Immobilized 4-aminophenyl 1-thio-β-
-galactofuranoside as a matrix for affinity purification of an exo-β-
-galactofuranosidase |
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Authors: | Luiz C Miletti Carla Marino Karina Mario Rosa M de Lederkremer Walter Colli Maria Júlia M Alves |
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Institution: | Luiz C. Miletti, Carla Marino, Karina Mariño, Rosa M. de Lederkremer, Walter Colli,Maria Júlia M. Alves, |
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Abstract: | An alternative and fast method for the purification of an exo-β-
-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β-
-galactofuranoside affinity chromatography system and specific elution with 10 mM
-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM
-galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β-
-galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 μmol h−1 μg−1 respectively, when 4-nitrophenyl β-
-galactofuranoside was employed as the substrate. |
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Keywords: | Affinity chromatography β -
-Galactofuranose exo-β -
-Galactofuranosidase Penicillium fellutanum 1-thio-β -
-Galactofuranoside |
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