Macroporous chitin affinity membranes for wheat germ agglutinin purification from wheat germ

Abstract

Macroporous chitin membranes of controlled porosity and pore sizes have been prepared. They have good mechanical properties and allow high flow rates of protein solutions at low pressure drops. Because of the numerous N-acetyl-D-glucosamine (GlcNAc) moieties they contain, the chitin membranes can be used for the separation of some valuable proteins both as affinity ligands and support matrix, without further modification. Due to their high porosity and high adsorption surface area, the chitin membranes provide a larger number of accessible binding sites for the wheat germ agglutinin than the chitin beads do. The adsorption capacity for wheat germ agglutinin (180 mg/g chitin membrane) is about 20 times larger than that of chitin beads. Because of the numerous binding sites, multiple-point bindings are involved in the protein adsorption. For this reason, a strong eluant, namely a 1 M acetic acid aqueous solution, had to be used to efficiently recover the wheat germ agglutinin from the membrane. The wheat germ agglutinin was extracted from wheat germ with 0.05 M HCl, precipitated with ammonium sulfate, dialyzed against 0.01 M Tris–HCl buffer (pH 8.5), and purified on the chitin membrane. A high purity (>99%) wheat germ agglutinin with high yield (∼50 mg/100 g wheat germ) was obtained.