Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42

Manish Kumar Tiwari, Hee-Jung Lee, Marimuthu Jeya, Jung-Kul Lee

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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 ∼34 kDa by sodium dodecyl sulfate–polyacrylamide gel and ∼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 Km=17.9 mM and kcat/Km=0.5 mM−1 s−1 for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 °C, respectively. Heat inactivation studies revealed a half life of the ReXDH at 40 °C of 120 min and a half denaturation temperature
(T1/2) of 53.1 °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
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Pages (from-to)571–581
Publication statusPublished - 2010
Externally publishedYes


  • Characterization
  • Homology modeling
  • Rhizobium etli
  • Thermostability
  • Xylitol dehydrogenase


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