Ammonium Sulfate

Following the classical approach in homogeneous nucleation theory, a general but simple expression for the homogeneous freezing rate is derived with account for solution and curvature effects, and applied to the examples of ammonium sulfate and sulfuric acid solutions. After showing that this method compares well with other approaches, a parameterization suitable for use in various cloud models is discussed.

The heterogeneous reaction between gas phase glyoxal and ammonium sulfate (AS) aerosols, a proxy for inorganic atmospheric aerosol, was studied in terms of the dependence of the optical, physical and chemical properties of the product aerosols on initial particle size and ambient RH. The reactions were studied under different relative 5 humidity (RH) conditions, varying from dry conditions (∼20 % RH) and up to 90 % RH, covering conditions prevalent in many atmospheric environments. At λ = 355 nm, the reacted aerosols demonstrate a substantial growth in optical extinction cross section, as well as in mobility diameter under a broad range of RH values (35-90 %). The ratio of the product aerosol to seed aerosol geometric cross section reached up to ∼3.5, 10 and the optical extinction cross-section up to ∼250. The reactions show a trend of increasing physical and optical growth with decreasing seed aerosol size, from 100 nm to 300 nm, as well as with decreasing RH values from 90 % to ∼40 %. Optically inactive aerosols, at the limit of the Mie range (100 nm diameter) become optically active as they grow due to the reaction. AMS analyses of the reaction of 300 nm AS at RH 15 values of 50 %, 75 % and 90 % show that the main products of the reaction are glyoxal oligomers, formed by acetal formation in the presence of AS. In addition, imidazole formation, which is a minor channel, is observed for all reactions, yielding a product which absorbs at λ = 290 nm, with possible implications on the radiative properties of the product aerosols. The ratio of absorbing substances (C-N compounds, including 20 imidazoles) increases with increasing RH value. A core/shell model used for the investigation of the optical properties of the reaction products of AS 300 nm with gas phase glyoxal, shows that the refractive index (RI) of the reaction products are in the range between 1.57-1.71 for the real part and between 0-0.02 for the imaginary part of the RI at 355 nm. The observed increase in the ratio of the investigated absorbing sub-25 stances is slightly indicated in the RI values found by the model, as the imaginary part of the product RI increases from 0.01 to 0.02 with increasing RH. The imaginary part is expected to increase further at higher RH and become more substantial in cloud 19224 ACPD

G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--A simple methodology for hyperimmune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched immunoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaC1 and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.

A keratinase enzyme was isolated and purified from a feather-degrading culture of Aspergillus oryzae. Fractional precipitation of the crude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at 75–85% ammonium sulfate saturation ...

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu 2 + -chelate Sepharose chromatography. Ostrich prothrombin exhibited a M r of 72 800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin b-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

We report the synthesis and characterization as well as cytotoxicity and biocompatibility studies of a poly(magnesium acrylate) hydrogel (PAMgA) developed for drug delivery applications. Two hydrogels with different mesh sizes, large and short, were synthesized (L-C PAMgA and S-C PAMgA). The hydrogels were characterized through swelling, FT-IR and DSC. Cytotoxicity in vitro was evaluated on cell line NIH-3T3 fibroblasts via direct contact and two indirect contact methods (MTT and flow citometry). Both PAMgA hydrogels exhibited low cytotoxicity with survival rates higher than 90%. To select their administration route, biocompatibility was evaluated after intraperitoneal, subcutaneous, and oral administration to mice of both hydrogels at different dose ranges. Swelling percentages obtained were 33.3 ± 4.2% and 166.7 ± 8.3% for L-C PAMgA and S-C PAMgA respectively, showing a great difference in both hydrogels. Among the administration routes assayed, the hydrogels were well tolerated after oral administration of a wide dose range (10-500 mg/kg), thereby indicating that both PAMgA hydrogels are excellent candidates for oral administration due to their in vitro biocompatibility and oral non-toxicity. These results together with the fact that their synthesis is simple and inexpensive make them good candidates for the design of oral drug delivery devices.

