Nadia Woitovich Valetti

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, 1g hydrate matrix adsorbed about 7mg of chymotrypsin. In the desorption process 80% of the biological activity of chymotrypsin was recovered under the condition of 50mM phosphate buffer, pH 7.00-500mM 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.

Molecular mechanism of lysozyme adsorption onto chemically modified alginate guar gum matrix

The equilibrium isotherms and adsorption kinetics of lysozyme (LZ) on epichlorohydrin (Epi) cross-linked alginate-guar gum (Alg-GG) matrix were studied. Adsorption kinetics followed a pseudo-first-order model while the equilibrium isotherm could be represented by the Freundlich equation. The maximal amount of LZ adsorbed onto this matrix was around 2.4mg per g of hydrated matrix at pH 7.00. The adsorption mechanism was associated to a simple diffusion process with a weak columbic interaction between LZ and the matrix. The presence of NaCl 0.3M induced a total displacement of the LZ from the matrix. Under this condition, the percentage of desorbed protein was 95%. Successive cycles of adsorption-washing-elution were performed and the results showed the reversibility of the process and the usefulness of the method for enzyme purification and separation. A last successful step was carried out for the purification of LZ from egg white as natural source. The model proved to be useful applied as a platform design in the isolation and purification of proteins.

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).

Influence of pH on viscoelastic properties of heat-induced gels obtained with a β-Lactoglobulin fraction isolated from bovine milk whey hydrolysates

A β-Lactoglobulin fraction (r-βLg) was isolated from whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the hydrolysis process on the r-βLg structure and the rheological properties of heat-induced gels obtained thereafter were studied at different pH values. Differences were observed between r-βLg and commercial β-Lg used as control. Higher values for the fluorescence emission intensity and red shifts of the emission wavelength of r-βLg suggested changes in its tertiary structure and more solvent-exposed tryptophan residues. Circular dichroism spectra also supported these evidences indicating that hydrolysis yielded an intermediate (non-native) β-Lg state. The thermal history of r-βLg through the new adopted conformation improved the microstructure of the gels at acidic pH. So, a new microstructure with better rheological characteristics (higher conformational flexibility and lower rigidity) and greater water holding ability was founded for r-βLg gel. These results were reflected in the microstructural analysis by scanning electron microscopy.

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.

Chemically modified alginate bead matrix for efficient adsorptive recovery of trypsin from fresh bovine pancreas

The adsorption of commercial trypsin (Try) onto epichlorohydrin cross‐linked alginate–guar gum matrix has been studied at equilibrium in batch and in fixed bed column. Experiments were conducted to study the effect of ionic strength, temperature and to obtain a thermodynamic characterization of the adsorption process. The resulting adsorption isotherm fitted the Hill equation. Experimental breakthrough curve profiles were compared with the theoretical breakthrough profiles obtained from the mathematical model, bed depth service time. At pH 5.0, 1.0 g hydrated matrix adsorbed 480.0 milligram of Try per gram of dried bed. The desorption process showed 80% of Try recovery in 50 mM phosphate buffer, pH 7.00—500 mM NaCl—20% propylene glycol. The obtained results were applied to an adsorption/washing/desorption process with fresh pancreas homogenate yielded 20% of recovery and 5.7 purification factor of Try. The matrix remained functional until the fifth cycle of repeated batch enzyme adsorption.

Adsorption of peroxidase from Raphanus sativus L onto alginate–guar gum matrix: Kinetic, equilibrium and thermodynamic analysis

This work explores the kinetics, equilibrium and thermodynamics of peroxidase adsorption onto spherical guar gum–alginate matrices. The effect of contact time, solution pH, initial protein concentration and temperature was studied in batch experiments. The results show that peroxidase adsorption increased with rising contact time and initial enzyme concentration, and was higher at pH 4.0. The kinetic processes can be predicted by both the pseudo-first-order rate kinetics and the pseudo-second-order rate kinetics. Equilibrium adsorption data were analyzed with different isotherm models. The experimental data fitted to the Freundlich model in agreement with the low energy activation, demonstrating the presence of a high physical and unspecific interaction between the enzyme and the matrix.

Complexes Formation Between Proteins and Polyelectrolytes and Their Application in the Downstream Processes of Enzyme Purification

Natural and synthetic polyelectrolytes have acquired notable importance in recent years due to their increasing application in different areas. One of these is downstream process methods which include the recovery, separation, concentration and purification of target enzymes from their natural sources. Polyelectrolytes interact with proteins to form soluble or non-soluble complexes. The interaction is driven by experimental variables of media such as pH, protein isoelectrical value, polyelectrolyte pKa, ionic strength and the presence of salts. The concentration of polyelectrolytes necessary to precipitate a protein completely is of the order of 10-4 – 10-2 % p/v. Precipitation of protein by PE is a novel technique integrating clarification, concentration and initial purification in a single step. This chapter presents some properties of aqueous solutions of natural and synthetic PE as a tool to use them in the protein downstream process.