Immobilized 4-aminophenyl 1-thio-beta-D-galactofuranoside as a matrix for affinity purification of an exo-beta-D-galacofuranosidase

An alternative and fast method for the purification of an exo-beta-D-galact ofuranosidase has been developed using a 4-aminophenyl I-thio-beta-D-galact ofuranoside 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 fol lowed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM. D-galactono-1,it -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 mix ture of the single bands excised from the gel were capable of immunoprecita ting 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 ascertain ed through binding to Concanavalin A-Sepharose as well as by specific react ion with Schiff reagent in Western blots. The purified enzyme has an optimu m acidic pH (between 3 and 6), and K-m and V-max values of 0.311 mM and 17 mu mol h(-1) mu g(-1) respectively, when 4-nitrophenyl beta-D-galactofurano side was employed as the substrate. (C) 1999 Elsevier Science Ltd. All righ ts reserved.