The catalytic performance and the formation of carbon in Fe-BEA and Fe-MFI catalysts prepared by three methods (hydrothermal synthesis, ion-exchange at room temperature with an iron-nitrate solution and ion-exchange at 80 • C with an iron ammonium sulfate solution) are analyzed with the aim to develop catalysts having larger dimensions of the cavities with respect to the MFI structure (such as ZSM-5 and silicalite) and which could be applied in the selective hydroxylation of molecules having larger kinetic diameters than benzene. The results show that, apart from minor differences, Fe-BEA catalysts have comparable catalytic performances in selective benzene hydroxylation to phenol with respect to Fe-MFI catalysts. However, all the Fe-BEA samples show a higher formation of carbon than the corresponding Fe-MFI samples as well as the formation of carbon having a higher degree of condensation. This is connected to the larger cavities in the BEA framework, especially at the intersections of the channels, with respect to the MFI framework. Highly active samples were obtained by ion-exchange at 80 • C with the iron ammonium sulfate solution. The phenol productivity in both Fe-MFI and Fe-BEA samples is about 60-70% higher than that of comparable samples prepared by hydrothermal synthesis.

Simple reproducible procedures for purification of the main soluble (S) and ionically bound (IB) cationic peroxidase isoenzymes from turnip roots were established. The procedures included ammonium sulfate precipitation of the isoenzymes, chromatographic separation of the main isoenzymes using cellulose phosphate columns and purification to homogeneity by hydrophobic interaction chromatography on phenyl Sepharose columns. The specific activity of the phenyl Sepharose purified S and IB isoenzymes were 2760 and 896 units/mg protein with 140 and 4.8 fold increase over the crude extract and 38 and 13% recovery. The pH maxima and K,, for phenol and H20: of purified S and IB were determined.

Since the early 1980s, it has been known that high Prostate-specific antigen (PSA) is a widely used serum prostate-specific antigen (PSA) 2 levels are well marker for screening and monitoring prostate cancer. correlated with the presence of prostate cancer (1, 2). Because PSA levels are normally quite low, an anti-By using simple immunometric assays it is now possible body-based assay must be used to detect PSA. However, to quantitate serum PSA levels in less than an hour (3, not all PSA-specific antibodies bind equally well to PSA 4). However, current problems with many commercial or to its different isoforms. Therefore, a better undertests point to the lack of understanding regarding how standing of how PSA interacts with PSA-specific anti-

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227fold. The V max and K m of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose-and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC 50 of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K i of 0.012 ± 0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K i of 0.026 ± 0.015.

Microcalorimetry was applied to assess and compare the toxic effect of heavy metals, such as As, Cu, Cd, Cr, Co, Pb and Zn, on the soil microbial activities and community. About 1.0 g soil spiked 5.0 mg glucose and 5.0 mg ammonium sulfate, the microbial activities were recorded as power-time curves, and their indices, microbial growth rate constant k, total heat evolution Q T , metabolic enthalpy H met and mass specific heat rate J Q/S , were calculated. Comparing these thermodynamic parameters associated with growth yield, a general order of toxicity to the soil was found to be Cr > Pb > As > Co > Zn > Cd > Cu. When soil was exposed to heavy metals, the amount of bacteria and fungi decreased with the incubation time, and the bacterial number diminished sharply. It illustrates that fungi are more tolerant, and bacteria-fungi ratio would be altered under metal stress. To determine the status of the glucose consumed, a glucose biosensor with eggshell membrane was used to measure the remaining glucose in soil sample. Results showed that the time at which glucose was consumed completely was agreed with the microcalorimetric time to a large extent, and depended on the toxicity of heavy metals as well.

Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 µg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 µg pDNA/ mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4°C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (≈100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process.

