Characterization of chymotrypsin-ι-carrageenan complex in aqueous solution: A solubility and thermodynamical stability study

The aim of this study is to report the results of research work on the molecular mechanism of complex formation between chymotrypsin and a negatively charged natural strong polyelectrolyte, ι-carrageenan, using spectroscopy techniques. The carrageenan-chymotrypsin complex showed a maximal non-solubility at pH around 4.50 with a stoichiometric ratio between 8 and 33. g of chymotrypsin per g of carrageenan. These values were depended on the enzyme concentration, pH and ionic strength medium. The insoluble complex was redissolved by modifying the pH and by a NaCl concentration around 0.2. M in agreement with a coulombic mechanism of complex formation. The non-soluble complex formation showed biphasic kinetics. A fast step was carried out around 10. s and a coulombic mechanism takes place, and a slower step of around 120. s, where participate only Van der Waals forces. The enzymatic activity of chymotrypsin was maintained even in the presence of carrageenan (0.005%, w/v).Fil: Woitovich Valetti, Nadia. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Boeris, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Control of the adsorption properties of alginate - guar gum matrix functionalized with epichlorohydrin through the addition of different flexible chain polymers as toll for the chymotrypsinogen isolation

This work addresses the obtaining and characterization of alginate-guar gum matrix, cross-linked with epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinyl pyrrolidine and Pluronic® F68. These matrixes were used for the adsorption of chymotrypsinogen and showed an increasing uptake in presence of the flexible chain polymer in the sense: none < Pluronic 68 < polyvinyl pyrrolidine < polyvinyl alcohol. The adsorption process was found to follow a first order kinetics model and was not influenced by the polymer type. It was found that Freundlich model was more suitable for our data. Polyvinyl alcohol and polyvinyl pyrrolidine addition increase the adsorption capacity of the original bed due to an increment in the rigidity of the gel caused by the formation of hydrogen bound between the polysaccharides and synthetics polymers.Fil: Brassesco, Maria Emilia. Universidad Nacional de Rosario; ArgentinaFil: Woitovich Valetti, Nadia. Universidad Nacional de Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.Fil: Gomez, Gabriela Noemi. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Nerli, Bibiana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentin

Adsorption isotherms, kinetics and thermodynamic studies towards understanding the interaction between cross-linked alginate-guar gum matrix and chymotrypsin

The adsorption kinetics of chymotrypsin, a pancreatic serine protease, onto an alginate-gum guar matrix cross-linked with epichlorohydrin has been performed using a batch-adsorption technique. The effect of various experimental parameters such as pH, salt presence, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression. The Langmuir, Freundlich and Hill adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Hill model was more suitable for our data because the isotherm data showed a sigmoidal behavior with the free enzyme concentration increasing in equilibrium. At 8 °C and at pH 5.0, 1 g hydrate matrix adsorbed about 7 mg of chymotrypsin. In the desorption process 80% of the biological activity of chymotrypsin was recovered under the condition of 50 mM phosphate buffer, pH 7.00-500 mM NaCl. When successive cycles of adsorption/washing/desorption were performed, it was observed that the matrix remained functional until the fourth cycle of repeated batch enzyme adsorption. These results are important in terms of diminishing of cost and waste generation.Fil: Woitovich Valetti, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentin

A friendly method for Raphanus sativus L (wild radish) peroxidase purification by polyelectrolyte precipitation

The separation of radish peroxidase from a fresh Raphanus sativus L extract was carried out using precipitation with two commercially available negatively charged synthetic polyelectrolytes: Eudragit® L 100 and Eudragit® S 100. The enzyme was precipitated by polyelectrolyte addition at pH 4.00. The non-soluble complex formed was separated by simple centrifugation and re-dissolved by a pH change. The recovery of radish peroxidase biological activity was 50% of the initial activity in the homogenate for EuL and 45% for EuS, with 1.5-fold increase in its specific activity. The total Eudragit® concentration to precipitate the enzyme was very low: about 2 × 10 -3% w/v. The volume of the final product decreased to 10% of the feedstock, concentrating the sample up to 10 times.Fil: Woitovich Valetti, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentin

