Search

Abstract: 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 33g 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 0.2M in agreement with a coulombic mechanism of complex formation. The non-soluble complex formation showed biphasic kinetics. A fast step was carried out around 10s and a coulombic mechanism takes place, and a slower step of around 120s, where participate only Van der Waals forces. The enzymatic activity of chymotrypsin was maintained even in the presence of carrageenan (0.005%, w/v). [Copyright &y& Elsevier]

Abstract: The condition of chymotrypsin (ChTRP)–Eudragit® (Eu) insoluble complex formation was studied with the aim of applying this information to the separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate. The optimal pH of the complex precipitation was 4.60 for ChTRP–Eudragit® L100 and 5.40 for ChTRP–Eudragit® S100. The polyelectrolyte concentration necessary for the commercial enzyme precipitation was lower than 0.1% (w/v). The complex formation was inhibited by NaCl for both polyelectrolytes. The method was applied to recover the enzyme from bovine homogenate; ChTRP was precipitated by Eudragit® addition. The non-soluble complexes were separated by simple centrifugation and re-dissolved by a pH change to 8.20. The best conditions to recover ChTRP brought about a purification factor of around 4 and 90% yield. [Copyright &y& Elsevier]

Abstract: 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 6.9g 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. [Copyright &y& Elsevier]

Abstract: The combination of two bioseparation techniques, partition in aqueous two-phase systems and polyelectrolyte precipitation of the target enzyme from the phase where it is present, was assayed to purify pepsin from bovine abomasum homogenate. Pepsin was partitioned in favor of the polyethyleneglycol-rich phase in an aqueous two-phase system of polyethyleneglycol 600 and 1450-sodium phosphate; however, a great amount of impure proteins were present. Chitosan (a cationic natural polyelectrolyte) was added to precipitate this acid enzyme as a form of insoluble complex. The addition of this second step increased the purity of the enzyme significantly while the yield was not significantly decreased. The combination of both partition in polyethyleneglycol 1450-phosphate system and chitosan precipitation produced a pepsin recovery of 48.5% with a purification factor of 9.0. The biological activity of the recovered enzyme remained unaltered. [Copyright &y& Elsevier]

Abstract: Interactions between catalase and the cationic polyelectrolytes: chitosan and Eudragit E100 have been investigated owing to their scientific and technological importance. These interactions have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It was found that the catalase conformation does not change significantly during the chain entanglements between the protein and the polyelectrolytes. The effects of pH, ionic strength and anions which modify the water structure were evaluated on the polymer–protein complex formation. A net coulombic interaction force between them was found since the insoluble complex formation decreased after the NaCl addition. Both polymers were found to precipitate around 80% of the protein in solution. No modification of the tertiary and secondary protein structure or the enzymatic activity was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan and Eudragit E100 proved to be a useful framework to isolate catalase or proteins with a slightly acid isoelectrical pH by means of precipitation. [Copyright &y& Elsevier]

Abstract: The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity. [Copyright &y& Elsevier]

Abstract: The formation of non-soluble complexes between a positively charged protein and a strong anionic polyelectrolyte, chymotrypsin, and poly vinyl sulfonate, respectively, was studied under different experimental conditions such as pH (1–3.5), protein concentration, temperature, ionic strength, and the presence of anions that modifies the water structure. Turbidimetric titration and dynamic light scattering approaches were used as study methods. When low protein–polyelectrolyte ratio was used, the formation of a soluble complex was observed. The increase in poly vinyl sulfonate concentration produced the interaction between the soluble complex particules, thus inducing macro-aggregate formation and precipitation. Stoichiometry ratios of 500 to 780 protein molecules were found in the precipitate per polyelectrolyte molecule when the medium pH varied from 1.0 to 3.5. The kinetic of the aggregation process showed to be of first order with a low activation energy value of 4.2±0.2 kcal/mol. Electrostatic forces were found in the primary formation of the soluble complex, while the formation of the insoluble macro aggregate was a process driven by the disorder of the ordered water around the hydrophobic chain of the polymer. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Study of the interaction of tannase (TAH) with three polycations. [•] Effect of the interaction on the secondary and tertiary structure of TAH. [•] TAH recovery from Aspergillus niger culture broth. [•] Precipitation with chitosan (CS): 53% yield, 10 times concentration. [•] Adsorption onto CS beads: 72% yield, 2.28-fold purification, 5 times concentration. [Copyright &y& Elsevier]

Abstract: The separation of chymotrypsin from a crude filtrate of fresh bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged natural strong polyelectrolyte: ι-carrageenan. The zymogen form of the enzyme was activated by addition of trypsin (0.0001%, w/w), then, the enzyme was precipitated by polyelectrolyte addition at pH 4.50. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.20. The recovery of chymotrypsin biological activity was 60% of the initial activity in the homogenate with 3-fold increase in its specific activity. The volume of the final product decreased to 10% of the feedstock, concentrating the sample up to 10 times. [Copyright &y& Elsevier]