Agustin A. Navarro del Cañizo

Immobilized metal ion affinity hollow-fibre membranes obtained by the direct grafting technique

Abstract An immobilized metal ion affinity hollow fibre was prepared by radiation-induced direct grafting of glycidylmethacrylate (GMA) on hydrophilized polyethylene membranes. The epoxy group was converted into an iminodiacetate by iminodiacetic acid treatment. The effect of the radiation dose, salt inhibitors, methanol and GMA concentration, on the grafting degree was studied. The degree of grafting was closely related to the GMA concentration. Salt inhibitors failed in the production of a differential effect on the grafting and homopolymerization processes. Between 30 and 35% of methanol, there is a maximum yield, and no grafting was obtained with a methanol concentration above 50%. Water flux dropped exponentially with the increase in the grafting degree. Scanning-electron microscopy showed that the graft branches are formed on the pores. The pectinesterase adsorption capacity of the membranes was of the same order as of the commercial chelating soft gels.

Protein Adsorption onto Tentacle Cation-Exchange Hollow-Fiber Membranes

A sulfonic group (up to 200 μmol/mL) membrane was incorporated to epoxy‐activated microporous hollow fibers to obtain high‐capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 μmol/mL, the membrane lysozyme maximum binding capacity was 84 ± 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7.0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min−1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic‐to‐static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

Direct lysozyme separation from egg white by dye membrane affinity chromatography

An affinity membrane from hydrophylised polyethylene hollow fibre as the support matrix was prepared. Red HE-3B was immobilised on the membrane and the adsorption behaviour of pure lysozyme solutions and homogenised egg white was investigated. Dye density (1.7 μmol ml−1) and maximum binding capacity (26 mg lysozyme ml−1) are comparable to those of commercial gel matrices. Dynamic binding capacity did not change when residence time was reduced from 3 to 1 min. A method for direct lysozyme separation from egg white was developed, with a productivity of 12 kg lysozyme m−3 h−1. The purity of the eluted lysozyme, as determined by HPLC, was 88%, with a recovery of 92%. Dynamic capacity was kept constant at 70% of the maximum binding capacity for at least 10 cycles through membrane regeneration with 0.1 M NaOH and 1 M NaCl. Functional properties of egg white, as judged by viscosity and foaming capacity measurements, did not change after the chromatographic lysozyme depletion. © 1999 Society of Chemical Industry

Foot and mouth disease virus concentration and purification by affinity chromatography

Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A4R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the Chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.

Purification of Neutral Protease by Dye Affinity Chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Abstract Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5 h at 85 °C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75 °C, whereas that of the free enzyme was 55 °C. After two months at 4 °C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180 min and, after five cycles of use, the activity loss was only 12.8%.