Citrate-phosphate Buffer Research Articles

Overexpression of methyl parathion hydrolase and its application in detoxification of organophosphates

The coding region of mpd gene corresponding to mature methyl parathion hydrolase (MPH) was heterologously overexpressed in Escherichia coli BL21 (DE3) by using pET expression system. The lactose-induced expression yield of MPH is increased 2-fold compared with IPTG as inducer. Furthermore, it was found that specific activity of MPH increased 48% by reducing the induction temperature to 22 degrees C. The addition of 25 mM lactose at 22 degrees C, the MPH activity of fermentation broth had a specific activity of 1.4 x 10(4) U/mg protein. Plasmid was no significant decrease in the modified medium. The optimal pH and temperature of MPH were 8.0 and 30 degrees C, respectively. Over a period of 5 months, the dried cells showed no significant decrease in the activity of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade about 98% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.

Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives.

The effects of different heating systems on the heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were investigated. Spores were heated in distilled water, Sorensen buffer (0.18 mol 1-1), McIlvaine buffer (0.0025-0.18 mol 1-1), and several solutions containing sodium chloride (0.06-12%), sodium nitrite (125 ppm), potassium sorbate (0.1%) and sodium benzoate (0.1%) over a wide range of temperatures (115-140 degrees C). D-values obtained for McIlvaine and Sorensen buffers, at the same molarities, were not significantly different (P > 0.05), but decimal reduction times increased as phosphate concentrations in the solutions decreased. The concentrations, in which statistically significant differences (P < 0.05) were obtained, varied among strains. Among the additives assayed, only sodium chloride reduced heat resistance, being effective at concentrations as low as 0.06%. The z-values calculated in this study ranged from 6.99 to 8.40 with a mean value of 7.60 +/- 0.45. Although z-values observed for salt and buffers (180 mol 1-1) were slightly higher than obtained in the other conditions assayed, the difference was not statistically significant (P > 0.05).

Optimized extraction of a lectin from Crataeva tapia bark using AOT in isooctane reversed micelles

Crataeva tapia bark lectin was extracted from a crude extract into a reversed micelle phase of the anionic surfactant AOT in isooctane and back-extracted, to a final aqueous phase by addition of butanol. The effects of pH, ionic strength and surfactant concentration on the protein transfer process from the aqueous to the organic phase were characterized, being the best results obtained after 5 min of contact, under agitation, between the two phases, at pH 5.5 (10 mM citrate-phosphate buffer), 30 mM NaCl, and 5 mM AOT. Recovery to a new aqueous phase was performed with 5 min of contact, under agitation, 10 mM citrate-phosphate buffer at pH 5.5, 500 mM KCl and 5% of butanol. The overall yield obtained for the process was 80% for lectin activity and 56% for protein recovery. The efficiency of the process was confirmed by SDS-PAGE analysis.

Electrode passivation by reaction products of the electrochemical and enzymatic oxidation of p-phenylenediamine

The electrochemical and enzymatic oxidation of p-phenylenediamine (PPD) was studied under various conditions to evaluate its reversibility and stability when used as a laccase redox mediator. In accordance with published results, PPD oxidation during cyclic voltammetry showed that a passivation occurred with cycling in static systems without laccase. Such passivation was observed both in McIlvaine and acetate buffers on glassy carbon and platinum electrodes. Our results suggest that the oxidised form of PPD reacts with other PPD molecules in their reduced state to form a polymer on the surface of the electrode. When laccase is present in solution, PPD is only found in its oxidised state and another behaviour is observed at the electrode, with the appearance of a redox couple with an E’ centered at about −25 mV vs. Ag/AgCl in 0.1 M acetate buffer. These redox waves are attributed to the formation of soluble PPD oligomers. RRDE experiments showed no passivation, meaning that in order to form a film, the reaction products from the first oxidation of PPD must be further oxidised in a slow, homogeneous reaction. Results from these experiments have a significant importance in the design of efficient enzyme-modified electrodes since PPD diffusion in the thick polymer layers used to immobilise enzymes is a much slower process than in free solution, which allows enough time for the oxidation products to further react at the electrode and to polymerise on its surface. Passivation can therefore be observed with modified electrodes, even under hydrodynamic conditions, while similar conditions will provide a reversible system on bare electrodes. An example of this consequence of the reactivity of PPD oxidation products on the mediator's reversibility is given through experiments with a modified glassy carbon RDE functionalised with a thick layer of poly(ethyleneimine) microcapsules. In such case, the cyclic voltammograms showed that the oxidation and reduction processes are broader than at a bare electrode and that higher scan rates or rotation rates must be used to avoid passivation.

