ values corresponding to the silver nanocrystal. The size-distribution of nanoparticles was determined using a particle-size analyzer and the average particle size was found to be 50 nm. This study also demonstrates that particle size could be controlled by varying the parameters such as temperature, pH and concentration of AgNO₃.     

Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichia coli

Introduction Nerve growth factor(NGF) was first discovered and purified by Rita Levi-Montalcini and Stanley Cohen in the 1950s[1,2]. It represents the first cellular growth factor ever discovered and involved in the growth, survival, and differentiation of specific nerve cell popula-tions[3].     

On the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer–dimer equilibrium where a Mg²⁺-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg²⁺) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 °C, 5 mM pyruvate (with 2 mM Mg²⁺) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg²⁺ is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from >10⁸ to <5 × 10⁵ or 3 × 10⁷ M⁻¹, respectively. With 2 mM Mg²⁺ at 15–25 °C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K_A′10⁶ M⁻¹ (ΔG′=−33.7±0.2 kJ mol⁻¹ and ΔH=+16.3 kJ mol⁻¹ at 20 °C with ΔC_p=−1.4 kJ K⁻¹ mol⁻¹). The binding of PEP to EI(H189A) is synergistic with that of Mg²⁺. Thus, physiological concentrations of PEP and Mg²⁺ increase, whereas pyruvate and Mg²⁺ decrease the amount of dimeric, active, dephospho-enzyme I.     

Influence of the medium electrolyte concentration on the electric polarizability of bacteria Escherichia coli in presence of ethanol

The electric polarizability is an important parameter of bacteria, giving information about the electric properties of the cells. In our previous works [A.M. Zhivkov, A.Y. Gyurova, Colloids Surf. B: Biointerfaces 66 (2008) 201; A.Y. Gyurova, A.M. Zhivkov, Biophys. Chem., 139 (2009) 8; A.M. Zhivkov, A.Y. Gyurova, J. Phys. Chem. B, 113 (2009) 8375] we have applied an experimental approach to distinguish the contribution of the components of the two types of interface electric polarizability—surface charge dependent (ChD) and Maxwell–Wagner (MW) polarizability. It is based on electro-optical study of the separate influence of the outer and inner medium electrolyte concentration, which changes the external ChD and internal MW components of polarizability; the last effect is reached by the membrane permeability increase in low ethanol concentration. In the present work we investigate the behavior of electric polarizability of Escherichia coli K12 at increasing the outer KCl concentration in presence of 10 vol.% ethanol in order to check if the polarizability components change independently from one another. The conclusion is that the outer electrolyte concentration influence indirectly the internal MW component by the trans-membrane concentration gradient, but the polarizability components themselves change independently.     

Functional Expression of phaCAB Genes from Cupriavidus taiwanensis Strain 184 in Escherichia coli for Polyhydroxybutyrate Production

Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66∼70% of total cell material of these recombinant strains.     

Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains. Received: 28 December 1998 / Revised: 22 April 1999 / Accepted: 24 April 1999     

Adhesion of Escherichia coli onto quartz, hematite and corundum: Extended DLVO theory and flotation behavior

The adhesion of Escherichia coli onto quartz, hematite and corundum was experimentally investigated. A strain of E. coli was used that had the genes for expressing protein for silica precipitation. The maximum cell adhesion was observed at pH <4.3 for quartz and at pH 4.5–8.5 for corundum. For hematite, cell adhesion remained low at all pH values. The microbe–mineral adhesion was assessed by the extended DLVO theory approach. The essential parameters for calculation of microbe–mineral interaction energy (Hamaker constants and acid–base components) were experimentally determined. The extended DLVO approach could be used to explain the results of the adhesion experiments. The effect of E. coli on the floatability of three oxide minerals was determined and the results showed that E. coli can act as a selective collector for quartz at acidic pH values, with 90% of the quartz floated at 1.5 × 10⁹ cells/ml. However, only 9% hematite and 30% corundum could be floated under similar conditions. By using E. coli and no reagents, it was possible to separate quartz from a hematite–quartz mixture with Newton's efficiency of 0.70. Removal of quartz from the corundum mixture was achieved by E. coli with Newton's efficiency of 0.62.     

