Saturated Ammonium Sulfate Solution Research Articles

PREPARATION OF A SPECIFIC PEROXIDASE CONJUGATE TO THE CAUSATIVE AGENT OF CAMPYLOBACTERIOSIS

Immunoglobulins are protein compounds in blood plasma - glycoproteins, the main function of which is to protect the body from infections. Immune antibodies are divided into five classes - IgA, IgG, IgM, IgD, IgE. Great importance in diagnostic studies is attached to immunoglobulins IgG, IgA, IgM. During the study, their qualitative and quantitative content is determined. The first determines the presence of infection in the blood, the second determines the level of antibodies in the blood of the organism affected by the disease. For each infection there is a certain level of antibodies M and G in the blood, and some infections are not accompanied by an increase in the level of immunoglobulin. IgG antibodies are large molecules with a mass of about 150 kDa. These are specific antibodies produced by cells of the immune system in response to the attack of pathogens - causative agents of viral, bacterial, fungal and other diseases. G-class immunoglobulins (IgG) are dominant among all other serum immunoglobulins. They ensure the formation of long-term and stable, in some cases lifelong immunity against a number of serious pathologies such as measles, rubella, chickenpox. IgG makes up 80% of all immunoglobulins in the blood serum and up to 20% of its total proteins. Plasmocytes (mature B-lymphocytes) synthesize IgG. G-immunoglobulin (IgG). Immunoglobulins of this isotype have the classic monomeric form and provide the bulk of the antibodies synthesized during the second immune response. Because of its low molecular weight, IgG readily penetrates tissues and is the most studied immunoglobulin with the ability to penetrate the placental barrier. It passes through the placenta from mother to fetus and protects the baby from infection, many different viruses, providing the baby with humoral immunity until the first (until about 4-6 months of age) of its own immunity. Consequently, a newborn baby will have the same antibodies as its mother for the first five to six months of life, and thus will be able to protect itself from all the dangerous pathogens the baby encounters in the mother's life until these antibodies break down and disappear. This set of immunoglobulins is very important for the newborn, who is very susceptible to infections, especially in the respiratory and digestive systems that can activate the complement system. The main purpose of this work is to isolate and purify G-immunoglobulins from rabbit serum immunized with Campylobacteriosis pathogen antigen. As a result of precipitation and purification of rabbit blood serum immunized with Campylobacteriosis antigen-causing agent by saturated ammonium sulfate solution by chromatography method, G-immunoglobulin with a total volume of 30 ml and protein concentration of about 1 mg/ml was isolated. The obtained G-immunoglobulin will be used in the preparation of peroxidase conjugate specific for campylobacteriosis pathogen.

Extraction and purification of polyphenol oxidase from edible mushroom (Agaricus bisporus) and its use in the manufacture of pastries

This study aimed to extract and purify polyphenol oxidase enzyme (PPO) from mushrooms and use it in pastry industry. The enzyme was extracted from edible mushroom using different buffers and purified by several steps. Some Characterizations of the isolated PPO were studied. The activity of PPO was evaluated using spectrophotometric method and catechol as a substrate. Characterization studies indicated that the enzyme showed the highest specific activity 2087 U/mg protein at pH 6.0 using 0.2M phosphate buffer. The ratio of 1:2 (w:v) from mushroom: buffer was the best for obtaining the highest specific activity (2490 U/mg protein). The optimum time of extraction of PPO was 10 mins to obtain high specific activity (2625 U/mg protein). The precipitation of ammonium sulfate and ion-exchange chromatography techniques were performed to purify PPO. The precipitation with saturated solution of ammonium sulfate (70%) was used, the Specific activity was 4332.7 U/mg protein with purification fold of 1.7 and enzymatic yield of 65.5%. Ion exchange chromatography using DEAE cellulose column estimated that the purification times of the enzymatic extract were 6.01, with an enzymatic yield 22.3%. Storage study of the PPO in edible mushroom at 4oC for 3 months indicated that the PPO loss the stability gradually over time, 33% from its original activity retained after 32 days. It can be concluded that edible mushroom is a good source of PPO and the enzyme still active when the mushroom was stored for one month.

S-100 Proteins: Basics and Applications as Biomarkers in Animals with Special Focus on Calgranulins (S100A8, A9, and A12).