Two phosphorylases could be detected on gel-electropherograms of leaf-extracts of Spinacia oleracea and of immature cotyledons of Vicia faba. These two phosphorylases could be separated by means of ammonium sulfate fractionation. Both the slower migrating phosphorylases from spinach and from beans, but not the fast one from beans, could be adsorbed on amyloplasts. This process takes place only when the amyloplasts are suspended in a salt medium. The slow phosphorylases can also be adsorbed on chloroplasts. The specific activity of the amyloplast-adsorbable phosphorylase in spinach leaves is about ten times higher in the cytoplasmatic fraction than in chloroplasts, a fact which suggests that this phosphorylase is localised in the cytoplasma. The addition of ADP or ATP to the reaction mixture had no influence on the synthesizing activity of the slow phosphorylases when they were tested with soluble amylopectin as a primer or while they were adsorbed on amyloplasts. The presence of ADPG and UDPG was inhibitory. The results reported above suggest that phosphorylase could catalyse the synthesis of starch in the plastids when photophosphorylation or oxidative phosphorylation occurs. This starch synthesis could be controlled by the concentration of ADPG. When, on the other hand, the ATP/Pi ratio is low, phosphorylase would be involved in starch breakdown. This reverse reaction is also regulated by the concentration of glucosylnucleotides. Durch Umfällung mit Ammoniumsulfat konnten die zwei, auf Gelelektropherogrammen nachweisbaren Phosphorylasen aus Blättern von Spinacia oleracea und aus unreifen Kotyledonen von Vicia faba getrennt werden. Die im elektrischen Feld langsamer wandernde Phosphorylase von Vicia und Spinat kann in Gegenwart salzhaltiger Medien auf Amyloplasten oder auch an Chloroplasten adsorbiert werden und dort den Aufbau von Stärke aus Glucose-1-Phosphat katalysieren. Das schneller wandernde Enzym aus Vicia faba zeigt diese Adsorption jedoch nicht. Die spezifische Aktivität der an Amyloplasten adsorbierbaren Phosphorylase aus Spinatblättern its in der cytoplasmatischen Fraktion etwa zehnfach höher als in den Chloroplasten. Die synthetisierende Aktivität der langsam wandernden Phosphorylasen wird durch ADP oder ATP nicht beeinflußt, dagegen hemmen sowohl ADP-Glucose als auch UDP-Glucose. Es wird die Möglichkeit diskutiert, daß Phosphorylase nicht nur den Abbau von Stärke katalysiert, sondern auch an ihrer Synthese in den Plastiden beteiligt ist, wenn Photophosphorylierung oder oxydative Phosphorylierung stattfindet.

D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research.

Marine microbes are a rich source of bioactive compounds, such as drugs, enzymes, and biosurfactants. To explore the bioactive compounds from our marine natural product library, an oil emulsification assay was applied to discover biosurfactants and bioemulsifiers. A spore-forming bacterial strain from sea mud was found to produce bioemulsifiers with good biosurfactant activity and a broad spectrum of antimicrobial properties. It was identified as Bacillus velezensis H3 using genomic and phenotypic data analysis. This strain was able to produce biosurfactants with an optimum emulsification activity at pH 6.0 and 2% NaCl by using starch as the carbon source and ammonium sulfate as the nitrogen source. The emulsification-guided isolation and purification procedure led to the discovery of the biosurfactant components, which were mainly composed of nC 14 -surfactin and anteisoC 15 -surfactin as determined by NMR and MS spectra. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The critical micelle concentrations (CMCs) of C 14 -surfactin and C 15 -surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06×10 -5 and 2.03×10 -5 mol/L, respectively. The surface tension values at CMCs for C 14 -surfactin and C 15 -surfactin were 25.7 and 27.0 mM/m, respectively. In addition, the H3 biosurfactant exhibited antimicrobial activities against Staphyloccocus aureus, Mycobacterium, Klebsiella peneumoniae, Pseudomonas aeruginosa, and Candida albicans. Thus B. velezensis H3 is an alternative surfactin producer with potential application as an industrial strain for the lipopeptide production.