Physicochemical study of the formation of complexes between pancreatic proteases and polyanions

The formation of insoluble complexes between proteins and oppositely charged polyelectrolytes was assessed. Two pancreatic enzymes: trypsin and chymotrypsin, and two anionic synthetic polyelectrolytes: polyacrylate and polyvinylsulfonate, were used for the study at the pH range between 3.00 and 5.00. Two different titration curve shapes, representing two insoluble complexes formation mechanisms, were found. The turbidity of enzyme–polyelectrolyte mixtures is related to the increase either in the size or in the quantity of the insoluble complexes. Ionic strength destabilized insoluble complex formation. Finally, the kinetics of the process of insoluble complex formation at different conditions was studied.Fil: Lombardi, Julia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; ArgentinaFil: Boeris, Valeria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; Argentin

Purification of chymotrypsin from pancreas homogenate by adsorption onto non-soluble alginate beads

Chymotrypsin was purified from an activated homogenate of bovine pancreas by adsorption onto non-soluble alginate beads in 25 mM Tris-acetate-5 mM CaCl2 buffer at different adsorbate-adsorbent ratios and the pH values were assayed. Under all the experimental conditions, the enzyme has a positive net electrical charge whereas alginate is negatively charged. After performing steps of washing and desorption in 25 mM Tris-acetate-500 mM CaCl2 buffer ρH 7.0, chymotrypsin was purified 9 times with an enzyme recovery of 62%. The eluate fractions resulted in two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The method allows purification with suitable values from a raw sample like pancreas homogenate without a previous clarification step.Fil: Spelzini, Darío. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Relationship between the protein surface hydrophobicity and its partitioning behaviour in aqueous two-phase systems of polyethyleneglycol- dextran

In order to develop possible correlations to predict partioning behaviour of proteins, five mammalian albumins (goat, bovine, equine, human and pig ones) with similar physico-chemical properties (molecular mass and isoelectrical point) were chosen. Evaluation of the relationship between hydrophobicity and partitioning coefficient (Kr) in polyethylenglycol-dextran (PEG-DxT500) systems formed by polyethyleneglycols of different molecular mass (3350, 6000 and 10,000) was investigated by estimating relative surface hydrophobicity (So) with a fluorescent probe, 1 anilino-8-naphthalene sulfonate. No relationship between Kr and So was found for systems formed by PEG3350, while aqueous two-phase systems with PEG6000 and PEG10,000 gave better correlations. The results obtained may be explained on the basis of an increase in the interaction between the latter PEGs and the protein due to their higher hydrophobic character which increases as the PEG molecular mass does so. In this way, systems with PEGs of higher molecular mass give the highest resolution to exploit hydrophobicity in partitioning.Fil: Tubio, Gisela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Nerli, Bibiana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; ArgentinaFil: Picó, Guillermo Alfredo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Chitosan-bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin-BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan-BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.Fil: Boeris, Valeria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Farruggia, Beatriz Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química y Física. Área Fisicoquímica; Argentin

A new platform for chymotrypsin isolation from fresh bovine pancreas using an environmental friendly polyelectrolyte: Alginate

The separation of chymotrypsin from a crude filtrate of fresh bovinepancreas homogenate was carried out using precipitation with acommercially available negatively charged natural weak polyelectrolyte:sodium alginate. The zymogen form of the enzyme was activated by theaddition of trypsin at pH 8.2 in the absence of Ca++, then, the enzyme wasprecipitated by sodium alginate addition at pH 5.00. The non-solublecomplex was separated by simple centrifugation and re-dissolved by apH change to 8.20. The recovery of chymotrypsin biological activity was36.1 % of the initial activity in the pancreas homogenate with a 3.2 foldincrease in its specific activity. The volume of the final product decreasedto 6.25 % of the initial feedstock, concentrating the sample up to 16times.Fil: Rausch, Caren. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Tecnología; ArgentinaFil: Woitovich Valetti, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Tecnología; ArgentinaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Tecnología; Argentin