Himeno Nami, Saburi Wataru, Wakuta Shinji, Takeda Ryosuke, Matsuura Hideyuki, Nabeta Kensuke, Sansenya Sompong, Ketudat Cairns James R, Mori Haruhide, Imai Ryozo, Matsui Hirokazu
Research Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Japan.
Biosci Biotechnol Biochem. 2013;77(5):934-9. doi: 10.1271/bbb.120889. Epub 2013 May 7.
β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid β-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1 L of culture medium, 2.1 mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 °C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 °C. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid β-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid β-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.
β-葡萄糖苷酶（EC 3.2.1.21）可在糖苷和寡糖的非还原端切断β-糖苷键，释放出β-D-葡萄糖。植物β-葡萄糖苷酶的重要功能之一是使植物激素的无活性糖苷去糖基化，以调节活性激素的水平。块茎酸是一种与茉莉酸相关的化合物，在马铃薯中表现出诱导块茎形成的活性。我们已经鉴定出两种酶，即水稻（Oryza sativa L.）中的OsTAGG1和OsTAGG2，它们对块茎酸β-D-葡萄糖苷具有水解活性。OsTAGG2的表达受创伤和茉莉酸甲酯上调，这表明该同工酶参与了对生物胁迫和创伤的响应，但OsTAGG2的生理底物仍不明确。在本研究中，我们在毕赤酵母中生产了重组OsTAGG2（rOsTAGG2P），并详细研究了其底物特异性。通过硫酸铵沉淀和镍螯合柱色谱法，从1升培养基中获得了2.1毫克纯化的重组酶。rOsTAGG2P的比活性（182 U/mg）与天然酶（171 U/mg）接近，这与在大肠杆菌中产生的重组OsTAGG2不同，后者的比活性比天然酶低约3倍。rOsTAGG2P的最适pH和温度分别为pH 3.4和60℃。经过pH和热处理后，该酶在pH 3.4 - 9.8范围内以及55℃以下仍保留其原始活性。尽管OsTAGG2最初是基于对块茎酸β-D-葡萄糖苷（TAG）的水解活性从水稻中分离出来的，但天然OsTAGG2和rOsTAGG2P对水杨酸β-D-葡萄糖苷（水杨酸的一种无活性储存形式）的活性比对块茎酸β-D-葡萄糖苷（TAG）高4.5 - 4.7倍。