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

The densities and viscosities of binary and ternary solutions of the aqueous two-phase systems created by the polyethylene glycol 6000 + triammonium citrate + water system were measured at different temperatures [(25, 30, 35, 40, and 45)°C] and correlated with empirical equations. The density data were compared with those available in the literature. The density data show a linear variation with the mass fraction of the polymer for all temperatures. The density and viscosity of the top and bottom phases were also measured and reported.

Crude purified bromelain extracts were obtained from 0.26% protein pineapple juice using sequential batch membrane processing systems which included microfiltration (MF) and ultrafiltration (UF) followed by ammonium sulfate extraction, ultracentrifugation and freeze drying. The membrane treatments (with an 8 km mineral MF and a 10000 molecular weight cut-off (MWCO) organic UF membranes), combined with 60% ammonium sulfate extraction resulted in 0.75-0.8% protein concentration, with 99% protein rejection. A 70% ammonium saturation and ultracentrifugation process (27000 X g at 2-3 'C), prior to freeze drying, were used in the last step to remove the residual non-protein constituents. These processes achieved low-moisture freeze-dried, and light-colored extracts, free of non-protein constituents and which accounted for about 50% yielded extracts containing 98% protein. The extracts assayed for bromelain and proteolytic activity resulted in almost 100% potential recovered, at completion.

In order to determine the optimum conditions for reactivity in the ammonium sulphate precipitation (Farr) assay we have studied the DNA binding properties of two antibodies raised against ultraviolet single stranded DNA (UVssDNA) complexed with methylated bovine serum albumin. In general the buffer composition, pH, temperature, and ionic strength conditions described for binding to undamaged DNA were found to be appropriate for binding to UV-irradiated DNA. However, some differences in detail were noted which indicate the necessity for checking the physical conditions of binding of individual antibodies. Mouse monoclonal antibody and rabbit polyclonal antisera bound to UVssDNA very rapidly, even when DNA and ammonium sulphate were added simultaneously, whereas this procedure prevented binding of rabbit antisera to UVirradiated double stranded DNA. Incubation at 45°C for 30 rain inhibited binding by mouse antibody, and incubation at 37°C for 60 rain caused reversible dissociation of the DNA-antibody complex. The optimised Farr assay was used to define the antigen specificities of the antibodies. The mouse antibody specifically bound to UVssDNA, but not to ssDNA, double stranded (ds) DNA, or UVdsDNA, whereas the rabbit antisera bound to UVssDNA, ssDNA or UVdsDNA, but not dsDNA. The extent of binding of the mouse antibody was dependent on the UV dose to the antigen, as well as the antigen concentration, indicating that the Farr assay can form the basis of a quantitative assay for photoproducts in DNA.

Nitrogen (N) loss from irrigated cropland, particularly sandy soils, significantly contributes to nitrate (NO 3 ) contamination in surface and ground waters and increases N applications to crops. Ammonium-loaded clinoptilolite (A-Cp) may reduce N leaching from sandy soils while sustaining plant productivity. We conducted two greenhouse plant growth experiments with sweet corn (Zea mays L.). Pots containing a sandy soil planted with sweet corn were fertilized with either ammonium sulfate (AS) or one of three size fractions of A-Cp, small (<0.25 mm), medium (0.25 to 2 mm), or large (2 to 4 mm), at rates of 112, 224, or 336 kgN ha" 1 (Experiment #1) and 112 or 224 kg N ha" 1 (Experiment #2). We compared ammonium (NH 4 + ) and NO 3 " leaching from these pots and compared corn relative growth rates (RGR), leaf area ratio (LAR), and net assimilation rate (NAR) among all N fertilizers. Soil amended with AS leached 10 to 73% of the added N, depending on N rate, whereas <5% of the added N leached from the A-Cp amended soil regardless of N rate and Cp particle size. No differences in corn growth were observed among N fertilizers, however, 'Corresponding author.

