Preparation of stable, highly active and immobilized glucose oxidase usingthe anti-enzyme antibodies and F(ab)'(2)

Immobilization of the Aspergillus niger glucose oxidase in high yield was a chieved using an immunoaffinity-based procedure. For this purpose IgGs, iso lated from the sera of rabbits immunized with glucose oxidase, were favoura bly oriented by binding on to cobalt-charged iminodiacetate-Sepharose. Larg e amounts of glucose oxidase could be immobilized by incubating the IgG-bou nd matrix alternately with the enzyme and either intact IgG or F(ab)(2)(') derived thereof, leading to the formation of multiple enzyme layers. After three incubation cycles using anti-(glucose oxiclase) IgG, an 8-fold increa se in the amount of enzyme immobilized was observed, while the increase was 11-fold when the F(ab)(2)(') replaced intact IgG. The preparations obtaine d thus were highly active, as also indicated by the high effectiveness fact or, eta. Immunoaffinity-layered immobilized preparations were markedly more resistant to inactivation induced by exposure to 60 degreesC, 4.0 M urea o r storage at 4 degreesC. The preparations also exhibited a remarkable resis tance against inactivation induced by the water-miscible organic solvents t etrahydrofuran, dioxan or acetone. Immobilized glucose oxidase preparations obtained using F(ab)(2)(') were generally observed to be superior in stabi lity compared with those immobilized with the help of intact IgG.