Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.
Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.
Karmali
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Departamento de Engenharia Química do Instituto Superior de Engenharia de Lisboa, Lisbon, Portugal.
Oliveira
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D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001487:Basidiomycota / Q000201:enzymology*; D015153:Blotting, Western; D002237:Carbohydrate Dehydrogenases / Q000276:immunology* / Q000302:isolation & purification; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005949:Glucose Oxidase / Q000276:immunology* / Q000302:isolation & purification; D006825:Hybridomas; D008031:Lignin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008031:Lignin; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005949:Glucose Oxidase