Purificação e caracterização de uma β-glucuronidase extraída do molusco mesogastropoda ampularidade Pomacea sp

This work studies the involved enzymatic way in the metabolism of glycosaminoglycans sulfateds in the mollusc Pomacea sp. Had been identified endoglycosidases and exoglycosidases in the enzymatic extract of the mollusc Pomacea sp by means of hydrolysis activity in condroitim sulphate of whale cartilage and of the p-Nitrofenil-β-glucuronide, respectively. The enzymatic extracts qere obtained of Pomacea sp. being used of 0.1 sodium acetate buffer, pH 5.0 and later centrifugated the 8,000 x g and the presents proteins in the sobrenadante were submitted to the fractionament with two crescents ammonium sulphate concentrations, the visualized activity biggest in the F2 fraction (50-80%). The β-glucuronidase (F3) was isolated in gel chromatography filtration Biogel 1.5m, the purification degree was ratified in Chromatography Liquid of high efficiency (HPLC). The enzyme was purificated 6.362,5 times with 35,6% yield. The β -glucuronidase isolated in this work showed a molecular mass of 100 kDa, determined for eletroforese in poliacrilamida gel . The determination of the ideal kinetic parameters for the catalysis of the p-nitrofenil- β -glucuronide for β-glucuronidase, showed excellent activity in pH 5,0 and temperature 65ºC for 6 hours and apparent Km of 72 x 10-2 mM. It is necessary for the total degradation of 3mM of p-N-β-glucoronide, the amount of 1,2μg of ss-glucuronidase. The BaCl2 increased the activity of ss-glucuronidase, and the activity was inhibited completely by the composites SDS and NaH2PO4

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Síntese e impregnação de peneiras moleculares Fe MCM-41 derivada de sílica da casca do arroz

The mesoporous molecular sieves of the MCM-41 and FeMCM-41 type are considered promissory as support for metals used as catalysts in oil-based materials refine processes and as adsorbents for environmental protection proposes. In this work MCM-41 and FeMCM41 were synthesized using rice husk ash - RHA as alternative to the conventional silica source. Hydrothermal synthesis was the method chosen to prepare the materials. Pre-defined synthesis parameters were 100°C for 168 hours, later the precursor was calcinated at 550°C for 2 hours under nitrogen and air flow. The sieves containing different proportions of iron were produced by two routes: introduction of iron salt direct synthesis; and a modification post synthesis consisting in iron salt 1 % and 5% impregnation in the material followed by thermal decomposition. The molecular sieves were characterized by X ray diffraction XRD, Fourier transform infrared spectroscopy FT-IR, X ray fluorescence spectroscopy XFR, scanning electronic microscopy SEM, specific surface area using the BET method, Termogravimetry TG. The kinetic model of Flynn Wall was used with the aim of determining the apparent activation energy of the surfactant remove (CTMABr) in the MCM- 41 porous. The analysis made possible the morphology characterization, identifying the presence of hexagonal structure typical for mesoporous materials, as well as observation of the MCM41 and iron of characteristic bands.

Pirólise rápida catalítica do capim elefante utilizando materiais mesoporosos e óxidos metálicos para deoxigenação em bio-óleo

The fast pyrolysis of lignocellulosic biomass is a thermochemical conversion process for production energy which have been very atratactive due to energetic use of its products: gas (CO, CO2, H2, CH4, etc.), liquid (bio-oil) and charcoal. The bio-oil is the main product of fast pyrolysis, and its final composition and characteristics is intrinsically related to quality of biomass (ash disposal, moisture, content of cellulose, hemicellulose and lignin) and efficiency removal of oxygen compounds that cause undesirable features such as increased viscosity, instability, corrosiveness and low calorific value. The oxygenates are originated in the conventional process of biomass pyrolysis, where the use of solid catalysts allows minimization of these products by improving the bio-oil quality. The present study aims to evaluate the products of catalytic pyrolysis of elephant grass (Pennisetum purpureum Schum) using solid catalysts as tungsten oxides, supported or not in mesoporous materials like MCM-41, derived silica from rice husk ash, aimed to reduce oxygenates produced in pyrolysis. The biomasss treatment by washing with heated water (CEL) or washing with acid solution (CELix) and application of tungsten catalysts on vapors from the pyrolysis process was designed to improve the pyrolysis products quality. Conventional and catalytic pyrolysis of biomass was performed in a micro-pyrolyzer, Py-5200, coupled to GC/MS. The synthesized catalysts were characterized by X ray diffraction, infrared spectroscopy, X ray fluorescence, temperature programmed reduction and thermogravimetric analysis. Kinetic studies applying the Flynn and Wall model were performed in order to evaluate the apparent activation energy of holoceluloce thermal decomposition on samples elephant grass (CE, CEL and CELix). The results show the effectiveness of the treatment process, reducing the ash content, and were also observed decrease in the apparent activation energy of these samples. The catalytic pyrolysis process converted most of the oxygenate componds in aromatics such as benzene, toluene, ethylbenzene, etc

