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Applied Microbiology and Biotechnology

, Volume 76, Issue 6, pp 1297–1307 | Cite as

Cloning, sequencing, overexpression and characterization of l-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production

  • Wayoon Poonperm
  • Goro TakataEmail author
  • Hiromi Okada
  • Kenji Morimoto
  • Tom Birger Granström
  • Ken Izumori
Biotechnologically Relevant Enzymes and Proteins

Abstract

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K m) and catalytic efficiency (k cat/K m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 M−1 min−1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.

Keywords

l-Rhamnose isomerase Bacillus pallidus Rare sugar 

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Wayoon Poonperm
    • 1
  • Goro Takata
    • 1
    Email author
  • Hiromi Okada
    • 1
  • Kenji Morimoto
    • 1
  • Tom Birger Granström
    • 1
  • Ken Izumori
    • 1
  1. 1.Rare Sugar Research Center and Faculty of AgricultureKagawa UniversityMikiJapan

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