Titanium dioxide (i.e. TiO 2 ) in nano-form is a constituent of many nanomaterials that are used in sunscreens, cosmetics, industrial products and in biomedical applications. Quantification of TiO 2 nanoparticles in various matrixes is a topic of great interest for researchers studying the potential health and environmental impacts of nanoparticles. However, analysis of TiO 2 as Ti 4+ is difficult because current digestion techniques require use of strong acids that may be a health and safety risk in the laboratory. To overcome this problem, we developed a new method to digest TiO 2 nanoparticles using ammonium persulfate as a fusing reagent. The digestion technique requires short times to completion and optimally requires only 1 g of fusing reagent. The fusion method showed >95% recovery of Ti 4+ from 6 g mL −1 aqueous suspensions prepared from 10 g mL −1 suspension of different forms of TiO 2, including anatase, rutile and mixed nanosized crystals, and amorphous particles. These recoveries were greater than open hotplate digestion with a tri-acid solution and comparable to microwave digestion with a tri-acid solution. Cations and anions commonly found in natural waters showed no significant interferences when added to samples in amounts of 10 ng to 110 mg, which is a much broader range of these ions than expected in environmental samples. Using ICP-MS for analysis, the method detection limit (MDL) was determined to be 0.06 ng mL −1 , and the limit of quantification (LOQ) was 0.20 ng mL −1 . Analysis of samples of untreated and treated wastewater and biosolids collected from wastewater treatment plants yielded concentrations of TiO 2 of 1.8 and 1.6 ng mL −1 for the wastewater samples, respectively, and 317.4 ng mg −1 dry weights for the biosolids. The reactions between persulfate ions and TiO 2 were evaluated using stoichiometric methods and FTIR and XRD analysis. A formula for the fusing reaction is proposed that involves the formation of sulfate radicals.

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of a-lactalbumin, blactoglobulin, and bovine serum albumin from cowÕs milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and b-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which b-lactoglobulin was separated from a-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure b-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of a-lactalbumin, b-lactoglobulin, and bovine serum albumin thus purified.

Abbreviations: β-Gal, β-galactosidase; CE, capillary electrophoresis; GOS, galactooligosaccharides; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; MUGAbstract A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from the crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a 5 specific activity of 97 U oNPG /mg protein. The K m , k cat and k cat /K m values for lactose and oNPG were 38 mM, 20 s -1 , 530 M -1 ·s -1 and 1.67 mM, 540 s -1 , 325,000 M -1 ·s -1 , respectively. The temperature optimum of β-galactosidase activity was 60-65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg 2+ ions enhanced both activity and stability 10 significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D-15 Glc, β-D-Galp-(1→3)-D-Glc, β-D-Galp-(1→6)-D-Gal, β-D-Galp-(1→3)-D-Gal, and the trisaccharides β-D-Galp-(1→3)-D-Lac, β-D-Galp-(1→6)-D-Lac.

The aim of the present work was to investigate the use of cashew apple juice as a low cost substrate for Lactobacillus casei B-442 cultivation and lactic acid production. Ammonium sulfate was employed as the only exogenous nitrogen source. The effect of cashew apple juice reducing sugars and ammonium sulfate concentration and the fermentation pH and temperature on biomass formation, lactic acid production, and productivity were evaluated. The highest productivity (2.36 g/L.h) was obtained applying 50 g/L of reducing sugar from the cashew apple juice supplemented with 6 g/L of ammonium sulfate. The process yield was about 95% when fermentation was carried out at 37 °C with pH controlled at pH 6.5 using NaOH (120 g/L).

