热嗜木糖异构酶的特性研究来自于热纤梭菌 ATCC 43494。
Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494.
机构信息
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.
出版信息
J Agric Food Chem. 2011 Aug 24;59(16):8702-8. doi: 10.1021/jf201428b. Epub 2011 Aug 1.
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 Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied 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。在本研究中，从嗜热纤维梭菌 ATCC 43494 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 20°C 诱导 20 h 后，获得了 3010 U/g 湿细胞的高活性 L-RhI 的产量。该酶通过热处理、核酸沉淀和离子交换色谱法依次进行纯化。纯化的 L-RhI 显示出明显的最佳 pH 值为 7 和最佳温度为 90°C。该酶在 pH 值为 4 到 11 的范围内稳定，并在 80°C 和 pH 7-8 下孵育 6 小时后保留了>90％的活性。与其他先前表征的 L-RhIs 相比，来自 C. saccharolyticus ATCC 43494 的 L-RhI 具有良好的热稳定性、最宽的 pH 稳定范围和最高的对 L-鼠李糖、L-木酮糖、L-甘露糖、D-阿洛糖和 D-核糖的催化效率（k(cat)/K(M)），表明该酶有可能应用于稀有糖的生产。
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