A membrane-, mediator-, cofactor-less glucose/oxygen biofuel cell

We report the fabrication and characterisation of a non-compartmentalised, mediator and cofactor free glucose-oxygen biofuel cell based on adsorbed enzymes exhibiting direct bioelectrocatalysis, viz. cellobiose dehydrogenase from Dichomera saubinetii and laccase from Trametes hirsuta as the anodic and cathodic bioelements, respectively, with the following characteristics: an open-circuit voltage of 0.73 V; a maximum power density of 5 microW cm(-2) at 0.5 V of the cell voltage and an estimated half-life of > 38 h in air-saturated 0.1 M citrate-phosphate buffer, pH 4.5 containing 5 mM glucose.

Analysis of Trace Residues of Tetracyclines in Dark-Colored Honeys by High-Performance Liquid Chromatography Using Polymeric Cartridge and Metal Chelate Affinity Chromatography

An efficient clean-up procedure was developed for the trace residue determination of tetracyclines (TCs) in dark-colored honeys. TCs were extracted from samples with McIlvaine buffer (pH 4.0) containing 0.01 mol/L Na(2)EDTA. The extracts were treated with both a polymeric cartridge (GL-Pak PLS-2) and a metal chelate affinity column (MCAC) preloaded with copper(II). TCs were eluted and analyzed by high-performance liquid chromatography (HPLC) using fluorescence detection. The method was evaluated for the determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in buckwheat honey, because its color is the darkest. The mean recoveries of OTC, TC and CTC from spiked samples, at three fortification levels, were >70%, and the relative standard deviations (RSDs) were <10%. Limits of quantitation (LOQs) of OTC, TC, and CTC were estimated to be 0.015 mg/kg, 0.019 mg/kg, and 0.024 mg/kg, respectively.

Purification and Characterization of Alcohol Dehydrogenase from Gluconobacter suboxydans

Purification and characterization of alcohol dehydrogenase (ADH) from Gluconobacter suboxydans was done in order to biotechnological and industrial application. Solubilization of enzyme from bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and Hydroxyapatite was successful in enzyme purification. Enzyme assay reaction mixture contained potassium ferricyanide 0.1 M, McIlvaine buffer 0.1 M (pH 5.5), Triton X-100 10%, ethanol 1 M and enzyme solution. The purified ADH Optimum pH activity was 5.5. The enzyme was in maximum stability in pH 5.8. The substrate specificity of the enzyme was determined using the same enzyme assay method as described above, except that various substrates (100 mM) were used instead of ethanol. The relative activity of the ADH for ethanol was higher than the others. The effects of metal ions and inhibitors on the activity of the enzyme were examined by measuring the activity using the same assay method as described above. Activity of purified enzyme was increased in the presence of Ca(+2) and was decreased in presence the of ethylenediamine tetra acetic acid (EDTA). Because the proper structure and function of the enzyme is related to structural Ca(+2) and EDTA can chelate Ca(+2). An apparent Michaelis constant for ethanol were examined to be 1.7 x 10(-3) M for ethanol as substrate.

Estudo do comportamento eletroquímico da enzima peroxidase na presença de peróxido de hidrogênio e ácido 5-aminossalicílico

O comportamento eletroquímico da enzima peroxidase (HRP) foi estudado utilizando o peróxido de hidrogênio como substrato enzimático e o ácido 5-aminossalicílico (5-ASA) como mediador de elétrons sobre eletrodo de grafite. Diversos parâmetros foram otimizados, tais como, o potencial aplicado à técnica amperométrica fixado em -0,125V, a solução tampão fosfato-citrato 0,1 mol L-1 pH 5,0 como eletrólito suporte e a proporção entre o 5-ASA e H2O2 em 1:7, entre outros. Foi observada a catálise da reação de oxidação do peróxido de hidrogênio na presença da enzima HRP e do mediador 5-ASA. O produto dessa oxidação foi reduzido na superfície do eletrodo, evidenciando um significativo aumento na intensidade da corrente catódica.

Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.

Citric Acid Interferes with Adenosine Triphosphate Determination by Bioluminescence

Adenosine triphosphate (ATP) measurement by bioluminescence is used in microbial adsorption studies for which colony-forming units (CFU) may underestimate true microbial numbers. We observed that citric acid profoundly affected ATP measurement by luciferin-luciferase assay, and we assessed the effect of citric acid on ATP detection in water and buffer systems in the pH range 4.9 to 7.4. Bioluminescence depended on the buffer system, increasing as pH increased in each buffer system and decreasing as the buffer system changed from Na-phosphate to citrate-phosphate. Citrate-phosphate buffer decreased bioluminescence by 82% relative to water at the same pH. Consequences of poor buffer selection are potentially reduced luciferase activity in the bioassay system and underestimation of microbial numbers.

Tilletia indica

<i>Tilletia indica</i>

Formulation and development of floating capsules of celecoxib: in vitro and in vivo evaluation.

The objective of the present study was to develop a hydrodynamically balanced system for celecoxib as single-unit floating capsules. Various grades of low-density polymers were used for formulation of these capsules. The capsules were prepared by physical blending of celecoxib and the polymer in varying ratios. The formulation was optimized on the basis of in vitro buoyancy and in vitro release in citrate phosphate buffer pH 3.0 (with 1% sodium lauryl sulfate). Capsules prepared with polyethylene oxide 60K and Eudragit RL100 gave the best in vitro percentage release and were used as the optimized formulation. By fitting the data into zero-order, first-order, and Higuchi models, we concluded that the release followed zero-order kinetics, as the correlation coefficient (R value) was higher for zero-order release. For gamma scintigraphy studies, celecoxib was radiolabeled with technetium-99m by the stannous reduction method. To achieve the maximum labeling efficiency the process was optimized by studying the reaction at various pH conditions and stannous concentration levels. The radiolabeled complex was added to the optimized capsule, and dissolution studies were performed to ensure that there was no leaching of radioactivity from the capsules. Gamma imaging was performed in rabbits to assess the buoyancy of the optimized formulation. The optimized formulation remained buoyant during 5 hours of gamma scintigraphic studies in rabbits.

Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestis

In this study, thermal inactivation parameters ( D- and z-values) of Alicyclobacillus acidoterrestris spores in McIlvaine buffers at different pH, apple juice and apple nectar produced with and without ascorbic acid addition were determined. The effects of pH, temperature and ascorbic acid concentration on D-values of A. acidoterrestris spores were also investigated using response surface methodology. A second order polynomial equation was used to describe the relationship between pH, temperature, ascorbic acid concentration and the D-values of A. acidoterrestris spores. Temperature was the most important factor on D-values, and its effect was three times higher than those of pH. Although the statistically significant, heat resistance of A. acidoterrestris spores was not so influenced from the ascorbic acid within the concentration studied. D-values in apple juice and apple nectars were higher than those in buffers as heating medium at similar pH. The D-values ranged from 11.1 (90 °C) to 0.7 min (100 °C) in apple juice, 14.1 (90 °C) to 1.0 min (100 °C) in apple nectar produced with ascorbic acid addition, and 14.4 (90 °C) to 1.2 min (100 °C) in apple nectar produced without ascorbic acid addition. However, no significant difference in z-values was observed among spores in the juices and buffers at different pH, and it was between 8.2 and 9.2 °C. The results indicated that the spores of A. acidoterrestris may survive in fruit juices and nectars after pasteurization treatment commonly applied in the food industry.

