title: Purification and enzymatic characterization of a novel β-1,6-glucosidase from Aspergillus oryzae.
abstract: In this study, among the 10 genes that encode putative β-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding β-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 β-d-glucopyranoside as the fluorogenic substrate for β-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 β-glucosidases, and BglH was stable over a wide temperature range (4°C-60°C). BglH was inhibited by Hg(2+), Zn(2+), glucono-δ-lactone, glucose, dimethyl sulfoxide, and ethanol, but not by ethylenediaminetetraacetic acid. Interestingly, BglH preferentially hydrolyzed gentiobiose rather than other oligosaccharides and aryl β-glucosides, thereby demonstrating that this enzyme is a β-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of β-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable β-glucosidase such as BglH for reducing the bitter taste of gentiobiose, and thus for controlling the sweetness of starch hydrolysates in the food industry via genome mining.
authors: Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: awatanabe@biochem.tohoku.ac.jp.
authors: Akira
authors: A
authors: Watanabe
authors: Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors: Moe
authors: M
authors: Suzuki
authors: Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors: Seiryu
authors: S
authors: Ujiie
authors: Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors: Katsuya
authors: K
authors: Gomi
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism*; D004795:Enzyme Stability; D019649:Food Industry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004187:Disaccharides; D001617:beta-Glucosidase; C100052:gentiobiose
keywords: Aspergillus oryzae; Gentiobiose; Glycoside hydrolase family 3; β-1,6-Glucosidase; β-Glucosidase