Purification and enzymatic characterization of a novel ß-1,6-glucosidase from Aspergillus oryzae.

In this study, among the 10 genes that encode putative ß-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding ß-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 ß-d-glucopyranoside as the fluorogenic substrate for ß-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 ß-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 ß-glucosides, thereby demonstrating that this enzyme is a ß-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of ß-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable ß-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.

Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl ß-glucosidases screened from Aspergillus oryzae genome.

By a global search of the genome database of Aspergillus oryzae, we found 23 genes encoding putative ß-glucosidases, among which 10 genes with a signal peptide belonging to glycoside hydrolase family 3 (GH3) were overexpressed in A. oryzae using the improved glaA gene promoter. Consequently, crude enzyme preparations from three strains, each harboring the genes AO090038000223 (bglA), AO090103000127 (bglF), and AO090003001511 (bglJ), showed a substrate preference toward p-nitrophenyl-ß-d-glucopyranoside (pNPGlc) and thus were purified to homogeneity and enzymatically characterized. All the purified enzymes (BglA, BglF, and BglJ) preferentially hydrolyzed aryl ß-glycosides, including pNPGlc, rather than cellobiose, and these enzymes were proven to be aryl ß-glucosidases. Although the specific activity of BglF toward all the substrates tested was significantly low, BglA and BglJ showed appreciably high activities toward pNPGlc and arbutin. The kinetic parameters of BglA and BglJ for pNPGlc suggested that both the enzymes had relatively higher hydrolytic activity toward pNPGlc among the fungal ß-glucosidases reported. The thermal and pH stabilities of BglA were higher than those of BglJ, and BglA was particularly stable in a wide pH range (pH 4.5-10). In contrast, BglJ was the most heat- and alkaline-labile among the three ß-glucosidases. Furthermore, BglA was more tolerant to ethanol than BglJ; as a result, it showed much higher hydrolytic activity toward isoflavone glycosides in the presence of ethanol than BglJ. This study suggested that the mining of novel ß-glucosidases exhibiting higher activity from microbial genome sequences is of great use for the production of beneficial compounds such as isoflavone aglycones.