Biomedical subjects

Francisco Batista-Viera

Publications and source records attributed to Francisco Batista-Viera.

4 recordsLinked to original sources

PubMed2004

Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots.

Preparation of Concanavalin A-adsorbents by immobilization on Sepharose activated with 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP-reagent) is reported. High immobilization yields of lectin (above 90%) were attained using an optimized CDAP-activating protocol. The effect of ligand density on the performance of the adsorbent for specific binding of glycoproteins was studied using horseradish peroxidase (HRP) as a model. Adsorption yields of pure HRP exceeding 90% were obtained with Con A-derivatives containing not < 20 mg of immobilized Con A/ml of packed gel. With lectin content of 2 mg/(ml of packed gel), only 20% of HRP was adsorbed. Purification of peroxidase from horseradish roots extract was successfully accomplished on Con A-Sepharose with high Con A content.

Adsorption↗

PubMed2003

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation.

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.

Chromatography, Affinity↗

PubMed2003

Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins.

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae beta-galactosidases and beta-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 micromol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis beta-galactosidase) and fourfold (for A. oryzae beta-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis beta-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae beta-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in beta-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.

Aspergillus oryzae↗

PubMed2002

Generating favorable nano-environments for thermal and solvent stabilization of immobilized beta-galactosidase.

beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.

Carbohydrate Metabolism↗