S100 proteins are a group of calcium-binding proteins which received this name because of their solubility in a 100% saturated solution of ammonium sulphate. They have a similar molecular mass of 10-12 KDa and share 25-65% similarity in their amino acid sequence. They are expressed in many tissues, and to date 25 different types of S100 proteins have been identified. This review aims to provide updated information about S100 proteins and their use as biomarkers in veterinary science, with special emphasis on the family of calgranulins that includes S100A8 (calgranulin A; myeloid-related protein 8, MRP8), S100A9 (calgranulin B; MRP14), and S100A12 (calgranulin C). The proteins SA100A8 and S100A9 can be linked, forming a heterodimer which is known as calprotectin. Calgranulins are related to the activation of inflammation and the immune system and increase in gastrointestinal diseases, inflammation and sepsis, immunomediated diseases, and obesity and endocrine disorders in different animal species. This review reflects the current knowledge about calgranulins in veterinary science, which should increase in the future to clarify their role in different diseases and potential as biomarkers and therapeutic targets, as well as the practical use of their measurement in non-invasive samples such as saliva or feces.

Isolation of Mycosporine-like Amino Acids from Red Macroalgae and a Marine Lichen by High-Performance Countercurrent Chromatography: A Strategy to Obtain Biological UV-Filters

Marine organisms have gained considerable biotechnological interest in recent years due to their wide variety of bioactive compounds with potential applications. Mycosporine-like amino acids (MAAs) are UV-absorbing secondary metabolites with antioxidant and photoprotective capacity, mainly found in organisms living under stress conditions (e.g., cyanobacteria, red algae, or lichens). In this work, five MAAs were isolated from two red macroalgae (Pyropia columbina and Gelidium corneum) and one marine lichen (Lichina pygmaea) by high-performance countercurrent chromatography (HPCCC). The selected biphasic solvent system consisted of ethanol, acetonitrile, saturated ammonium sulphate solution, and water (1:1:0.5:1; v:v:v:v). The HPCCC process for P. columbina and G. corneum consisted of eight separation cycles (1 g and 200 mg of extract per cycle, respectively), whereas three cycles were performed for of L. pygmaea (1.2 g extract per cycle). The separation process resulted in fractions enriched with palythine (2.3 mg), asterina-330 (3.3 mg), shinorine (14.8 mg), porphyra-334 (203.5 mg) and mycosporine-serinol (46.6 mg), which were subsequently desalted by using precipitation with methanol and permeation on a Sephadex G-10 column. Target molecules were identified by HPLC, MS, and NMR.

Investigating purification and activity analysis of urease enzyme extracted from jack bean source: A green chemistry approach

Urease is an enzyme of historical importance in the field of biochemistry, generally microbial and plant urease is the primary sources of urease. The significant applications of urease enzyme are found to be foremost in food industry, medical equipment's and biosensors. In this work, urease has been extracted from Jack bean meal using ammonium sulphate and acetone precipitation. A significant amount of urease was precipitated and concentrated at 60% saturated solution of ammonium sulphate. The obtained precipitates were dissolved in 50 mM phosphate buffer (pH 8) after centrifugation, and subjected to sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the molecular weight of urease. Results obtained from the SDS-PAGE were validated using Zymography. Anion exchange chromatography was used to separate the desired protein at different pH (7.0, 7.5 and 8.0). The eluted fractions were assessed for urease activity using phenol-nitroprusside dependent ammonia release assay. Under these assay conditions, one unit of urease activity was calibrated as the amount of enzyme liberating 1 μM of NH3 from urea per unit time. The eluted fraction and Zymography analysis show the purified urease observed at 90 kDa and activity of purified urease, respectively. The obtained results for specific activity (173.67Units mg) and % purification (99.71%) for urease has been compared with the available literature, which are found to be in close relation with existing results. The proposed method is a novel approach which has recorded highest % purification and specific activity. Furthermore, it can be suitable for extracting urease from jack bean source for various industrial applications.

