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 (NH(4))(2)SO(4) 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 glycosyl-specific IgG, the amount of enzyme immobilized could be raised 30-fold. The K (m) value of immunoaffinity-layered glucose oxidase preparations remained unaltered, while the V (max) 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.
Jan
Ulfat
U
Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh-202002, India.
Husain
Qayyum
Q
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D001234:Aspergillus niger / Q000201:enzymology; D002241:Carbohydrates / Q000737:chemistry* / Q000276:immunology; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D005949:Glucose Oxidase / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011817:Rabbits; D013696:Temperature; D014508:Urea / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002241:Carbohydrates; D004800:Enzymes, Immobilized; D006023:Glycoproteins; D014508:Urea; D005949:Glucose Oxidase