Purification and characterization of acetate kinase from Clostridium thermocellum

Acetate kinase (EC 2.7.2.1), an enzyme involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridium thermocellum, was purified 144-fold. The enzyme has an Mr of 84 kD by non-denaturing gradient gel electrophoresis, and an Mr of 46 kD when estimated with a denaturing gel; thus it appears to be a homodimer. Optimum enzyme activity occurs at 50°C and between pH 7.2 and 8.0. Acetate kinase is stable to temperatures up to 60°C, but is completely inactivated at 80°C after two h. The enzyme is stable between pH 7.0 and 9.0 when incubated at 50°C for two h. Optimum acetate kinase activity occurs at a MgCl₂:ATP ratio of 2:1, which indicates an interaction between Mg²⁺ and ATP and that between Mg²⁺ and acetate kinase. Enzyme activity is partially inhibited by KCl, an inorganic salt frequently used in chromatography and fermentation media, losing 60% activity in the presence of 0.2 M KCl. Sigmoidal enzyme kinetics were observed from the velocity plot of acetate kinase when either the acetate (S_0.5 = 285 mM) or ATP (S_0.5 = 11 mM) concentration was varied, suggesting cooperative binding of the two substrates.     

Phototransformation of monovinyl and divinyl protochlorophyllide by NADPH:Protochlorophyllide oxidoreductase of barley expressed in Escherichia coli

The chlorophyll precursors monovinyl protochlorophyllide (MV-PChlide) and divinyl protochlorophyllide (DV-PChlide) were extracted from mutant C-2A' of the unicellular green alga Scenedesmus obliquus which accumulates both protochlorophyllide derivatives in the dark. The two pigments were characterized by absorption and fluorescence spectroscopy and by plasma desorption mass spectrometry. The molecular masses of MV-PChlide and DV-PChlide were determined as 612 and 610 atomic mass units (amu) respectively. Both MV-PChlide and DV-PChlide were accepted as substrates and photoconverted to chlorophyllides in vitro by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.     

Reduction of Escherichia coli 0157:H7 in shredded iceberg lettuce by chlorination and gamma irradiation

Lettuce was inoculated with a six-strain cocktail of acid-adapted Escherichia coli 0157:H7 at a level of 1×10⁷ CFU/g. Following chlorination at 200 μg/ml, the lettuce was irradiated at 0.15, 0.38, or 0.55 kGy using a ⁶⁰Co source. Survival of E. coli 0157:H7, aerobic mesophiles and yeast and molds were measured over a period of 10 days. For quality analysis, chlorinated lettuce was subjected to irradiation at 0.33 and 0.53 kGy and stored at 1.0°C, 4.0°C or 7.0°C. Changes in texture and color were determined by instrumental means and changes in flavor, odor, and visual quality were determined by sensory testing.

Chlorination plus irradiation at 0.55 kGy produced a 5.4−log reduction in E. coli 0157:H7 levels. Chlorination alone reduced the E. coli 0157:H7 counts by 1–2 logs. Irradiation at 0.55 kGy was also effective in reducing standard plate counts and yeast and mold counts. Irradiation at this level did not cause softening of lettuce and sensory attributes were not adversely affected. In general, appearance and flavor were affected more by the length of storage than by temperature conditions. The 5+log reduction in E. coli counts and lack of adverse effects on sensory attributes indicate that low-dose irradiation can improve the safety and shelf-life of fresh-cut iceberg lettuce for retail sale or food service.     

The effect of frequency and power density on the ultrasonically-enhanced killing of biofilm-sequestered Escherichia coli

Infection on implanted medical devices is a critical concern because the bacteria are recalcitrant to antibiotic therapy; currently the only way to eliminate the infection is to remove the device. We have found that low-frequency ultrasound renders bacteria more susceptible to antibiotics. The effect of low-intensity ultrasound on the enhancement of antibiotic action against biofilm bacteria was measured by subjecting thick E. coli biofilms for 2 h at 37°C to one of four conditions: (1) incubation in nutrient broth; (2) incubation in nutrient broth with antibiotic; (3) ultrasonication in nutrient broth without antibiotic; and (4) ultrasonication in nutrient broth with antibiotic. Two frequencies (70 and 500 kHz) and several ultrasonic intensities were examined, ranging from 2 to 200 mW/cm². It was determined that low-intensity ultrasound significantly enhanced killing of biofilm E. coli by gentamicin. This enhancement increased with increasing ultrasonic intensity and decreased with increasing frequency. A mathematical model of ultrasonically-enhanced transport in cylindrical pores and channels shows that gentamicin transport increases with ultrasonic intensity and decreases with increasing frequency. However, the magnitude of increased transport is so small that it is difficult to attribute enhanced killing of bacteria to enhanced antibiotic transport through the pores and channels of the biofilm; therefore, other mechanisms must play a role. The use of low-intensity ultrasound in conjunction with antibiotic treatment may prove to be a viable clinical method of eliminating biofilm infections from the surfaces of implanted medical devices.     