Detection of atomoxetine and its metabolites in the urine by thin-layer chromatography and mass spectrometry

The aim of the study was the detection of atomoxetine and its biotransformation products in the urine under TLC screening conditions and identification of the metabolites using mass spectrometry method. Materials and methods. The volunteer’s urine samples after taking a single therapeutic dose of atomoxetine (2 capsules of 60 mg each of Strattera®) were studied. Sample preparation included diluting acid hydrolysis followed by the native compound and metabolites extraction with chloroform from the saturated solution of ammonium sulfate at pH of 11–12. Thin-layer chromatography studies of the extracts were carried out in 18 mobile phases including those proposed by The International Association of Forensic Toxicologists for general drug screening, and those widely used in forensic toxicological studies. The color reactions were carried out using a range of chromogenic reagents. A Varian 1200 L mass spectrometer (Netherlands) equipped with a dual quadrupole mass analyzer was applied for analysis of the eluates from chromatograms. Identification was undertaken at the direct introduction of the sample into the ion chamber, electron-impact ionization (70 eV), and full ion scanning mode. Results. The spot of the native drug on the chromatogram was identified by the Rf, value. Two atomoxetine biotransformation products were identified by the molecular weights that correspond to the molecular ion peaks in the mass spectra. Conclusions. Atomoxetine and its biotransformation products were detected in the urine under TLC screening conditions and identified using mass spectrometry method. Chromatographic mobility of the native compound, hydroxyatomoxetine, and dihydroxyatomoxetine in the TLC screening systems as well as the results of their visualization using chromogenic reagents applied for toxicological drug screening in the systematic toxicological analysis have been determined.

Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum

Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography - tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4–6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3–200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.

Development of enzyme immunoassay for detecting I and II types of shiga-like toxins

Introduction. The aim of the work was development of enzyme immunoassay for detecting I and II types of shiga-like toxins and assessment of it diagnostic properties. Materials and methods. For the research, we used hybridomas producing monoclonal antibodies to shiga-like toxins of types I and II, obtained at the branch of the Federal State Budgetary Institution “48 Central Research Institute” of the Ministry of Defense of Russian Federation (Kirov); BALB/c mice; shiga-like toxins of types I and II. Hybridoma cells were cultured in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids by precipitation with a saturated solution of ammonium sulfate, followed by purification by ion exchange chromatography. The obtained preparations of monoclonal antibodies were used to develop enzyme immunoassay for the detection of shiga-like toxins of types I and II. Specific components of enzyme immunoassay were freeze-dried in a protective environment. Results. As a result of research, preparative quantities of monoclonal antibodies against I and II types of shiga-like toxins were obtained and purified; selection of monoclonal antibodies for sorption on the solid phase and for the synthesis of immunoperoxidase conjugates was carried out. Conclusion. experimental enzyme immunoassay allowing to identify 1 ng/ml I and II types of shiga-like toxins in «sandwich»-ELISA was developed.

Determination of fluoroquinolones illegally added in traditional prostate medicines by ultrasonic-assisted dispersive liquid liquid micro-extraction based on deep eutectic solvent combined with quantitative 19F nuclear magnetic resonance method

We optimized the combination of deep eutectic solvent (DES)-based ultrasonic-assisted dispersive liquid-liquid microextraction method and 19F qNMR for quantitation of 5 kinds of fluoroquinolone antibiotics illegally added to drugs for prostatitis treatments (Ciprofloxacin, Levofloxacin, Sparfloxacin, Gatifloxacin, Enrofloxacin). In this study, methyltrioctylammonium bromide and n-capric acid were mixed at a 2:1 M ratio to prepare hydrophobic DES. The newly prepared DES was used as the extract. Fluoroquinolone antibiotics were extracted from saturated ammonium sulfate solution and detected by 19F qNMR. In this study, the effects of extraction conditions such as DES volume, salt dosage, ultrasonic time, centrifugal time, and vortex time on extraction efficiency were systematically investigated. Under the optimal conditions, good linearity (correlation coefficient r: 0.9993–0.9998) was obtained with a detection limit of 0.1 μg mL−1. The extraction reproducibility (RSD) of intra-day and inter-day were below 5.01 and 5.98%, respectively. This study showed feasibility of detection of fluoroquinolone antibiotics illegally added in traditional medicines for prostatitis treatments by DES-based ultrasonic-assisted dispersion-liquid microextraction combined with 19F qNMR method.

