Immobilized 4-aminophenyl 1-thio-beta-D-galactofuranoside as a matrix for affinity purification of an exo-beta-D-galactofuranosidase.
An alternative and fast method for the purification of an exo-beta-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-beta-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-beta-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 Km and Vmax values of 0.311 mM and 17 mumol h-1 microgram-1 respectively, when 4-nitrophenyl beta-D-galactofuranoside was employed as the substrate.
Miletti
L C
LC
Departamento de Bioquímica, Universidade de São Paulo, Brazil.
Marino
C
C
Mariño
K
K
de Lederkremer
R M
RM
Colli
W
W
Alves
M J
MJ
D002846:Chromatography, Affinity / Q000379:methods*; D005697:Galactosides; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010407:Penicillium / Q000201:enzymology; D001616:beta-Galactosidase / Q000302:isolation & purification*
C403129:4-aminophenyl 1-thio-galactofuranoside; D005697:Galactosides; D008024:Ligands; D006026:Glycoside Hydrolases; C023112:exo-beta-D-galactofuranosidase; D001616:beta-Galactosidase