Effects of vacuum UV and UVC radiation on dry Escherichia coli plasmid pUC19 II. Mutational specificity at the lacZ gene

The mutational spectra at the lacZ gene, induced either by vacuum UV at 160 nm or UVC at 254 nm in vacuum-dried preparations of Escherichia coli plasmid pUC19 DNA, have been characterized from 72 E. coli-propagated mutants by DNA sequencing. In plasmids irradiated in vacuum, vacuum UV is five times more mutagenic than UVC. In the UV-induced mutants, base substitutions largely predominate, with GC → AT (G, guanine; C, cytosine; A, adenine; T, thymine) transitions being the most abundant type of base change for vacuum UV (61%) and UVC (47%). Most of the GC → AT transitions appear to occur at dipyrimidine sites, which are located at the non-transcribed DNA strand. Some, but not all, hot spots for GC → AT transitions are identical for vacuum UV and UVC. Frameshifts, resulting from a loss of the thymine residue, are specific for UVC (22%), and were not detected after treatent with vacuum UV. They occur predominantly at thymine runs of the transcribed DNA strand. Only a few deletions were detected following irradiation with vacuum UV (7.5%) and UVC (2%); however, their frequency is not enhanced compared with the spontaneous mutation spectrum. The data confirm the important role of base substitution mutations in UV-induced mutagenesis, which is not only valid for the UVC range, but extends towards the vacuum UV range.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range

Several monoclonal antibodies (mABs) have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli (EC 3.5.1.11), an enzyme without S---S bridges and a pH stability range of 4–9. During the immobilization the fluorescence emission maxima of the mABs were found to change from 336 nm to ca. 350 nm. Only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range. This mAB was immobilized on different spherical supports (Eupergit C 250 L and Sepharose) and one perfusible support (Knauer Quick Disc) and used for analytical and preparative bioaffinity chromatography. Under isocratic conditions the plate height for the perfusible biospecific adsorbent was found to be an order of magnitude lower than for the other supports. The different forms of this proteolytically processed bacterial enzyme could not be separated, however, by the biospecific adsorbents. At the mAB density used in the adsorbents (10–3O μM), less than 30% of the theoretical binding capacity of the immobilized mABs could be used to adsorb the enzyme.     

Zirconia supported undecatungstophosphate: synthesis and characterization of a bifunctional catalyst

The synthesized supported undecatungstophosphate has been proven to be successful for acid catalyzed as well as oxidation reactions, especially, in obtaining 98.5% conversion of styrene and 100% selectivity for benzaldehyde.     

An inorganic phosphate (Pi) sensor triggers ‘turn-on’ fluorescence response by removal of a Cu ion from a Cu-ligand sensor: determination of Pi in biological samples

A fluorescent ligand that displays a high selectivity for Cu²⁺ has been synthesized. On complexation with Cu²⁺, the fluorescence of the ligand is quenched. Inorganic phosphate ions decomplex Cu²⁺ displaying a fluorescence enhancement that can even be seen with naked eyes. The method was successfully used in quantitative determination of inorganic phosphates in serum, urine, and saliva samples.     