Copyright © 2005 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/05/125/0127/$30.00 ... *Author to whom all correspondence and reprint requests should be addressed. ... MUSTAFA ERAT,1 HÜLYA DEMiR,2 AND HALiS S¸AKiROG˘LU*,2

Lipases are the most widely used biocatalysts, because they can catalyze several unnatural and remarkable reactions in non-aqueous media, such as bio-fuel production, production of value-added products such as esters, organic acids, food, beverage, cosmetics and pharmaceutical materials. Solid-state fermentation (SSF) represents an interesting alternative to produce industrial enzymes at lower costs due to the possibility of using inexpensive agro-industrial residues as culture media. This review aims to explore various agriculture byproducts like husk, straw, agricultural raw materials, waste of the oil industry, among others that are locally available and are also costeffective requiring low nutrient supplementation to produce microbial lipase(s) in SSF. Enzyme production is associated with the growth of the bacterial culture. The physico-chemical fermentation parameters such as pH of the medium, moisture content, particle-size, nature of particles and microbial inoculum level play crucial role(s) in lipase production. SSF has gained renewed interest and fresh attention of researchers to develop processes to achieve large-scale enzyme production by solid waste treatment and in its application in the industry to synthesize the products of commercial value.

Assessments of the efficacy of mitigation of greenhouse gas (GHG) emissions from paddy rice systems have typically been analyzed based on field studies. Extrapolation of the mitigation potential of alternative management practices from field studies to a national scale may be enhanced by spatially explicit process models, like the DeNitrification and DeComposition (DNDC) model. Our objective was to analyze the impacts of mitigation alternatives, management of water, fertilizer, and rice straw, on net GHG emissions (carbon dioxide, methane, and nitrous oxide fluxes), yields, and water use. After constructing a GIS database of soil, climate, rice cropping area and systems, and management practices, we ran DNDC with 21-yr alternative management schemes for each of the approximately 2500 counties in China. Results indicate that, despite large-scale adoption of midseason drainage, there is still large potential for additional methane reductions from Chinese rice paddies of 20 to 60% over 2000-2020. However, changes in management for reducing CH 4 emissions simultaneously affect soil carbon dynamics as well as N 2 O emissions and can thereby reorder the ranking of technical mitigation effectiveness. The order of net GHG emissions reduction effectiveness found here is upland rice . shallow flooding . ammonium sulfate . midseason drainage . off-season straw . slow-release fertilizer . continuous flooding. Most of the management alternatives produced yields comparable to the baseline; however, continuous flooding and upland rice significantly reduced yields. Water management strategies appear to be the most technically promising GHG mitigation alternatives, with shallow flooding providing additional benefits of both water conservation and increased yields.

The present paper describes an efficient single step chromatographic method for purification of C-Phycocyanin from three cyanobacterial species, i.e., Spirulina sp. (freshwater), Phormidium sp. (marine water) and Lyngbya sp. (marine water). C-Phycocyanin from these cyanobacterial species was purified to homogeneity and some of their properties were investigated. The purification involves a multistep treatment of the crude extract by fractional precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-Sepharose CL-6B column. Pure C-Phycocyanin was finally obtained from Spirulina, Phormidium, and Lyngbya spp. with purity ratio (A 620 /A 280 ) 4.42, 4.43, and 4.59, respectively, further the purity and homogeneity were confirmed by native and SDS-PAGE. The estimated molecular weights of purified C-PC from Spirulina, Phormidium, and Lyngbya spp. were 112, 131, and 81 kDa, respectively. SDS-PAGE of pure C-Phycocyanin yielded two bands corresponding to a and b subunits. The results of SDS-PAGE demonstrate the same molecular weight of b subunits (24.4 kDa) for all the three cyanobacterial species, whereas the molecular weight of the a subunit is different for all (17 kDa Spirulina sp., 19.1 kDa Phormidium sp., 15.2 kDa Lyngbya sp.). Thus, the C-Phycocyanin was characterized as (ab) 3 for Spirulina and Phormidium spp., while as (ab) 2 for Lyngbya sp.

We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays.