Features of isothermal crystallization of ammonium sulphate

Soil fertility is rapidly decreasing due to irrational land use, erosion and soil pollution and adverse weather conditions. The use of composite granular organic-mineral fertilizers that contain mineral and organic components allows the soil fertility to be increased in the most effective and safe manner for the environment. In the production of granular composite fertilizers in a fluidized bed granulator, the process of isothermal mass crystallization is the main parameter that affects the quality.
 The effect of impurities on the morphological structure of ammonium sulfate microcrystals during mass crystallization on the surface was investigated by means of microscopic analysis. The influence of temperature and pH of the solution on the structure and shape of the microcrystal layer was studied.
 The process of mass crystallization depends on the presence of impurities, pH and temperature. Given the fact that there is no analytical or theoretical dependence describing the influence of impurities on the morphological structure, the influence is determined experimentally. The presence of even a small content of surfactants and other impurities in the ammonium sulfate solution has a significant effect on the morphological structure of ammonium sulfate microcrystals, which is revealed as reduction in the concentration of impurities in ammonium sulfate obtained by coke-chemical method. The pH level of the medium affects the structure and shape of microcrystals: smaller crystals with a layered structure are formed in the case of weakly alkaline medium with pH = 8 and smaller microcrystals with a granular structure are formed in the case of pH = 4. Therefore, it is important to maintain the required pH level of the medium during the mass crystallization of ammonium sulfate.
 In isothermal mass crystallization of saturated aqueous solutions of ammonium sulfate with humic compounds and bone meal proceeding on a surface with temperature 90 °C, small elongated crystals with microcrystal sizes from 10 to 100 μm are formed. Impurities of bone meal and humic compounds in the form of inclusions are placed between the crystals of ammonium sulfate: namely, the endosegregation of impurities in the microcrystalline framework is observed. As the concentration of organic matter in the solution increases, which crystallizes as droplets on the surface, rubble and dendrites are formed outside the initial droplet placement. This phenomenon is explained by the increase of diffusion resistance at the center of the drop; as a result, ammonium sulfate crystallizes in the zone with lower resistance.
 The process of isothermal mass crystallization of saturated aqueous solutions of ammonium sulfate with admixtures of humic compounds, bone meal and other target components in the specified ratios will create new highly effective fertilizers. The obtained results allow formulating theoretical bases for the formation of solid structures based on ammonium sulfate with admixtures of organic and mineral components.

Разработка и валидация методики изолирования ставудина из биологических жидкостей

Objective - study of isolating Stavudine from biological fluids (urine, blood plasma). Materials and methods. Objects of research: substance and tablets of 30 mg Stavudine. Reagents: purified water, chloroform, ethyl acetate, dichloromethane, ether, heptane, toluene, acetone, 95% ethyl alcohol, 0.1 M hydrochloric acid solution, 10% ammonium hydroxide solution, 5%solutions of sodium sulfate, 20%sodium sulfate saturated, sodium chloride, sodium chloride saturated, 20%ammonium sulfate, ammonium sulfate saturated. Universal ionomer IT-1101. Stavudine was isolated by liquid-liquid extraction. To detect and quantify Stavudine, thin - layer sorbent chromatography (TLC) and UV spectrophotometry were used. Statistical data processing was performed using the software package for Windows XP (Microsoft Excel), and using the Student's t-test. Results. When using thin-layer chromatography to identify Stavudine, it was found that the Rf value of the spots of Stavudine extracts from biological material corresponded to the Rf value range of 0.67-0.69. The Rf spot value of the standard Stavudine witness sample corresponded to (0.68±0.01). The stability study showed that the Stavudine solution is stable in a hydrochloric acid solution of 0.1 M, in which the absorption spectrum of Stavudine is characterized by absorption maxima at wavelengths of 209±1 and 267±1 nm. The wavelength of 267±1 nm was chosen as the analytical wavelength for the quantitative determination of Stavudine. In the course of the study, various factors affecting the degree of extraction of Stavudine from aqueous solutions were studied. The extractant is ethyl acetate, pH 3, the electrolyte is a saturated solution of ammonium sulfate. The extraction time is 7 minutes, twice. Conclusion. Methods of identification and quantitative determination of Stavudine in extracts from urine and blood plasma by TLC and UV spectrophotometry have been developed. Methods of isolation of Stavudine from urine and blood plasma using the liquid-liquid extraction method have been developed (93.33±2.02%; 89.77±2.02% respectively).

