Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: Bioconversion of D-galactose to D-tagatose using the enzyme

Byoung Chan Kim, Yoon Hee Lee, Han Seung Lee, Dong Woo Lee, Eun Ah Choe, Yu Ryang Pyun

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Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56 677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85°C, and required divalent cations such as Co2+ and Mn2+ for its activity and thermostability. The apparent Km values of the enzyme for L-arabinose and D-galactose were 116 mM (vmax, 119 μmol min-1 mg-1) and 250 mM (vmax, 14.3 μmol min-1 mg-1), respectively, that were determined in the presence of both 1 mM Co2+ and 1 mM Mn2+. A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.

Original languageEnglish
Pages (from-to)121-126
Number of pages6
JournalFEMS Microbiology Letters
Issue number1
Publication statusPublished - 2002 Jun 18


All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Genetics

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