Caracterización de nuevos materiales para su aplicación en el aislamiento y purificación de macromoléculas

Translated title of the contribution: Characterization of new materials for application in the isolation and purification of macromolecules

Research output: Types of ThesisDoctoral Thesis

Abstract

Nowadays, there is a great need to develop new tools and methods in the area of bioseparation. One of the main demands of the food, textile, paper, cosmetics and pharmaceutical industries is the availability of large quantities of enzymes and, in some cases, with high degrees of purity, as these have advantages in terms of production speed and cost, as well as process performance and low negative impact on the environment. Thus, the search for new bioseparative techniques to obtain enzymes has arisen.
In recent years, adsorption processes have been widely developed for use in the recovery of macromolecules. This technique makes use of the adsorption capacity of a solid phase to bind molecules present in a solution. Adsorption can be carried out in a stirred tank, where the system reaches equilibrium adsorbate concentration in both the adsorbent and the solution; or in a column where, depending on the flow direction of the solution, it can be classified as packed bed, expanded bed or fluidised bed. The main problem with this technique is that existing commercial adsorption matrices are expensive and have a short lifetime, so it is desirable to design new cost-effective systems with high adsorption capacity. The use of natural polymers to obtain matrices with protein adsorption capacity is an excellent option, as they are easy to prepare, cost-effective and non-polluting.
In the first part of this work, matrices of Alginate and Guar Gum, two natural polymers, were developed from the gelation of a solution of these polysaccharides when in contact with a CaCl2 solution. The obtained spheres were covalently cross-linked with Epichlorohydrin, in order to increase their stability and avoid dissolution. They were morphologically and structurally characterised, verifying their average size of 2 mm, their high hydrophilicity or capacity to retain water and the successful reaction between the OH groups of Alginate and the epoxy groups of Epichlorohydrin from the analysis of the Infrared spectrum (FT-IR) of the chemically treated matrices.
The kinetics and adsorption isotherms of Lysozyme on the polymer matrix were studied. The adsorption kinetics followed a pseudo first order model, while the adsorption isotherm at equilibrium could be represented by the Freundlich model. The maximum amount of Lysozyme adsorbed on this matrix was approximately 2.4 mg per g of hydrated matrix at pH 7.0. The adsorption mechanism was associated with a simple diffusion process with a weak Coulombic interaction between the protein and the matrix. The presence of 0.3 M NaCl induced a total displacement of Lysozyme from the matrix. Under this condition, the percentage of desorbed protein was 95%. Successive washing adsorption-desorption cycles were performed and the results showed the reversibility of the process and the usefulness of the method for enzyme purification and separation. Also, the adsorption and desorption of Lysozyme in packed bed using the Alginate-Guar Gum matrix was studied. It was concluded that the Thomas and Bed Height Duty Time models fitted correctly to the experimental data of the breakthrough curves with high correlation coefficients. A final step was carried out for the purification of Lysozyme from egg white as a natural source, obtaining a 75% yield and a purification factor of about 15 when working in packed bed column.
In a second part, the obtaining and characterisation of the Alginate-Guar Gum matrix cross-linked with Epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinylpyrrolidine and Pluronic® F68. The matrices obtained were used for the adsorption of Lysozyme and Chymotrypsinogen, and showed, for both proteins, an increasing uptake in the presence of the flexible chain polymers in the sense: none < Pluronic® F68 < polyvinylpyrrolidine < polyvinyl alcohol. It was found that, in all cases, the adsorption process follows a pseudo first order kinetic model and is not influenced by the type of polymer and that the Freundlich model is the most suitable for fitting the experimental data. It was confirmed that, the addition of polyvinyl alcohol and polyvinylpyrrolidine showed the highest adsorption capacity for both proteins compared to the Alginate-Guar Gum matrix. This is due to an increase in gel stiffness caused by the formation of hydrogen bridge bonds between the polysaccharides and the synthetic polymers. Finally, a 79% recovery and a 17-fold purification factor of Lysozyme from egg white was obtained in a packed bed system with the Alginate-Guar Gum-Polyvinyl alcohol matrix. This verified that the presence of this polymer causes structural changes in the matrix that benefits the adsorption of macromolecules from their natural source.
Finally, Alginate microparticles were obtained by Spray Drying and chemically cross-linked with Epichlorohydrin. The microparticles were characterised by size, surface morphology (SEM), zeta potential, thermal analysis (TGA/DSC) and Raman spectroscopy. The resulting microparticles showed average sizes of 700 nm and a zeta potential value of -84 mV. The adsorption capacity of the matrix was studied using Lysozyme and Chymotrypsinogen as model proteins. The equilibrium isotherms were described by Langmuir and Hill models, respectively. The maximum adsorption capacities for Lysozyme and Chymotrypsinogen were 1880 and 3034 mg/g of Alginate microparticles, respectively. Finally, the sorption capacity of Alginate microparticles for both proteins from a complex protein mixture was studied. After a cycle of adsorption, washing and desorption, the separation of the model proteins from the total proteins present in the system was verified by SDS-PAGE.
Translated title of the contributionCharacterization of new materials for application in the isolation and purification of macromolecules
Original languageSpanish
Supervisors/Advisors
  • Picó, Guillermo A., Supervisor, External person
Thesis sponsors
Award date15 Jan 2015
Publication statusPublished - 11 Mar 2019
Externally publishedYes

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