Nihon Shokuhin Kako Co., Ltd., Shizuoka, Japan.
Appl Environ Microbiol. 2012 Mar;78(6):1836-45. doi: 10.1128/AEM.07514-11. Epub 2012 Jan 6.
An α-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl α-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30°C and pH 6.5. Monovalent cations such as K(+), Rb(+), Cs(+), and NH(4)(+) increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40°C in the presence of 10 mM NH(4)(+), although temperature stability was not affected. The apparent K(m) and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K(m) for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of α-d-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger α-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 α-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 α-glucosidase, respectively.
从盐单胞菌属 H11 菌株中分离到一种具有以下独特性质的α-葡萄糖苷酶（HaG）：（i）高转葡糖苷活性，（ii）一价阳离子激活，和（iii）非常窄的底物特异性。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）纯化的 HaG 的分子量估计为 58 kDa。HaG 对麦芽糖、蔗糖和对硝基苯-α-D-葡萄糖苷（pNPG）具有高水解活性，但对其他二糖或高于三糖的麦芽低聚糖几乎没有活性。HaG 在 30°C 和 pH 6.5 下对麦芽糖表现出最佳活性。一价阳离子，如 K(+)、Rb(+)、Cs(+)和 NH(4)(+)，将酶活性提高到原始活性的 2-9 倍。这些离子将活性-pH 曲线移向碱性侧。在 10 mM NH(4)(+)存在下，最适温度升高到 40°C，尽管温度稳定性没有受到影响。一价阳离子显著改善了麦芽糖和 pNPG 的表观 K(m)和 k(cat)值。令人惊讶的是，它们的存在使 pNPG 的 k(cat)/K(m)提高了 372-969 倍。HaG 在转葡糖苷化中用作一些醇的受体底物，并且在高效合成α-d-葡萄糖基甘油方面非常有用。从副产物的少量来看，生产水平的效率优于先前报道的黑曲霉α-葡萄糖苷酶。HaG 的序列分析表明，它被归类于糖苷水解酶家族 13。它的氨基酸序列与野油菜黄单胞菌 WU-9701α-葡萄糖苷酶、野油菜黄单胞菌 pv. 萝卜 756C 寡-1,6-葡糖苷酶、恶臭假单胞菌 DSM 4166 寡-1,6-葡糖苷酶和根癌农杆菌 F2α-葡萄糖苷酶的同源性分别为 60%、58%、57%和 56%。
