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Preparation of a highly stable, very active and high‐yield multilayered assembly of glucose oxidase using carbohydrate‐specific polyclonal antibodies
Author(s) -
Jan Ulfat,
Husain Qayyum
Publication year - 2004
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20030092
Subject(s) - glucose oxidase , affinity chromatography , chemistry , polyclonal antibodies , sepharose , chromatography , biochemistry , glutaraldehyde , concanavalin a , enzyme , antibody , biology , immunology , in vitro
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.