Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase
An alternative and fast method for the purification of an exo-β-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Pe...
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todo:paper_00086215_v320_n3-4_p176_Miletti2023-10-03T14:06:54Z Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase Miletti, L.C. Marino, C. Mariño, K. De Lederkremer, R.M. Colli, W. Alves, M.J.M. β-D-Galactofuranose 1-thio-β-D-Galactofuranoside Affinity chromatography exo-β-D-Galactofuranosidase Penicillium fellutanum 4 aminophenyl 1 thio beta dextro galactofuranoside concanavalin a dextro galactono 1,4 lactone enzyme antibody exo beta dextro galactofuranosidase galactofuranosidase glycoprotein immobilized enzyme microbial enzyme sepharose unclassified drug affinity chromatography article controlled study enzyme activity enzyme isolation enzyme kinetics enzyme purification enzyme substrate immunoaffinity chromatography immunoprecipitation nonhuman penicillium pH polyacrylamide gel electrophoresis priority journal technique western blotting beta-Galactosidase Chromatography, Affinity Galactosides Glycoside Hydrolases Hydrogen-Ion Concentration Kinetics Ligands Penicillium Penicillium fellutanum An alternative and fast method for the purification of an exo-β-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-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 D-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-D-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 K(m) and V(max) values of 0.311 mM and 17 μmol h-1 μg-1 respectively, when 4-nitrophenyl β-D-galactofuranoside was employed as the substrate. Copyright (C) 1999 Elsevier Science Ltd. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00086215_v320_n3-4_p176_Miletti |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
β-D-Galactofuranose 1-thio-β-D-Galactofuranoside Affinity chromatography exo-β-D-Galactofuranosidase Penicillium fellutanum 4 aminophenyl 1 thio beta dextro galactofuranoside concanavalin a dextro galactono 1,4 lactone enzyme antibody exo beta dextro galactofuranosidase galactofuranosidase glycoprotein immobilized enzyme microbial enzyme sepharose unclassified drug affinity chromatography article controlled study enzyme activity enzyme isolation enzyme kinetics enzyme purification enzyme substrate immunoaffinity chromatography immunoprecipitation nonhuman penicillium pH polyacrylamide gel electrophoresis priority journal technique western blotting beta-Galactosidase Chromatography, Affinity Galactosides Glycoside Hydrolases Hydrogen-Ion Concentration Kinetics Ligands Penicillium Penicillium fellutanum |
| spellingShingle |
β-D-Galactofuranose 1-thio-β-D-Galactofuranoside Affinity chromatography exo-β-D-Galactofuranosidase Penicillium fellutanum 4 aminophenyl 1 thio beta dextro galactofuranoside concanavalin a dextro galactono 1,4 lactone enzyme antibody exo beta dextro galactofuranosidase galactofuranosidase glycoprotein immobilized enzyme microbial enzyme sepharose unclassified drug affinity chromatography article controlled study enzyme activity enzyme isolation enzyme kinetics enzyme purification enzyme substrate immunoaffinity chromatography immunoprecipitation nonhuman penicillium pH polyacrylamide gel electrophoresis priority journal technique western blotting beta-Galactosidase Chromatography, Affinity Galactosides Glycoside Hydrolases Hydrogen-Ion Concentration Kinetics Ligands Penicillium Penicillium fellutanum Miletti, L.C. Marino, C. Mariño, K. De Lederkremer, R.M. Colli, W. Alves, M.J.M. Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| topic_facet |
β-D-Galactofuranose 1-thio-β-D-Galactofuranoside Affinity chromatography exo-β-D-Galactofuranosidase Penicillium fellutanum 4 aminophenyl 1 thio beta dextro galactofuranoside concanavalin a dextro galactono 1,4 lactone enzyme antibody exo beta dextro galactofuranosidase galactofuranosidase glycoprotein immobilized enzyme microbial enzyme sepharose unclassified drug affinity chromatography article controlled study enzyme activity enzyme isolation enzyme kinetics enzyme purification enzyme substrate immunoaffinity chromatography immunoprecipitation nonhuman penicillium pH polyacrylamide gel electrophoresis priority journal technique western blotting beta-Galactosidase Chromatography, Affinity Galactosides Glycoside Hydrolases Hydrogen-Ion Concentration Kinetics Ligands Penicillium Penicillium fellutanum |
| description |
An alternative and fast method for the purification of an exo-β-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-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 D-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-D-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 K(m) and V(max) values of 0.311 mM and 17 μmol h-1 μg-1 respectively, when 4-nitrophenyl β-D-galactofuranoside was employed as the substrate. Copyright (C) 1999 Elsevier Science Ltd. |
| format |
JOUR |
| author |
Miletti, L.C. Marino, C. Mariño, K. De Lederkremer, R.M. Colli, W. Alves, M.J.M. |
| author_facet |
Miletti, L.C. Marino, C. Mariño, K. De Lederkremer, R.M. Colli, W. Alves, M.J.M. |
| author_sort |
Miletti, L.C. |
| title |
Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| title_short |
Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| title_full |
Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| title_fullStr |
Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| title_full_unstemmed |
Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase |
| title_sort |
immobilized 4-aminophenyl 1-thio-β-d-galactofuranoside as a matrix for affinity purification of an exo-β-d-galactofuranosidase |
| url |
http://hdl.handle.net/20.500.12110/paper_00086215_v320_n3-4_p176_Miletti |
| work_keys_str_mv |
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| _version_ |
1807318941145497600 |