Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.
J Agric Food Chem. 2010 Oct 13;58(19):10431-6. doi: 10.1021/jf102063q.
L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.
L-鼠李糖异构酶（EC 5.3.1.14，L-RhI）催化 L-鼠李糖和 L-鼠李酮之间的醛糖-酮糖的可逆异构化。在这项研究中，从嗜热厌氧杆菌 NTOU1 中通过 PCR 克隆了编码 L-Rhi 的 L-Rhi 基因，并在大肠杆菌中表达。在存在 0.2 mM IPTG 诱导的情况下，获得了 9780 U/g 湿细胞的高活性 L-RhI 的产量。使用热处理、核酸沉淀和阴离子交换色谱法依次对 L-RhI 进行纯化。纯化的 L-RhI 显示出明显的最适 pH 值为 7 和最适温度为 75°C。该酶在 pH 值为 5 到 9 的范围内稳定，在 40-70°C 孵育 2 小时后，活性完全保留。来自嗜热厌氧杆菌 NTOU1 的 L-RhI 是迄今为止最耐热的 L-RhI，它具有高比活性（163 U/mg）和可接受的热处理纯度，表明该酶有潜力用于稀有糖的生产。
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