Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

Recuperação e purificação de proteínas do soro de queijo tipo coalho usando cromatografia de troca iônica e interação hidrofóbica em leito na forma expandida

Expanded Bed Adsorption plays an important role in the downstream processing mainly for reducing costs as well as steps besides could handling cells homogenates or fermentation broth. In this work Expanded Bed Adsorption was used to recover and purify whey proteins from coalho cheese manufacture using Streamline DEAE and Streamline SP both ionic resins as well as a hydrophobic resin Streamline Phenyl. A column of 2.6 cm inner diameter with 30 cm in height was coupled to a peristaltic pump. Hydrodynamics study was carried out with the three resins using Tris-HCl buffer in concentration of 30, 50 and 70 mM, with pH ranging from 7.0 to 8.0. In this case, assays of the expansion degree as well as Residence Time Distribution (RTD) were carried out. For the recovery and purification steps, a whey sample of 200 mL, was submitted to a column with 25mL of resin previously equilibrated with Tris/HCl (50 mM, pH 7.0) using a expanded bed. After washing, elution was carried out according the technique used. For ionic adsorption elution was carried out using 100 mL of Tris/HCl (50 mM, pH 7.0 in 1M NaCl). For Hydrophobyc interaction elution was carried out using Tris/HCl (50 mM, pH 7.0). Adsorption runs were carried out using the three resins as well as theirs combination. Results showed that for hydrodynamics studies a linear fit was observed for the three resins with a correlation coefficient (R2) about 0.9. In this case, Streamline Phenyl showed highest expansion degree reaching an expansion degree (H0/H) of 2.2. Bed porosity was of 0.7 when both resins Streamline DEAE and Streamline SP were used with StremLine Phenyl showing the highest bed porosity about 0.75. The number of theorical plates were 109, 41.5 and 17.8 and the axial dipersion coefficient (Daxial) were 0.5, 1.4 and 3.7 x 10-6 m2/s, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. Whey proteins were adsorved fastly for the three resins with equilibrium reached in 10 minutes. Breakthrough curves showed that most of proteins stays in flowthrough as well as washing steps with 84, 77 and 96%, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. It was observed protein peaks during elution for the three resins used. According to these peaks were identified 6 protein bands that could probably be albumin (69 KDa), lactoferrin (76 KDa), lactoperoxidase (89 KDa), β-lactoglobulin (18,3 KDa) e α-lactoalbumin (14 KDa), as well as the dimer of beta-lactoglobulin. The combined system compound for the elution of Streamline DEAE applied to the Streamline SP showed the best purification of whey proteins, mainly of the α-lactoalbumina

Recuperação e purificação de ramnolipídeos produzidos por pseudomonas aeruginosa P029-GVIIA utilizando melaço de cana como substrato

Biosurfactants are molecules produced by microorganisms mainly bacteria as Pseudomonas and Bacillus. Among the biosurfactants, rhamnolipids play an important role due to their tensoactive as well as emulsifying properties. Besides can be produced in a well consolidated way the production costs of biosurfactants are quite expansive mainly if downstream processing is goning to be considered. Actually, attention has been given to identification of biosurfactants as well as optimization of its fermentative processes including downstream ones. This work deals with the development of strategies to recovery and purification of rhamnolipids produced by Pseudomonas aeruginosa P029-GVIIA using sugar-cane molasses as substrate. Broth free of cells was used in order to investigate the best strategies to recovery and purification produced by this system. Between the studied acids (HCl and H2SO4) for the acid precipitation step, HCl was the best one as has been showed by the experimental design 24. Extraction has been carried out using petroleum ether and quantification has been done using the thioglycolic acid method. Adsorption studies were carried out with activated carbon in a batch mode using a 24 experimental design as well as combined with an hydrophobic resin Streamline Phenyl aiming to separate the produced biosurfactant. Biosurfactant partial identification was carried out using High Performance Liquid Chromatography (HPLC). Experiments in batch mode showed that adsorption has been controlled mainly by pH and temperature. It was observed a reduction of 41.4% for the liquid phase and the solid phase it was possible to adsorb up to 15 mg of rhamnolipd/g of activated carbon. The kinetics of adsorption has been well fitted to a pseudo-first order reaction with velocity constant (k1) of 1.93 x 10-2 min-1. Experiments in packed bed ranging concentration on eluent (acetone) has been shown the highest recovery factor of 98% when pure acetone has been used. The combined effect if using activated carbon with an hydrophobic resin Streamline Phenyl has been shown successful for the rhamnolipids purification. It has been possible to purify a fraction of the crude broth with 98% of purity when the eluted of activated carbon packed bed was used with pure acetone