G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--A simple methodology for hyperimmune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched immunoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaC1 and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K m 5.6 mg/mL, and V max 925 μmol mg−1 min−1 with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.

Gradients of ammonium sulfate in liposomes [(NH4)2SO4]lip.> [(NH4)2SO4]med. were used to obtain 'active' loading of amphipathic weak bases into the aqueous compartment of liposomes. The loading is a result of the base exchange with the ammonium ions. This approach was applied to encapsulate anthracyclines and acridine orange inside the liposomes at very high efficiency (> 90%). Doxorubicin was accumulated in the aqueous phase of the liposomes where it reached a level as high as 100-fold the doxorubicin concentration in the remote loading medium. Most of the intraliposomal doxorubicin was present in an aggregated state. The active entrapment and loading stability were dependent on liposome lipid composition, lipid quality, medium composition and temperature, as well as on the pK a and hydrophobicity of the base. The ammonium sulfate gradient approach differs from most other chemical approaches used for remote loading of liposomes, since it neither requires preparation of the liposomes in acidic pH, nor to alkalinize the extraliposomal aqueous phase. The stability of the ammonium ion gradient is related to the low permeability of its counterion, the sulfate, which also stabilizes anthracycline accumulation for prolonged storage periods (> 6 months) due to the aggregation and gelation of anthracycline sulfate salt.

Recent efforts to improve hydrophobic interaction chromatography (HIC) for use in monoclonal antibody (mAb) purification have focused on two approaches: optimization of resin pore size to facilitate mAb mass transport, and use of novel hydrophobic charge induction (HCIC) mixed mode ligands that allow capture of mAbs under low salt conditions. We evaluated standard HIC and new generation HIC and HIC-related chromatography resins for mAb purification process efficiency and product quality both as isolated chromatography steps and in purification process trains. We find that HIC resins with optimized pore size have significantly improved binding capacity which can increase HIC purification unit operation efficiency. The HCIC Mercapto-Ethyl-Pyridine (MEP) resin, which shows a different salt impact trend and impurity resolution pattern from standard HIC resin, can not only capture mAb from crude CHO fermentation supernatant but also substantially enhance mAb purification process flow efficiency when serving as a polishing role.

Pure culture of Bacillus amyloliquefaciens previously isolated and sequenced from our lab has been maintained on nutrient agar slant at 4°C. Amylase activity of pure culture was assayed by starch hydrolysis test. The enzyme production was done by using amylase production media at 37°C in shake flask culture. The enzyme was extracted by centrifugation, and then it was purified by ammonium sulfate precipitation 10 to 65%. The precipitated protein sample has been subjected to dialysis by using phosphate buffer. The enzyme activity of crude protein extract was determined by DNS method and maximum activity was 0.32 AU/ml. Crude protein and purified samples were subjected to 10% SDS-PAGE.

Objective: Effects of metamizol and magnesium sulfate on erythrocyte glucose 6-phosphate dehydrogenase enzyme activity were investigated in in vitro and in vivo conditions. Methods: For in vitro studies, glucose 6-phosphate dehydrogenase was purified from human erythrocyte and rats were used for in vivo studies. Enzyme activity was determined according to the Beutler method by using a spectrophotometer at 340 nm.

Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121؇C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and ␣-chymotrypsin but insensitive to ␣-amylase, lysozyme, catalase, and lipase. The 4.5-kDa bacteriocin was purified and characterized and designated lactobin A. Lactobin A was isolated as a floating pellicle from culture supernatant brought to 35% saturation with ammonium sulfate. Upon this ammonium sulfate treatment, crude lactobin A was incorporated, together with Tween 80 as a major contaminant, in high-molecular-mass complexes sized at approximately 670 kDa by gel filtration chromatography. Contaminating fatty acids were removed from these micelles by a simple one-step methanol-chloroform extraction without loss of activity. Both inhibitory peptides were separated in an isocratic isopropanol gradient on a PepRPC 5/5 reversed-phase column, and both peptides retained activity towards Lactobacillus helveticus ATCC 15009 upon separation. Lactobin A has a molecular mass determined by electrospray mass spectrometry of 4,879 ؎ 0.69 Da. Its peptide chain contains 50 unmodified amino acids, of which 26% are glycine residues and 40% are hydrophobic residues (A, V, L, I, and P). It displays the highest structural homology (42% identity and 28% similarity) with the lafX gene product, encoded by the second open reading frame of the lactacin F operon. These data strongly indicate that lactobin A belongs to the class IIb bacteriocins according to the classification of Klaenhammer.

Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg 2+ , Zn 2+ , Ca 2+ and Cu 2+ could promote the keratinase production, while exogenous urea, NH 4 Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL −1 , at (g L −1 ) sucrose 16.8, MgCl 2 ·6H 2 O 1.9, feather keratin 40.0, NaH 2 PO 4 ·2H 2 O 6.0 and K 2 HPO 4 ·6H 2 O 1.0, where dry cell weight nearly had a minimum 8.58 g L −1 . Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 • C and pH 8.0 with K + , Zn 2+ or Co 2+ . Compared with Savinase 16 L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres.

The present study aims at evaluating the performance of the relative importance of di erent types of interactions in yeast cell occulation. The yeasts isolated from several sources, puriÿed and identiÿed by morphological and biochemical methods. About 50 strains of yeast were isolated on PDA medium, among those strains six strains were occulent and one of them was identiÿed as Saccharomyces cerevisiae. The in uence of di erent additives on the occulation was investigated and it was shown that pistacia gum (turpentine) increases the occulation up to 93.8%. Addition of ammonium sulfate (0:2 g l −1 ) increases the production of alcohol to 6.19%. ?

Fibrin glue (FG) is used to control bleeding, to adhere tissues together, and to seal tissue defects. FG is prepared from platelet-rich plasma or by mixing concentrated fibrinogen solutions with thrombin. Concentrated fibrinogen solutions are produced by cryoprecipitation or by chemical precipitation of plasma. The literature on FG preparation is reviewed in order to compare the advantages and disadvantages of the different products reported and to summarize the clinical applications. It is concluded that additional studies are needed to fully evaluate the advantages and disadvantages of fibrinogen concentrated using cryoprecipitation and chemical precipitation and that specific advantages exist for use of both pooled homologous and autologous blood.

Carnobacterium piscicola CP5, isolated from a French mold-ripened soft cheese, produced a bacteriocin activity named carnocin CP5, which inhibited Carnobacterium, Enterococcus and Listeria spp. strains, and among the Lactobacillus spp. only Lactobacillus delbrueckii spp. . The activity was purified by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography followed by reverse-phase high-performance liquid chromatography (RP-HPLC). This latter step separated two peaks with anti-listerial activity (CP51 and CP52). Carnocin CP51 was partially sequenced, and the N-terminal part revealed the presence of the ''pediocin-like consensus'' sequence-Tyr-Gly-Asn-Gly-Val-. Then, a degenerated 24-mer oligonucleotide probe was constructed from the N-terminal sequence and used to detect the structural gene. It was localized on a plasmid of about 40 kb. Cloning of restriction fragments of this one, followed by DNA sequencing, revealed the presence of the second anti-Listeria bacteriocin gene (CP52). By comparing sequences in data banks and confirming results with PCR reactions, carnocin CP51 shared homologies with carnobacteriocin BM1, and carnocin CP52 was similar to carnobacteriocin B2, both produced by C. piscicola LV17 [2]. However, carnobacteriocin A from C. piscicola LV17 gene was lacking in C. piscicola CP5, and the two microorganisms have been isolated from different ecological environments: C. piscicola CP5 and C. piscicola LV17 were isolated from soft cheese and vacuum-packed meat respectively. This fact could allow different application perspectives for C. piscicola CP5.