Synthesis and characterization of Co/CdSe core/shell nanocomposites: bifunctional magnetic-optical nanocrystals

Nanocomposite materials provide the possibility for multifunctional properties in contrast with their more-limited single-component counterparts. Here, we report the synthesis and characterization of the first all-inorganic core/shell hybrid magnetic-optical nanoparticle, cobalt/cadmium selenide. The core/shell nanocrystals are prepared in a facile one-pot reaction, and their microstructure is analyzed using low- and high-resolution transmission electron microscopy. Using magnetic and optical characterization, we demonstrate bifunctional behavior, whereby the core retains the magnetic properties of the starting Co nanoparticle, and the shell emits similarly to a single-component CdSe nanoparticle. Interestingly, while the coercivity was found to be unchanged by shell formation, the blocking temperature for the composite structure was observed to be substantially lower (Co: >350 K; Co/CdSe: 240 K). In addition, we observed that at low temperatures (20 K) shell CdSe photoluminescence (PL) decay was very rapid (<1 ns). In contrast, nanocrystalline CdSe PL decay is typically much slower at such temperatures (>50 ns). Finally, we propose possible explanations for the unusual magnetic and optical behavior of the core/shell hybrid nanostructures.     

Comparison of the effects of visible femtosecond laser pulses and continuous wave laser radiation of low average intensity on the clonogenicity of Escherichia coli

To evaluate the contribution of local pulsed heating of light-absorbing microregions to biochemical activity, irradiation of Escherichia coli was carried out using femtosecond laser pulses (λ = 620 nm, τ_p=3 × 10⁻¹³ s, f_p = 0.5 Hz, E_p = 1.1 × 10⁻³J cm⁻², I_av = 5.5 × 10⁻⁴ W cm⁻², I_p = 10⁹ W cm⁻²) and continuous wave (CW) laser radiation (λ = 632.8 nm, I = 1.3 W cm⁻²). The irradiation dose required to produce a similar biological effect (a 160%–190% increase in the clonogenic activity of the irradiated cells compared with the non-irradiated controls) is a factor of about 10³ lower for pulsed radiation than for CW radiation (3.3 × 10⁻¹ and 7.8 × 10² J cm⁻² respectively). The minimum size of the microregions transiently heated on irradiation with femtosecond laser pulses is estimated to be about 10 Å, which corresponds to the size of the chromophores of hypothetical primary photoacceptors—respiratory chain components.     

Fusoxysporone - a new type of diterpene from fusarium oxysporum

A novel diterpene, fusoxysporone 1, was isolated from liquid cultures of the fungus Fusarium oxysporum IMB FO 18;2. The skeleton of 1 - related to the viscidane type - is described for the first time and named fusoxysporane. Additionally, fusaric acid and dehydrofusaric acid were identified in extracts of the culture medium.     

Reactions of N - and C -alkenylanilines: VIII. Synthesis of functionalized cycloalka[ b ]indoles from o -(cycloalk-2-en-1-yl)anilines

N-Acyl-2-(cyclohex-2-en-1-yl)anilines react with molecular iodine to give the corresponding N-acyl-1-iodo-1,2,3,4,4a,9a-hexahydrocarbazoles which undergo isomerization into 1-R-2a,3,4,5,5a,10a-hexahydro[1,3]oxazolo[5,4,3-j,k]carbazol-10-ium iodides; no isomerization occurs with N-acetyl-3-iodo1,2,3,3a,4,8b-hexahydrocyclopenta[b]indole. The reaction of N-p-tolylsulfonyl-3,4,4a,9a-tetrahydrocarbazoles with hydrogen peroxide leads to the formation of a single 1,2-epoxy derivative with trans orientation of the nitrogen-and oxygen-containing rings. N-p-Tolylsulfonyl-1,3a,4,8b-tetrahydrocyclopenta[b]indoles give rise to the corresponding 2,3-epoxy derivatives with both trans and cis orientation of the dihydropyrrole and oxirane fragments. The resulting epoxides undergo trans-opening with formation of N-p-tolylsulfonyl-1-hydroxy-2-methoxy-1,2,3,4,4a,9a-hexahydrocarbazoles and N-p-tolylsulfonyl-3-hydroxy-2-methoxy-1,2,3,3a,4,8b-hexahydrocyclopenta[b]indoles on heating in methanol in the presence of KU-2 cation exchanger. Mutual orientation of the oxirane and nitrogen-containing rings in the epoxides derived from cyclopenta[b]-indoles was proved by X-ray analysis. Original Russian Text ? R.R. Gataullin, N.A. Likhacheva, K.Yu. Suponitskii, I.B. Abdrakhmanov, 2007, published in Zhurnal Organicheskoi Khimii, 2007, Vol. 43, No. 9, pp. 1316–1326. For communication VII, see [1].