Hidrólise e caracterização de poliacrilamida hidrofobicamente modificada: avaliação da aplicabilidade na recuperação de petróleo

The hydrolysis reaction in alkaline conditions of the commercial polymer poly(acrylamide-co-metacrylate of 3,5,5-trimethyl-hexane) called HAPAM, containing 0.75 % of hydrophobic groups, was carried out in 0.1 M NaCl and 0.25M NaOH solutions, varying the temperature and reaction time. The polymers were characterized by 1H and 13C Nuclear Magnetic Resonance (NMR), Elemental Analysis and Size Exclusion Chromatography (SEC). The values of the hydrolysis degree were obtained by 13C NMR. The viscosity of HAPAM and HAPAM-10N-R solutions was evaluated as a function of shear rate, ionic strength and temperature. At high polymer concentration (Cp), the viscosity of HAPAM solutions increased with the ionic strength and decreased with the temperature. The viscosity of HAPAM-10N-R solutions increased significantly in distilled water, due to repulsions between the carboxylate groups. At high Cp, with the increase of ionic strength and temperature, occurred a decrease of viscosity, due to mainly the high hydrolysis degree and the low amount of hydrophobic groups. These results indicated that the studied polymers have properties more suitable for the application in Enhanced Oil Recovery (EOR) in low salinity and moderate temperature reservoirs

Aplicação da parafina e do óleo de linhaça como hidrofobizantes da vermiculita, para remoção de derivados de óleo diesel resentes em água produzida sintética

Produced water has lately aroused interest due to their high degree of salinity, suspended oil particles, chemicals added in various manufacturing processes, heavy metals and radioactivity sometimes. Along with oil and due to its high volume production, water production is one of the pollutants of most concern in the process of oil extraction. PAHs due to their ubiquity and their characteristics carcinogenic or mutagenic and teratogenic even have attracted the attention of every scientific society. Formed from the incomplete combustion of organic matter may be natural or anthropogenic. Some materials have been researched with the goal of cleaning up environmental matrices that may be contaminated by hydrocarbons. Among these materials researched various clays have been employed, of which highlights the vermiculite. The family of phyllosilicates, vermiculite for its potential and its high hydrophobic surface area has been a tool widely used in the decontamination of water in processes of oil spills. However, when it loses its capacity expanded hydrophobic having the necessity of using a hidrofobizante to make it organophilic. Among the numerous hidrofobizantes researched and used the linseed oil was the pioneer. In this study sought to evaluate the capacity of removal of PAHs using the vermiculite hydrofobized with linseed oil and wax also, for it was made use of the 24 full factorial design as the main tool for the experiments. We also evaluated the clay grain size (-20 +48 and -48 +80 #), the percentage of hidrofobizante applied (5 and 10%) and salinity of the water produced synthesized in our laboratory (35,000 and 55,000 ppm). The molecular fluorescence spectroscopy due to its sensitivity and speed was used to verify the adsorption capacity of clay, as well as gas chromatography served as an auxiliary technique to identify and quantify the PAHs in solution. In order to characterize the vermiculite was made use of X-ray fluorescence and X-ray diffraction. The infrared and thermogravimetry were essential to note hydrophobization and the amount of coating of clay. According to the fluorescence analysis showed that the test 12 was the best result in about 98% adsorption of fluorescent compounds, however the high salinity, the smallest particle size, the highest percentage of hidrofobizante and the use of linseed oil showed greater efficiency in the removal capacity of these hydrocarbons, in accordance with the trend followed by the analysis of the major factors of the factorial design. To verify the adsorption capacity of clay using a fixed volume of water produced synthetically, used as the test base 12, at their respective levels and factors. Thus, it was observed that after adding about 1 ½ liters of water solution produced synthetically, about 300 times its volume in mass, the vermiculite was able to adsorb 80% of fluorescent species present in solution

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa