Isolamento de bactérias resistentes a cromo hexavalente e purificação parcial da enzima redutora de cromo do Bacillus sp. ES29

A redução do Cr(VI) para Cr(III) diminui a toxidade deste metal no ambiente uma vez que, o Cr(III) é insolúvel às membranas biológicas. Assim a redução microbiana do Cr(VI) é uma alternativa para reduzir os impactos ambientais causados por este metal, utilizado em diversos processos industriais. O objetivo deste trabalho foi selecionar microrganismos a partir de solo contaminado com cromo, caracterizar sua capacidade de redução do Cr(VI) durante o crescimento celular e purificar parcialmente a enzima cromo redutase do Bacillus sp. ES29, através da precipitação com sulfato de amônio (45-75%), cromatografia de gel filtração (Sephadex G-25) e cromatografia de interação hidrofóbica (Octyl Sepharose). A atividade de redução do Cr(VI) pelos isolados foi quantificada com o reagente de s-difenilcarbazida. No isolamento, foram obtidas 20 bactérias resistentes a cromo(VI). Seis destas foram capazes de reduzir 100 mg L-1 Cr(VI) em 24 horas. Um dos isolados foi identificado, através de testes bioquímicos, como pertencete ao gênero Bacillus, sendo tolerante a 750 mg L-1 Cr(VI) e reduzindo mais de 40% do Cr(VI) durante o crescimento celular. Na purificação parcial da enzima foi obtido um fator de purificação de 11,2, aumentando a atividade específica da enzima acima de 11 vezes, porém se faz necessário mais passos de purificações para obtenção desta enzima pura. As bactérias selecionadas e a enzima parcialmente purificada, foram eficientes na redução do Cr(VI) e apresentam potencial para outros estudos, visando a aplicação em processos de biorremediação.

Determinação de carbono orgânico por oxidação pelo dicromato de potássio e titulação do excesso do oxidante por iodometria

Aula Prática 7

De Ilhéus a Canga, de Horácio Bento de Gouveia: a narrativa e as suas (re)escritas (com uma proposta de edição crítico-genética e com uma tradução parcial do romance para francês)

Orientação de: Jacqueline Penjon e João David Pinto Correia

Purificação e caracterização parcial de uma B-D glicosidade de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Purificação e caracterização parcial de uma Fucana C de Dictyota mentrualis e estudo do seu efeito antiinflamatório e nociceptivo

In recent years, sulfated polysaccharides from marine algae have emerged as an important class of natural biopolymers with potential application in human and veterinary health care, while taking advantage of the absence of potential risk of contamination by animal viruses. Among these, fucans isolated from the cell walls of marine brown alga have been study due to their anticoagulant, antithrombotic, anti-inflammatory and antiviral activities. These biological effects of fucans have been found to depend on the degree of sulfation and molecular size of the polysaccharide chains. In the present study, we examined structural features of a fucan extracted from brown alga Dictyota menstrualis and its effect on the leukocyte migration to the peritoneum. The sulfated polysaccharides were extracted from the brown seaweed by proteolytic digestion, followed by sequential acetone precipitation producing 5 fractions. Gel lectrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. Electrophoresis in agarose gel in three different buffers demonstrated that the fraction 2.0v have only one population of fucan. This compound was purify by exclusion molecular. It has shown composition of fucose, xilose, sulfate and uronic acid in molar ration of 1.0: 1.7: 1.1: 0.5 respectively. The effect of this heterofucan on the leukocyte migration was observed 6h after zymozan (mg/g) administration into the peritoneum. The heterofucan showed higher antimigratory activity, it decrease the migration of leukocyte in 83.77% to peritoneum. The results suggest that this fucan is a new antimigratory compound with potential pharmacological appications

Purificação e caracterização parcial de duas n-acetil-B-hexosaminidases do equinoderma marinho echinometra lucunter

Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-β-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 °C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate

Síntese e caracterização de óxidos de cério e cobalto obtidos por gel-combustão e Pechini aplicados às reações de oxidação de n-hexano

Oxide type spinel AB2O4 presents structure adjusted for application in the automobile industry. The spinel of cobalt has many practical applications had its excellent physical and chemical properties such as catalyst in hydrocarbon oxidation reaction. The CeO2 has been used in many of these processes because it assigns to a material with excellent thermal resistance and mechanics, high capacity of oxygen stockage (OSC) among others properties. This work deals with the synthesis, characterization and catalytic application of spinel of cobalt and CeO2 with fluorita structure, obtained for method of Pechini and method of Gel-Combustion. The process of Pechini, the puff was obtained at 300 ºC for 2 h in air. In the process of Gel-Combustion the approximately at 350 ºC material was prepared and burnt for Pyrolysis, both had been calcined at 500 ºC, 700 ºC, 900 ºC and 1050 ºC for 2 h in air. The materials of the calcinations had been characterized by TG/DTA, electronic microscopy of sweepings (MEV), spectroscopy of absorption in the infra-red ray (FTIR) and diffraction of X-rays (DRX). The obtained material reaches the phase oxide at 450 oC for Pechini method and 500 °C for combustion method. The samples were submitted catalytic reaction of n-hexane on superficies of materials. The reactor function in molar ration of 0, 85 mol.h-1.g-1 and temperature of system was 450 °C. The sample obtained for Pechini and support in alumine of superficial area of 178,63 m2.g-1 calcined at 700 ºC, give results of catalytic conversions of 39 % and the sample obtained for method of gel-combustion and support in alumina of 150 mesh calcined at 500 ºC result 13 % of conversion. Both method were selective specie C1

Caracterização de superfícies de titânio modificado por oxidação à plasma

Recent years have seen a significant growth in surface modifications in titanium implants, resulting in shorter healing times in regions with low bone density. Among the different techniques, subtraction by chemical agents to increase oxidation has been applied for surface treatment of dental implants. However, this technique is generally unable to remove undesirable oxides, formed spontaneously during machining of titanium parts, raising costs due to additional decontamination stages. In order to solve this problem, the present study used plasma as an energy source to both remove these oxides and oxidize the titanium surface. In this respect, Ti disks were treated by hollow cathode discharge, using a variable DC power supply and vacuum system. Samples were previously submitted to a cleaning process using an atmosphere of Ar, H2 and a mixture of both, for 20 and 60 min. The most efficient cleaning condition was used for oxidation in a mixture of argon (60%) and oxygen (40%) until reaching a pressure of 2.2 mbar for 60 min at 500°C. Surfaces were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), atomic force microscopy (AFM), adhesion and cell proliferation. SEM showed less cell spreading and a larger number of projections orfilopodia in the treated samples compared to the control sample. AFM revealed surface defects in the treated samples, with varied geometry between peaks and valleys. Biological assays showed no significant difference in cell adhesion between treated surfaces and the control. With respect to cell proliferation, the treated surface exhibited improved performance when compared to the control sample. We concluded that the process was efficient in removing primary oxides as well as in oxidizing titanium surfaces

Estudo do efeito dos metabólitos do benzeno e tolueno sobre as mitocôndris cerebrais e hepáticas de ratos

Aim: The aim of this work was to investigate the hypothesis that catechol and 3MC inhibit FADH2-linked basal respiration in mitochondria isolated from rat liver and brain homogenates. Moreover, catechol ability to induce DNA damage in rat brain cells through the comet assay (alkaline single-cell gel electrophoresis assay) was also observed. Methods: Two different catechols were evaluated: pirocatechol (derived from benzene) and 3-methylcatechol (derived from toluene); rat liver and brain homogenates were incubated with 1mM catechol at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate for a period of 12 minutes. In additional experiments, rat brain cells were treated with 1, 5 and 10mM pirocatechol for up to 20 minutes at 37º C, and submitted to electrophoresis. Results: Catechols (pirocatechol and 3methylcatechol) induced a time-dependent partial inhibition of FADH2-linked basal mitochondrial respiration. Indeed, pirocatechol was able to produce a dosedependent DNA oxidative damage in rat brain cells by 2 and 4 injury levels. These results suggest that reactive oxygen species generated by the oxidation of catechols, induced an impairment on mitochondrial respiration and a DNA damage, which might be related to their citotoxicity. Conclusion: Catechols produced an inhibition of basal respiration associated to FADH2 in isolated liver and brain mitochondria; 3-methylcatechol, at the same concentration, produced similar toxicity in the mitochondrial model. Indeed, pirocatechol induced a DNA damage in rat brain cells, mainly observed in comets formation and consequent DNA degradation

Digestão total e parcial de alguns componentes de dietas contendo diferentes níveis de casca de soja e Fontes de nitrogênio, em bovinos

Este experimento foi realizado para avaliar o efeito de dois níveis de casca de soja (CS) e de três fontes de nitrogênio sobre a digestão total e parcial aparentes no rúmen e nos intestinos. Seis novilhos Holandês x Zebu com idade média de 12 meses e 229 kg de PV foram fistulados no rúmen e no abomaso e distribuídos em quadrado latino 6x6 e alimentados individualmente ad libitum. O consumo e as digestibilidades totais e parciais dos nutrientes foram avaliados. Não houve efeito da fonte de proteína sobre o consumo, quantidades de matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), fibra em detergente neutro (FDN) e da fibra em detergente ácido (FDA) no rúmen, no abomaso e nas fezes e sobre os coeficientes de digestibilidades destes componentes. O aumento, porém, do nível de casca de soja elevou os coeficientes de digestibilidade de todos os componentes estudados nos locais do trato digestivo, com exceção nos intestinos, onde o nível de casca de soja não afetou a digestão da PB, FDN e da FDA. Os resultados indicam que a casca de soja pode substituir tanto o milho como, parcialmente, a fração volumosa nas rações de ruminantes. A farinha de subproduto avícola e a amiréia podem substituir o farelo de soja nas rações de bovinos sem reflexos negativos sobre a digestão de seus componentes nutritivos.