Effect of processing conditions on the prebiotic activity of commercial prebiotics

The functional prebiotic stability of fructooligosaccharides (FOS) and inulin was determined using a prebiotic activity assay. Prebiotic activity scores were determined based on the change in cell biomass of Lactobacillus paracasei 1195 on the prebiotic relative to that of Escherichia coli under equivalent conditions. Prebiotics were dissolved in citrate–phosphate buffer solutions (10% FOS or 2% inulin), and then exposed to each of three treatments simulating food processing conditions: low pH (pH 3–6), heat at low pH (30 min at 85 °C, pH 4–7), and Maillard reaction conditions (up to 6 h at 85 °C with 1% glycine, pH 7). Prebiotics were considered functionally stable if their score was unchanged after treatment. In general, only heating at low pH caused a significant reduction in prebiotic activity, with one of the FOS products being the least stable. The other conditions caused little change in activity. These results provide a basis for selecting prebiotics for use as functional food ingredients and for predicting the extent to which processing affects prebiotic activity.

Extractive Spectrophotometric Methods for Determination of Zolmitriptan in Tablets

Two simple and sensitive extractive spectrophotometric methods have been developed for determination of zolmitriptan (ZTP) in tablets. These methods are based on the formation of yellow ion-pair complexes between ZTP and tropaeolin OO (TPOO) and bromothymol blue (BTB) in citrate-phosphate buffer of pH 4.0 and 6.0, respectively. The formed complexes were extracted with dichloromethane and measured at 411.5 and 410 nm for TPOO and BTB, respectively. The best conditions of the reactions were studied and optimized. Beer's law was obeyed in the concentration ranges of 2-20 and 1.5-17 microg/mL with molar absorptivities of 1.42 x 10(4) and 1.60 x 10(4) L/mol/cm for the TPOO and BTB methods, respectively. Correlation coefficients were 0.9998 and 0.9999 for TPOO and BTB methods, respectively. Limits of detection of the TPOO and BTB methods were 0.341 and 0.344 microg/mL, respectively, and the limits of quantitation were 1.034 and 1.051 microg/mL, respectively. Sandell's sensitivity and stability constant were also calculated. The proposed methods have been applied successfully for the analysis of the drug in its dosage forms. No interference was observed from excipients present in tablets. Statistical comparison of the results with those obtained by a high-performance liquid chromatography method showed excellent agreement and indicated no significant differences in accuracy and precision.

Effects of different mixtures of fibrolytic enzymes on digestion and fermentation of bahiagrass hay

This study was designed to determine the best combination of ferulic acid esterase (FAE), cellulose (CEL), and xylanase (XYL) for hydrolyzing mature bahiagrass in the absence of rumen fluid, and to evaluate effects of the best multienzyme cocktails on digestibility and fermentation of bahiagrass in the presence of rumen fluid. In Experiment 1, 42 enzyme mixtures of pure CEL, XYL, and FAE were dissolved in a citrate phosphate buffer (pH 6.0) and added to 0.5 g of a 12-week regrowth of bahiagrass ( Paspalum notatum) hay. In each mixture, each enzyme was applied at 0, 0.5, 1, or 2 g/100 g of dry matter (DM) except that FAE was not applied at 2 g/100 g DM. Dry matter (DM) disappearance was measured after the solutions were incubated at 39 °C for 24 h. Each enzyme was applied in triplicate and the experiment was repeated thrice. The highest DM disappearance was with enzyme cocktails containing 2, 2, and 0 (2–2–0) or 2, 2, and 1 (2–2–1) g of XYL, CEL, and FAE, respectively/100 g of forage DM. In Experiment 2, the hay was treated with nothing (Control), a commercial enzyme preparation (Depol 740, D740L) containing all three enzymes, or the 2–2–0 and 2–2–1 enzyme cocktails from Experiment 1. The enzyme mixtures were dissolved in 1 ml of citrate–phosphate buffer (pH 6.0) and sprayed on 0.5 g of bahiagrass hay in triplicate in each of two runs. Samples were fermented in buffered-rumen fluid using a wireless automated gas production system for either 24 or 96 h in duplicate runs, and residues were analyzed for DM and volatile fatty acid concentrations. The kinetics of 96 h fermentation were determined by fitting a non-linear model to the data. For the 24 h fermentations, enzyme application did not increase DM or NDF digestibility but all enzyme mixtures decreased acetate concentration (P<0.001) and increased (P<0.04) propionate and butyrate concentrations. Consequently, all enzyme mixtures decreased (P<0.001) the acetate to propionate ratio. For the 96 h fermentations, all enzyme mixtures decreased the lag phase (P<0.05) but did not impact DM or NDF digestibility, gas pool size or fermentation rate. This study identified certain mixtures of XYL, CEL, and FAE that increased 24-h DM disappearance of bahiagrass in the absence of rumen fluid. Application of these multienzyme cocktails reduced the lag phase and improved efficiency of fermentation of mature bermudagrass in rumen fluid but did not improve extent of DM digestion.

