Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea.
Appl Microbiol Biotechnol. 2011 Feb;89(3):635-44. doi: 10.1007/s00253-010-2844-4. Epub 2010 Sep 18.
Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an L-rhamnose isomerase (L-RhI). The identified L-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k(cat) of 8,971 min⁻¹ and a k(cat)/K(m) of 17 min⁻¹mM⁻¹ for L-rhamnose. Although L-RhIs have been characterized from several other sources, B. halodurans L-RhI is distinguished from other L-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans L-RhI a good choice for industrial applications. This work describes one of the most thermostable L-RhI characterized thus far.
对巴氏芽孢杆菌 ATCC BAA-125 的全基因组序列分析揭示了一个异构酶基因（rhaA），该基因编码 L-鼠李糖异构酶（L-RhI）。从巴氏芽孢杆菌中克隆并在大肠杆菌中过表达了鉴定出的 L-RhI 基因。DNA 序列分析显示，一个开放阅读框为 1257bp，能够编码一个 418 个氨基酸残基的多肽，分子量为 48178Da。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计纯化酶的分子量约为 48 kDa，通过凝胶过滤层析估计分子量为 121 kDa，表明该酶是一个同源二聚体。该酶的最适 pH 和温度分别为 7 和 70°C，L-鼠李糖的 k(cat)为 8971 min⁻¹，k(cat)/K(m)为 17 min⁻¹mM⁻¹。尽管已经从其他几个来源鉴定出了 L-RhIs，但巴氏芽孢杆菌 L-RhI 与其他 L-RhIs 的区别在于其高温最适温度（70°C），热稳定性高，在 60°C 下保持 100%活性长达 10 小时。该酶的半衰期在 60°C 和 70°C 下分别超过 900 分钟和 25 分钟，使巴氏芽孢杆菌 L-RhI 成为工业应用的理想选择。这项工作描述了迄今为止鉴定出的最耐热的 L-RhI 之一。
