Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, 143-701, Korea.
Appl Microbiol Biotechnol. 2010 Jun;87(2):571-81. doi: 10.1007/s00253-010-2478-6. Epub 2010 Feb 23.
An NAD(+)-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST affinity chromatography. The molecular mass of the purified enzyme was estimated to be approximately 34 kDa by sodium dodecyl sulfate-polyacrylamide gel and approximately 135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols, xylitol was the preferred substrate of ReXDH with a K (m) = 17.9 mM and k(cat) /K (m) = 0.5 mM(-1) s(-1) for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 degrees C, respectively. Heat inactivation studies revealed a half life of the ReXDH at 40 degrees C of 120 min and a half denaturation temperature (T (1/2)) of 53.1 degrees C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability.
从 Rhizobium etli CFN42（ReXDH）中克隆并在大肠杆菌中过表达了一种依赖 NAD(+)的木糖醇脱氢酶。DNA 序列分析显示，开放阅读框为 1044bp，能够编码 347 个氨基酸残基的多肽，计算分子量为 35858Da。使用 GST 亲和层析将 ReXDH 蛋白以活性可溶性形式纯化。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 34kDa，通过凝胶过滤色谱法约为 135kDa，表明该酶是同源四聚体。在各种多元醇中，木糖醇是 ReXDH 的首选底物，K(m)为 17.9mM，k(cat)/K(m)为 0.5mM(-1)s(-1)。该酶的最适 pH 和温度分别为 9.5 和 70°C。热失活动力学研究表明，ReXDH 在 40°C 时的半衰期为 120min，半变性温度（T(1/2)）为 53.1°C。ReXDH 的最适温度和热稳定性均高于已知的 XDHs。ReXDH 的同源建模和序列分析揭示了导致其高耐热性的因素。尽管已经从其他几个来源鉴定了 XDHs，但 ReXDH 因其高耐热性而与其他 XDHs 区分开来。
