A Novel Magnetic Affinity Support for Protein Adsorption and Purification

Abstract

A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.

Horizontal gene transfer promoted evolution of the ability to propagate under anaerobic conditions in yeasts

“…This step was repeated once and the pellets from both centrifugations were dissolved in isolation medium and combined to give the pellet fraction. Hexokinase (Bergmeyer 1974), alcohol dehydrogenase (Tong et al 2001) and succinate dehydrogenase (Knecht et al 1996) activities were assayed as described previously. DHODase activity, using DHO as substrate and ferricyanide as electron acceptor, was measured as described above.…”

“…After fractionation of the cell lysate, the DHODase activities were determined using DHO as substrate and FeCy as electron acceptor. These activities were compared with the activities of marker enzymes; hexokinase (Bergmeyer 1974) and alcohol dehydrogenase (Tong et al 2001) as cytosolic markers, and succinate dehydrogenase (Knecht et al 1996) as a mitochondrial marker. The pellet fraction, containing mitochondrial markers, exhibited the same low DHODase activity regardless of whether it was prepared from aerobically or anaerobically grown cells.…”

Immobilization of lipase on surface modified magnetic nanoparticles using alkyl benzenesulfonate

“…Bioactive substances such as enzymes, proteins [2][3][4][5][6][7][8][9][10][11], have been bound to it. Iron oxide nanoparticles have been widely used as magnetic resonance (MR) contrast agents, and important studies such as cancer, gene expression, angiogenesis imaging, and cellular trafficking have been well performed [12][13][14][15][16].…”

Preparation and properties of magnetic nano‐ and microsized particles for biological and environmental separations

“…Briefly, they include magnetic dye-ligand chromatography, magnetic supports with chelates of transition metals and affinity ligands for the isolation of complementary substances. Magnetic supports with reactive dyes, e. g., Cibachrom Blue F3GA, or Procion Red 120, were used as group-selective ligands for the isolation and purification of lysozyme [218], albumin, alcohol dehydrogenase [218], and lactate dehydrogenase [219]. Magnetic supports with chelates of transition metals (IMAC -see Section 3.1) found widespread applications in the purification of cytochrome C, uricase, hemoglobin, etc.…”