In this study, among the 10 genes that encode putative beta-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding beta-1,6-glucosidase with a substrate specificity for gentiobiose. The transformant harboring AO090038000425, which we named bglH, was overexpressed under the control of the improved glaA gene promoter to form a small clear zone around the colony in a plate assay using 4-methylumbelliferyl beta-D-glucopyranoside as the fluorogenic substrate for beta-glucosidase. We purified BglH to homogeneity and enzymatically characterize this enzyme. The thermal and pH stabilities of BglH were higher than those of other previously studied A. oryzae beta-glucosidases, and BglH was stable over a wide temperature range (4 degrees C -60 degrees C). BgIH was inhibited by Hg2+, Zn2+, glucono-delta-lactone, glucose, dimethyl sulfoxide, and ethanol, but not by ethylenediaminetetraacetic acid. Interestingly, BgIH preferentially hydrolyzed gentiobiose rather than other oligosaccharides and aryl beta-glucosides, thereby demonstrating that this enzyme is a beta-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of beta-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable beta-glucosidase such as BgIH for reducing the bitter taste of gentiobiose, and thus for controlling the sweetness of starch hydrolysates in the food industry via genome mining. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.