Preparation of stable, highly active and immobilized glucose oxidase using the anti-enzyme antibodies and F(ab)'2.
Immobilization of the Aspergillus niger glucose oxidase in high yield was achieved using an immunoaffinity-based procedure. For this purpose IgGs, isolated from the sera of rabbits immunized with glucose oxidase, were favourably oriented by binding on to cobalt-charged iminodiacetate-Sepharose. Large amounts of glucose oxidase could be immobilized by incubating the IgG-bound 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 oxidase) IgG, an 8-fold increase in the amount of enzyme immobilized was observed, while the increase was 11-fold when the F(ab)2 replaced intact IgG. The preparations obtained thus were highly active, as also indicated by the high effectiveness factor, eta. Immunoaffinity-layered immobilized preparations were markedly more resistant to inactivation induced by exposure to 60 degrees C, 4.0 M urea or storage at 4 degrees C. The preparations also exhibited a remarkable resistance against inactivation induced by the water-miscible organic solvents tetrahydrofuran, dioxan or acetone. Immobilized glucose oxidase preparations obtained using F(ab)2 were generally observed to be superior in stability compared with those immobilized with the help of intact IgG.
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Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh-202002, India.
Husain
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Saleemuddin
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D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001234:Aspergillus niger / Q000201:enzymology; D002847:Chromatography, Agarose; D005949:Glucose Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D013696:Temperature; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D000906:Antibodies; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D014508:Urea; D005949:Glucose Oxidase