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochondral ossification

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A(1), thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins. (C) 1998 Elsevier B.V. B.V.

Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of different sampling intervals on apparent protein and energy digestibility of common feed ingredients by juvenile oscar fish (Astronotus ocellatus)

O presente estudo avaliou a digestibilidade aparente da proteína e da energia de ingredientes (farelo de soja, farinha de peixe, farelo de trigo e milho) por juvenis de apaiari (Astronotus ocellatus) usando dois diferentes intervalos de coleta (30 min. e 12h). Os 160 juvenis de apaiari utilizados (22,37 ± 3,06 g de peso corporal) foram divididos em quatro tanques rede plásticos e cilíndricos, cada um colocado em um tanque de alimentação de 1.000 L. O experimento foi inteiramente casualizado em esquema fatorial 2 x 4 (2 intervalos de coleta de fezes e 4 ingredientes foram) com quatro repetições. Os testes estatísticos não detectaram efeito da interação entre o intervalo de coleta e tipo de ingrediente nos coeficientes de digestibilidade. O intervalo de coleta não afetou a digestibilidade da proteína e da energia. As características físicas das fezes dos juvenis de apaiari aparentemente as tornam menos sensíveis à perda de nutrientes por lixiviação, permitindo intervalos de coleta maiores. A digestibilidade da proteína dos ingredientes avaliados foi semelhante, mostrando que a digestibilidade aparente de ingredientes animais e vegetais por juvenis de apaiari é eficiente. Os coeficientes de digestibilidade da energia foram maiores para a farinha de peixe e o farelo de soja comparado a farelo de trigo e milho. Ingredientes ricos em carboidratos (farelo de trigo e milho) apresentaram os piores coeficientes de digestibilidade da energia e, portanto, não são usados eficientemente pelos juvenis de apaiari.

Supercritical fluid extraction from Lippia alba: global yields, kinetic data, and extract chemical composition

In this work, experimental data for the system Lippia alba + CO2 is presented. The major constituents of the L. alba volatile oil are limonene and carvone. Thus, literature data for the systems limonene + CO2 and carvone + CO2, and the Peng-Robinson equation of state (PR-EOS) were used to select the operating temperature and pressure, which maximize the global yield in L. alba extract. Global yields were determined at 80, 100, and 120 bar and 40, 45, and 50 degrees C. L. alba extracts were also obtained by conventional processes (hydrodistillation, low-pressure ethanol extraction and Soxhlet ethanol). The chemical compositions of the extracts were determined by gas and thin layer chromatography (TLC). The secretor structures of L. alba were observed by scanning electron microscopy (SEM) before and after supercritical extraction. The largest yield (similar to 7%, mass of extract/mass of dry solid) of the CO2-extract was obtained at 318 K and 100 bar. The chemical compositions of the CO2-extracts were different from those of the extracts obtained by Soxhlet and low-pressure solvent extraction (LPSE) because of the co-extraction of heavy substances by ethanol. The operating conditions that maximized the carvone and limomene yields were 80 bar and 323 K (80 mass%) and 120 bar and 323 K (17 mass%), respectively. (c) 2004 Elsevier B.V All rights reserved.

