Mariano Grasselli

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.

From microspheres to monoliths: Synthesis of porous supports with tailored properties by radiation polymerization

Abstract Advanced functional materials, both in shape of beads and monoliths, are in high demand for a variety of applications ranging from catalysis, chromatography, diagnostics, sensors to combinatorial chemistry. Here we report the synthesis of functional supports of different size and shape by radiation co-polymerization in organic solvents using diethylene glycol dimethacrylate (DEGDMA) and glycidylmethacrylate (GMA) as co-monomers. With the increase in the GMA content, the particle diameter increases from 0.9 to about 3 μm, while the yield decreases from 80% to 50%. The usefulness of the microspheres for immobilization was tested with histidine and lysosyme. Monoliths were prepared in situ as chromatographic columns. Our results showed that when a 30% monomer solution was irradiated, a 100% conversion of the monomer was obtainable with doses higher than 15 kGy. We showed the effect of dose and dose rate as well as the irradiation temperature on the pore size of the monoliths and the flux. The effect of such different solvents as tetrahydrofuran, ethylpropionate, acetone, methanol, ethanol, propanol and butanol is also discussed. Our results showed that radiation synthesis of porous polymer supports with epoxy functionality is a viable alternative to either thermally initiated or photo polymerization.

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.

Synthesis and performance of 3D-Megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H+/g, 1.4 mmol H+/g, and 17 mmol H+/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg‐white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work.

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

Oriented immobilization of proteins on grafted porous polymers

Abstract The modification of polymers by radiation grafting has been utilized for several decades. The penetrability of gamma rays allows to modify the internal surfaces of porous materials retaining its mechanical properties. In recent years applications of these materials to obtain chromatographic supports and biocatalysts have been reported. In this work, we described the grafting of glycidyl methacrylate (GMA) onto a macroporous polysulfone polymer. Reproducible amount of grafting, from 10% to 60% was obtained by choosing favourable monomer concentration and gamma radiation doses from 6 kGy up. Afterwards, iminodiacetic acid (IDA) and amino phenyl arsine oxide (PAO) were covalently attached to the grafted polyGMA, in correspondence with the grafting degree. Later on, a recombinant histidin-patch thioredoxin protein (HP-rTrx) was immobilized onto this surface by two different ways, involving specific protein orientations. The first one involves an IDA–Ni 2+ complex and three HP-rTrx’s histidines and the other one involves a co-ordination site between PAO and two proximal HP-rTrx’s cysteines, which corresponds to the active site of the enzyme. Specific polyclonal antibodies recognize HP-rTrx on the polymer. Proper orientation of the protein was confirmed by HP-rTrx activity measurements. The described procedure allows the successful oriented immobilization of a protein onto a macroporous polysulfone material.

Preparation and characterisation of immobilised metal ion hollow-fibre polysulphone membranes. Their application in high-speed pectic enzyme fractionation

Abstract Chelating hollow-fibre membranes were prepared from epoxy-activated polysulphone microfiltration fibres by introducing iminodiacetic acid (IDA) groups in the presence of dimethyl sulphoxide. Fibres with 160, 350 and 620 μmol epoxy groups/ml provided ligand densities of 69, 134 and 203 μmol IDA/ml and pure water fluxes of 7.8, 5.8 and 0.42 cm/min, respectively. However, lysozyme capacity was close to 4 μmol/ml for all fibres. Adsorption isotherms for lysozyme and pectinesterase did not fit Langmuir-type curves and the existence of two types of ligand (A and B) with different accessibility to proteins was assumed. For pectinesterase, maximum capacities of 5100 and 2900 U/ml and dissociation constants of 25 and 316 U/ml were found, respectively, for ligands A and B. For lysozyme, maximum capacities were 2.9 and 0.9 μmol/ml and dissociation constants 5.0 and 102 μM, respectively, for said ligands. A cartridge assembled with IDA hollow fibres had a dynamic capacity for pectinesterase of 7509 U/ml. Productivity of this cartridge for pectic enzyme fractionation was 750 pectinesterase U/ml min, far higher than that obtained with a chelating soft gel (81 pectinesterase U/ml min).

Chromatographic characterization of immobilized metal ion hollow-fiber affinity membranes obtained by direct grafting

Abstract Iminodiacetic acid was immobilized onto membranes with different grafting degrees by reaction in phosphate buffer or water/dimethyl sulfoxide. Membranes subjected to conversion in water/dimethyl sulfoxide underwent greater conversion than those modified in phosphate buffer, despite their grafting degree. Copper saturation capacity increased consistently with the grafting degree and histidine saturation capacity was approximately half than that of copper. When working with proteins, membrane behavior was related to the molecular weight of the protein tested. Accessible sites for lysozyme decreased with the increase in the grafting degree and the rise in the conversion of epoxy groups in iminodiacetic groups in water/dimethyl sulfoxide while they remained practically unchanged when the conversion step was performed in phosphate buffer. When working with hemoglobin, this effect was the same but at lower capacities. For hollow fibres with 60 and 75% grafting, capacities were the same despite the conv...

High-Speed Pectic Enzyme Fractionation by Immobilised Metal Ion Affinity Membranes

Immobilised metal ion affinity polysulfone hollow-fibre membranes, with a high capacity for protein adsorption, were prepared and their utilisation for commercial pectic enzyme fractionation was studied. The pass-through fraction containing pectinlyase is useful for fruit-juice clarification without methanol production on account of pectinesterase being retained by the IDA-Cu2+ membrane.

Fungal pectic enzyme fractionation by dye affinity chromatography

A dye affinity fractionation of a commercial pectic enzyme preparation is described. From nine immobilized triazinic dyes assayed, only Blue R-HE and Yellow 4R-HE were able to retain pectin lyase thus allowing the obtention of a fraction not producing methanol during fruit juice clarification. While pectinesterase did not bind to the Chromatographic matrix, the retained pectin lyase could be quantitatively eluted with a salt gradient.