An extracellular, constitutive, and nonglucose repressed beta-glucosidase from a yeast strain of Debaryomyces hansenii was purified and immobilized using a one-step procedure on hydroxyapatite (HTP). Analysis of purified enzyme gave two bands both on SDS gel electrophoresis, native gel electrophoresis, and capillary electrophoresis. The two bands on SDS gels were positive for carbohydrate staining. Their apparent molecular mass was estimated to be 122 and 96 kDa with carbohydrates, and 109 and 81 kDa after carbohydrate removal, respectively. Amino acid analysis of electroblotted bands revealed that the N-terminus was blocked in both cases. Gel slices corresponding to the two bands, as obtained after native gel electrophoresis, were found to be reactive when incubated separately with p-nitro-phensyl-beta-D-glucopyranoside (pNPG) as substrate. The K-m of the two forms coeluted from HTP in the same fractions was 3.68 +/- 0.06 mM. The optimum pH was 5. The immobilized enzyme exhibited a lower activity than the purified free enzymes, but both were much more stable than the enzymes in cell-free supernatant. The two enzyme isoforms in the mixture were only active against few glycosides with beta-linkage configuration. Since the HTP-bound enzyme was found to be active, stable, easily separable from the substrate, and reusable, it could be potentially used in its immobilized form for the release of specific-bound aroma in wine and fruit juices.