Avaliação de catalisadores a base de carbeto e nitreto de molibdênio na obtenção de lactitol via hidrogenação da lactose

Seeking a greater appreciation of cheese whey was developed to process the hydrogenation of lactose for the production of lactitol, a polyol with high added value, using the catalyst Ni / activated carbon (15% and 20% nickel), the nitride Mo2N, the bimetallic carbide Ni-Mo/ activated carbon and carbide Mo2C. After synthesis, the prepared catalysts were analyzed by MEV, XRD, laser granulometry and B.E.T. The reactor used in catalytic hydrogenation of lactose was the type of bed mud with a pressure (68 atm), temperature (120 oC) and stirring speed (500 rpm) remained constant during the experiments. The system operated in batch mode for the solid and liquid and semi-continuous to gas. Besides the nature of the catalyst, we studied the influence of pH of reaction medium for Mo2C carbide as well as evaluating the character of the protein inhibitor and chloride ions on the activity of catalysts Ni (20%)/Activated Carbon and bimetallic carbide Ni-Mo/Activated Carbon. The decrease in protein levels was performed by coagulation with chitosan and adsorption of chloride ions was performed by ion exchange resins. In the process of protein adsorption and chloride ions, the maximum percentage extracted was about 74% and 79% respectively. The micrographs of the powders of Mo2C and Mo2N presented in the form of homogeneous clusters, whereas for the catalysts supported on activated carbon, microporous structure proved impregnated with small particles indicating the presence of metal. The results showed high conversion of lactose to lactitol 90% for the catalyst Ni (20%)/Activated Carbon at pH 6 and 46% for the carbide Mo2C pH 8 (after addition of NH4OH) using the commercial lactose. Monitoring the evolution of the constituents present in the reaction medium was made by liquid chromatography. A kinetic model of heterogeneous Langmuir Hinshelwood type was developed which showed that the estimated constants based catalysts promoted carbide and nitride with a certain speed the adsorption, desorption and production of lactitol

Conversão catalítica de clorometano em hidrocarbonetos

Amorphous silica-alumina and modified by incipient impregnation of iron, nickel, zinc and chromium were synthetized in oxide and metal state and evaluated as catalysts for the chloromethane conversion reaction. With known techniques their textural properties were determined and dynamics techniques in programmed temperature were used to find the acid properties of the materials. A thermodynamic model was used to determine the adsorption and desorption capacity of chloromethane. Two types of reactions were studied. Firstly the chloromethane was catalytically converted to hydrocarbons (T = 300 450 oC e m = 300 mg) in a fixed bed reactor with controlled pressure and flow. Secondly the deactivation of the unmodified support was studied (at 300 °C and m=250 g) in a micro-adsorver provided of gravimetric monitoring. The metal content (2,5%) and the chloromethane percent of the reagent mixture (10% chloromethane in nitrogen) were fixed for all the tests. From the results the chloromethane conversion and selectivity of the gaseous products (H2, CH4, C3 and C4) were determined as well as the energy of desorption (75,2 KJ/mol for Ni/Al2O3-SiO2 to 684 KJ/mol for the Zn/Al2O3-SiO2 catalyst) considering the desorption rate as a temperature function. The presence of a metal on the support showed to have an important significance in the chloromethane condensation. The oxide class catalyst presented a better performance toward the production of hydrocarbons. Especial mention to the ZnO/Al2O3-SiO2 that, in a gas phase basis, produced C3 83 % max. and C4 63% max., respectively, in the temperature of 450 oC and 20 hours on stream. Hydrogen was produced exclusively in the FeO/Al2O3-SiO2 catalysts (15 % max., T = 550 oC and 5,6 h on stream) and Ni/SiO2-Al2O3 (75 % max., T = 400 oC and 21,6 h on stream). All the catalysts produced methane (10 à 92 %), except for Ni/Al2O3-SiO2 and CrO/Al2O3-SiO2. In the deactivation study two models were proposed: The parallel model, where the product production competes with coke formation; and the sequential model, where the coke formation competes with the product desorption dessorption step. With the mass balance equations and the mechanism proposed six parameters were determined. Two kinetic parameters: the hydrocarbon formation constant, 8,46 10-4 min-1, the coke formation, 1,46 10-1 min-1; three thermodynamic constants (the global, 0,003, the chloromethane adsorption 0,417 bar-1, the hydrocarbon adsorption 2,266 bar-1), and the activity exponent of the coke formation (1,516). The model was reasonable well fitted and presented a satisfactory behavior in relation with the proposed mechanism

Study of the diclofenac/phospholipid interactions with liposomes and monolayers

The interaction of diclofenae sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting In[1 - (Gamma/Gamma(infinity))] versus t(1/2) (C) 2004 Elsevier B.V. All rights reserved.