Preparation of a highly stable, very active and high-yield multilayered assembly of glucose oxidase using carbohydrate-specific polyclonal antibodies

The purified oligosaccharide chains of Aspergillus niger glucose oxidase were coupled to BSA with the help of the cross-linking reagent glutaraldehyde. The neoglycoconjugate thus obtained was purified by concanavalin A–Sepharose chromatography and characterized by SDS/PAGE. Immunization of rabbits with the neoglycoprotein raised the glycosyl-specific anti-(glucose oxidase) polyclonal antibodies. Antibodies were purified by (NH4)2SO4 precipitation, followed by DEAE-cellulose chromatography. The IgG–Sepharose was prepared by covalently coupling the purified polyclonal antibodies to the CNBr-activated Sepharose 4B. The large assembly of glucose oxidase was made on the IgG–Sepharose by alternate incubation of glucose oxidase and glycosyl-specific anti-(glucose oxidase) polyclonal IgG. The immunoaffinity-layered assembled preparations were highly active and, after six alternate binding cycles with enzyme and glycosylspecific IgG, the amount of enzyme immobilized could be raised 30-fold. The Km value of immunoaffinity-layered glucose oxidase preparations remained unaltered, while the Vmax slightly decreased as compared with the soluble enzyme. A layer-by-layer immobilization of glucose oxidase resulted in significant improvement in stability against high temperature, 4.0 M urea and very high concentrations of water-miscible organic solvents such as acetone, dimethylformamide, dioxan and tetrahydrofuran.