Simultaneous determination of malachite green, gentian violet and their leuco-metabolites in aquatic products by high-performance liquid chromatography–linear ion trap mass spectrometry

A method has been developed for the simultaneous determination of malachite green, gentian violet and their leuco-metabolites in various aquatic products using isotope dilution liquid chromatography–linear ion trap mass spectrometry without post-column oxidation. Sample was extracted with McIlvaine buffer and acetonitrile, followed by partitioning with dichloromethane, purified on basic alumina and OASIS MCX SPE column, and finally analyzed by LC–ESI–MS/MS with the select reaction monitoring (SRM) mode. Decision limits (CC α, α = 0.01) and detection capability (CC β, β = 0.05) of the method were in the range of 0.02–0.09 and 0.04–0.13 μg/kg for MG, GV, LMG and LGV in grass carp, eel, salmon, shrimp and shellfish, respectively, recoveries of MG, GV, LMG and LGV at all fortification levels (0.25–10 μg/kg) were from 80.8% to 115.7%, inter-day relative standard derivations were from 1.9% to 18.4%. This method appeared suitable for the control of MG, GV, LMG and LGV residues in aquatic products.

Mitochondrial Fission and Fusion Mediators, hFis1 and OPA1, Modulate Cellular Senescence

The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated beta-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated beta-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.

Influence of the degree of polymerisation in the ability of catechins to act as anthocyanin copigments

The objective of this study was to evaluate the influence of the degree of polymerisation of flavanols in their ability to act as anthocyanin copigments. With this aim, ethyl-bridged catechin derivatives were produced by reaction between catechin and acetaldehyde. Further fractionation in Toyopearl HW-40 allowed the separation of five fractions of increasing molecular size containing mixtures of compounds in the range from monomers to hexamers as established by LC-MS. The copigmentation assays were carried out in citrate-phosphate buffer solutions in 12% ethanol at pH 3.6, using malvidin 3-O-glucoside as pigment and increasing concentrations of the different catechin fractions to obtain copigment to pigment ratios ranging 0:1–20:1. Copigmentation was assessed from the modifications in the visible spectra of the solutions; chromatic analysis in the CIELAB colour space was used to characterise the copigmentation effect. It was found that all the compounds assayed were able to interact with the anthocyanin and induced changes in the colour of the solutions. The greatest effect was induced by oligomers containing 2–3 elementary catechin units, whereas monomers were the poorest anthocyanin copigments among the compounds checked. An interesting observation was that the formation of the copigmentation complexes was not and immediate process, but it required some time to attain the equilibrium, suggesting that the phenomenon was controlled thermodynamically rather than kinetically.

Ascospore inactivation and germination by high pressure processing is affected by ascospore age

Abstract The effects of age on high pressure resistance of the ascospores of heat resistant moulds Byssochlamys fulva, B. nivea, Neosartorya fischeri and N. spinosa were determined. Ascospores were harvested from cultures grown for 3–15 weeks at 30 °C on malt extract agar. Following filtration and determination of concentration, the ascospores were subjected to high pressure processing (HPP) at 600 MPa for 10 min in 0.1 M citrate phosphate buffer (pH 4 and 6) and mango puree (pH 5). The results supported our hypothesis that age (maturity) affects high pressure resistance of ascospores of heat resistant moulds. A reduction of log10 2.5 cfu mL− 1 was achieved for three week old ascospores of B. nivea whereas for nine week old ascospores only a half log reduction was achieved. Similar results were observed for B. nivea and N. fischeri. The HPP treatment caused activation of ascospores of N. spinosa, with older ascospores showing increased activation. Industrial relevance The observation of activation of some ascospores by HPP, indicates that HPP alone is insufficient for elimination of these problematic spoilage microorganisms. HPP would need to be combined with other hurdles in order to produce high quality pressure-treated shelf-stable fruit products.