Analysis of the Mercury Distribution in Blood as a Potential Tool for Exposure Assessment \u2014 Results from Two Artisanal and Small-Scale Gold Mining Areas in Zimbabwe

People in artisanal and small-scale gold mining (ASGM) areas are frequently exposed to high levels of mercury (Hg). Hg analyses in urine and whole blood are the gold standard of biomonitoring, although this may not provide sufficient information about the source of exposure, e.g., due to the use of Hg for gold extraction or due to nutrition. To evaluate, whether the pharmacokinetic properties of individual Hg species may be useful for exposure assessment, we determined the Hg levels in different blood components from 199 participants. Therefore, whole blood was centrifuged on-site to yield erythrocytes and plasma. Globin was isolated from the erythrocytes by precipitation with ethyl acetate. Albumin was isolated from plasma by gradual precipitation with saturated ammonium sulfate solution. Hg levels in all samples were determined by using a direct Hg analyzer. Median Hg levels for whole blood, erythrocytes, and plasma were 2.7, 3.7, and 1.3 μg/l, respectively. In globin and albumin, median Hg levels were 10.3 and 7.9 μg/kg, respectively. The distribution of Hg was strongly correlated with whole blood Hg levels (p < 0.01) and the time between the last use of Hg and the date of the participation (p < 0.01). The results suggest that the distribution of Hg in blood is substantially affected by the extent and the frequency of the exposure to elemental Hg. Therefore, the analysis of Hg in erythrocytes and plasma may be a valuable tool for Hg exposure assessment in ASGM areas.

Proteinaceous constituents of red cabbage juice increase IL-10, but decrease TNF-α secretions using LPS-stimulated mouse splenocytes

This study investigates the anti-inflammatory effects of proteinaceous constituents from red cabbage (Brassica oleracea L. var.) juice (RC). The RC was first absorbed using activated charcoal (RCA), then partially purified using a Sephadex LH-20 column and finally precipitated using saturated ammonium sulfate solution to obtain the protein-rich fractions. The crude and protein-rich samples from RC were administered to lipopolysaccharide (LPS) - stimulated BALB/c mouse splenocyte cultures for 48 hours. The results showed that the lyophilized RC powder was rich in protein (30.2 ± 0.2%), carbohydrate (33.8 ± 6.0%), phenolics (2.8 ± 0.3%), and flavonoids (1.1 ± 0.1%). After the activated charcoal absorption treatment, the levels of protein (5.2 ± 0.0%), phenolics (0.2 ± 0.0%), and flavonoids (0.0 ± 0.1%) in RCA, except carbohydrate (39.2 ± 2.5%), were significantly decreased. The ammonium sulfate precipitated proteins from red cabbage juice, especially the chromatographic fraction 4 (F4) through the Sephadex LH-20 column, significantly (P < 0.01) increased an anti-inflammatory interleukin (IL-10) from 1,547 ± 106 to 2,060 ± 83 pg/mL, but significantly (P < 0.05) decreased a pro-inflammatory tumor necrosis factor (TNF-α) secretions using LPS-stimulated mouse splenocytes from 1,137 ± 71 to 789 ± 167 pg/mL. This study suggests that the proteinaceous constituents of red cabbage juice show an anti-inflammatory potential via increasing IL-10, but decreasing TNF-α secretions using LPS-stimulated mouse splenocytes.

Cross-linked α-galactosidase aggregates: optimization, characterization and application in the hydrolysis of raffinose-type oligosaccharides in soymilk.

Cross-linked enzyme aggregates (CLEAs) of α-galactosidase, partially purified from maize (Zea mays) flour, were prepared. The impact of various parameters on enzyme activity was examined to optimize the immobilization procedure. Biochemical characterization of the free and immobilized enzyme was carried out. Stability (thermal, pH, storage and operational stability) and reusability tests were performed. The potential use of the free enzyme and the CLEAs in hydrolysis processes of raffinose-type oligosaccharides present in soymilk was investigated. α-galactosidase CLEAs were prepared with 47% activity recovery under optimum conditions [1:5 (v/v) enzyme solution:saturated ammonium sulfate solution ratio; 7.5mg protein and 0.1% (v/v) glutaraldehyde, 6h, 4°C, 150 rpm]. α-galactosidase CLEAs exhibited increased stability in comparison to the free enzyme. The CLEAs and the free enzyme showed a maximum activity at 40°C and their optimal pH values were5.5 and 6.0, respectively. Kinetic constants (KM , Vmax and kcat ) were calculated for the free enzyme and the CLEAs in the presence of p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose and raffinose. The effect of various chemicals and sugars on enzyme activity showed that both enzyme forms were significantly inhibited by HgCl2 and galactose. The CLEAs hydrolyzed 85% of raffinose and 96% of stachyose. The α-galactosidase CLEAs, with their satisfactory enzymatic characteristics, have much potential for use in the food and feed industry. © 2019 Society of Chemical Industry.

Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Preparation and Optimisation of Cross-Linked Enzyme Aggregates Using Native Isolate White Rot Fungi Trametes versicolor and Fomes fomentarius for the Decolourisation of Synthetic Dyes.

The key to obtaining an optimum performance of an enzyme is often a question of devising a suitable enzyme and optimisation of conditions for its immobilization. In this study, laccases from the native isolates of white rot fungi Fomes fomentarius and/or Trametes versicolor, obtained from Czech forests, were used. From these, cross-linked enzyme aggregates (CLEA) were prepared and characterised when the experimental conditions were optimized. Based on the optimization steps, saturated ammonium sulphate solution (75 wt.%) was used as the precipitating agent, and different concentrations of glutaraldehyde as a cross-linking agent were investigated. CLEA aggregates formed under the optimal conditions showed higher catalytic efficiency and stabilities (thermal, pH, and storage, against denaturation) as well as high reusability compared to free laccase for both fungal strains. The best concentration of glutaraldehyde seemed to be 50 mM and higher efficiency of cross-linking was observed at a low temperature 4 °C. An insignificant increase in optimum pH for CLEA laccases with respect to free laccases for both fungi was observed. The results show that the optimum temperature for both free laccase and CLEA laccase was 35 °C for T. versicolor and 30 °C for F. fomentarius. The CLEAs retained 80% of their initial activity for Trametes and 74% for Fomes after 70 days of cultivation. Prepared cross-linked enzyme aggregates were also investigated for their decolourisation activity on malachite green, bromothymol blue, and methyl red dyes. Immobilised CLEA laccase from Trametes versicolor showed 95% decolourisation potential and CLEA from Fomes fomentarius demonstrated 90% decolourisation efficiency within 10 h for all dyes used. These results suggest that these CLEAs have promising potential in dye decolourisation.

Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain.

Fatty acid desaturation enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids. ω-3 desaturase has an important role in converting ω-6 fatty acids into ω-3 fatty acids. Although genes for this desaturase have been identified, the enzymatic activity of Δ-17 with or without transmembrane domain, and the function of the Δ-17 desaturase is poorly understood. In the present study, a transgenic microorganism was used to clone the Δ-17 full length (Δ-17FL) and Δ-17 without transmembrane domain (Δ-17NT), the expression efficiency was improved and western blotting was used to detect the protein expression level. The purification of Δ-17 was precipitated using saturated ammonium sulfate solution, dissolved in phosphate buffered saline buffer, and then filtered using a 10 kDa ultrafiltration cube. Gas chromatography analysis was used to measure the effect of Δ-17NT or Δ-17FL expression on Pichia pastoris fatty acid composition. Furthermore, the function of Δ-17NT in HepG2 cells was measured and the mechanism was explored. It was demonstrated that Δ-17NT decreased cell growth and increased apoptosis in hepatocellular carcinoma cell lines in vitro. In conclusion, successful expression of high levels of recombinant Δ-17NT represents a critical step towards the elucidation of the function of membrane fatty acid desaturases.

Purification and Characterization of a Novel Anti-Campylobacter Bacteriocin Produced by Lactobacillus curvatus DN317.

