Watanabe Akira, Suzuki Moe, Ujiie Seiryu, Gomi Katsuya
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.
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.
J Biosci Bioeng. 2016 Mar;121(3):259-64. doi: 10.1016/j.jbiosc.2015.07.011. Epub 2015 Aug 29.
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.
在本研究中，在米曲霉基因组中糖苷水解酶家族3（GH3）中编码推定的β-葡萄糖苷酶且带有信号肽的10个基因中，我们发现了一个新基因（AO090038000425），其编码对龙胆二糖具有底物特异性的β-1,6-葡萄糖苷酶。携带AO090038000425（我们命名为bglH）的转化体在改良的glaA基因启动子控制下过表达，在以4-甲基伞形酮基β-D-吡喃葡萄糖苷作为β-葡萄糖苷酶的荧光底物的平板试验中，在菌落周围形成一个小的透明圈。我们将BglH纯化至同质，并对该酶进行了酶学表征。BglH的热稳定性和pH稳定性高于其他先前研究的米曲霉β-葡萄糖苷酶，并且BglH在较宽的温度范围（4℃-60℃）内稳定。BglH受到Hg(2+)、Zn(2+)、葡萄糖酸δ-内酯、葡萄糖、二甲基亚砜和乙醇的抑制，但不受乙二胺四乙酸的抑制。有趣的是，BglH优先水解龙胆二糖而非其他寡糖和芳基β-葡萄糖苷，从而证明该酶是一种β-1,6-葡萄糖苷酶。据我们所知，这是首次从曲霉属真菌或其他真核生物中纯化和表征β-1,6-葡萄糖苷酶的报道。本研究表明，通过基因组挖掘，有可能找到一种更合适的β-葡萄糖苷酶，如BglH，以降低龙胆二糖的苦味，从而控制食品工业中淀粉水解产物的甜度。
