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Abstract:

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.

Registro:

Documento: Artículo
Título:Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase
Autor:Miletti, L.C.; Marino, C.; Mariño, K.; De Lederkremer, R.M.; Colli, W.; Alves, M.J.M.
Filiación:Departamento de Bioquímica, Inst. Quim., Univ. S. Paulo, C.P., São Paulo, Brazil
CIHIDECAR, Depto. Quim. Organ., Fac. Cie. E., Buenos Aires, Argentina
Palabras clave: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
Año:1999
Volumen:320
Número:3-4
Página de inicio:176
Página de fin:182
DOI: http://dx.doi.org/10.1016/S0008-6215(99)00158-5
Título revista:Carbohydrate Research
Título revista abreviado:Carbohydr. Res.
ISSN:00086215
CODEN:CRBRA
CAS:4-aminophenyl 1-thio-galactofuranoside; beta-Galactosidase, EC 3.2.1.23; exo-beta-D-galactofuranosidase, EC 3.2.1.-; Galactosides; Glycoside Hydrolases, EC 3.2.1.-; Ligands
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00086215_v320_n3-4_p176_Miletti

Referencias:

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Citas:

---------- APA ----------
Miletti, L.C., Marino, C., Mariño, K., De Lederkremer, R.M., Colli, W. & Alves, M.J.M. (1999) . Immobilized 4-aminophenyl 1-thio-β-D-galactofuranoside as a matrix for affinity purification of an exo-β-D-galactofuranosidase. Carbohydrate Research, 320(3-4), 176-182.
http://dx.doi.org/10.1016/S0008-6215(99)00158-5
---------- CHICAGO ----------
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" . Carbohydrate Research 320, no. 3-4 (1999) : 176-182.
http://dx.doi.org/10.1016/S0008-6215(99)00158-5
---------- MLA ----------
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" . Carbohydrate Research, vol. 320, no. 3-4, 1999, pp. 176-182.
http://dx.doi.org/10.1016/S0008-6215(99)00158-5
---------- VANCOUVER ----------
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. Carbohydr. Res. 1999;320(3-4):176-182.
http://dx.doi.org/10.1016/S0008-6215(99)00158-5