The lactic acid bacteria (LAB) microbiota of Saudi chicken ceca was determined. From 60 samples, 204 isolates of lactic acid bacteria were obtained. Three isolates produced antimicrobial activities against Campylobacter jejuni, Listeria monocytogenes, and Bacillus subtilis. The isolate DN317, which had the highest activity against Campylobacter jejuni ATCC 33560, was identified as Lactobacillus curvatus (GenBank accession numbers: KX353849 and KX353850). Full inhibitory activity was observed after a 2-h incubation with the supernatant at pH values between 4 and 8. Only 16% of the activity was conserved after a treatment at 121°C for 15min. The use of proteinase K, pepsin, chymotrypsin, trypsin, papain, and lysozyme drastically reduced the antimicrobial activity. However, lipase, catalase, and lysozyme had no effect on this activity. The active peptide produced by Lactobacillus curvatus DN317 was purified by precipitation with an 80% saturated ammonium sulfate solution, and two steps of reversed phase HPLC on a C18 column. The molecular weight of this peptide was 4448Da as determined by MALDI-ToF. N-terminal sequence analysis using Edman degradation revealed 47 amino acid residues (UniProt Knowledgebase accession number C0HK82) revealing homology with the amino acid sequences of sakacin P and curvaticin L442. The antimicrobial activity of the bacteriocin, namely curvaticin DN317, was found to be bacteriostatic against Campylobacter jejuni ATCC 33560. The use of microbial antagonism by LAB is one of the best ways to control microorganisms safely in foods. This result constitutes a reasonable advance in the antimicrobial field because of its potential applications in food technology.

Extraction of Peroxidase from Seeds of Some Plants belonging to Gramineae, Fabaceae and Solanaceae Families and Study of Biochemical Characteristics from the Selected Source

This study was conducted in order to extract peroxidase from seeds of 10 plants belonging to 3 families; Fabaceae (Mung bean, Lentils and Peanut), Solanaceae (Chili pepper, Bell pepper, Datura and wild eggplant),Poaceae (Oat, pearl millet and proso millet).the enzyme was qualified and its kinetic constants were assessed. Extract prepared from proso millet seeds using phosphate buffer 0.1M at pH7 contained the highest specific activity 3.35 U. mg.protein-1 as compared with seeds of other plants. In determination of optimum condition of enzyme extraction, three kinds of buffer solutions at two concentrations 0.1 and 0.2M within pH range 4.5-9 were used. Sodium acetate at pH 6 at 0.1M concentration gave the best results for enzyme extraction with specific activity of 8.06 U. mg.protein-1. The ratios of 1:4 recorded the best specific activity 5.98 U. mg.protein-1. Results showed that the optimum period of extraction was 30 min. with specific activity of 7.11 U. mg.protein-1. Precipitation with saturated solution of ammonium sulfate (30-90%) was used. the highest specific activity was noticed in saturation percent 80% and was 11.29 U. mg.protein-1 with purification fold of 1.66 and enzymatic yield of 70%.

Generation and selective isolation of IgG antibodies against Jurkat T-cell membrane proteins

Currently, Graft-versus-Host Disease (GvHD) has been the major barrier to the application of allogeneic bone marrow transplantation for hematopoietic disorders treatment, especially leukemia. GvHD is caused by donor mature T cells’ attack on recipient’s tissues and organs. Recently, T cell depletion using immunomagnetic nanoparticles is one of the most effective methods to prevent GvHD. In this present study, polyclonal antibodies against Jurkat T cell membrane were generated as a component of immunomagnetic particle. Firstly, Jurkat T cell membrane was fractionated by cloud point extraction using Triton X-114. Subsequently, the fractionated membrane was used to subcutaneously immunize rabbits for polyclonal antibodies production with a dose of 50 μg for priming and 25 μg for the next four boostings. Rabbit serum was collected and partially precipitated in 50 percent of saturated ammonium sulfate solution. Next, precipitated polyclonal antibodies were purified by using protein-A affinity chromatography column and the purity was determimed by SDS-PAGE. Afterwards, the purified antibodies were used in immune-fluorescent stainings and the recognition to Jurkat T cells was evaluated via fluorescent microscopic imaging. Finally, the purified antibodies were negatively selected by incubating with TF-1 cell, a hematopoietic stem cell, to eliminate cross-reacting antibodies. The immunocytofluorescent staining results showed that the purified and selected polyclonal antibodies weakly cross-reacted with TF-1 cells and strongly bound to Jurkat T cell