Stable Ammonia-specific NAD Synthetase from<i>Bacillus stearothermophilus</i>: Purification, Characterization, Gene Cloning, and Applications

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  • YAMAGUCHI Fumihiko
    <i>Planova Technology Development, Asahi-Kasei Corporation</i>
  • KOGA Shinji
    <i>Diagnostics Research and Development Department, Asahi-Kasei Corporation</i>
  • YOSHIOKA Issei
    <i>Diagnostics Research and Development Department, Asahi-Kasei Corporation</i>
  • TAKAHASHI Mamoru
    <i>Diagnostics Research and Development Department, Asahi-Kasei Corporation</i>
  • SAKURABA Haruhiko
    <i>Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima</i>
  • OHSHIMA Toshihisa
    <i>Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima</i>

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タイトル別名
  • Stable Ammonia-specific NAD Synthetase from Bacillus stearothermophilus: Purification, Characterization, Gene Cloning, and Applications.

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  Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60°C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60°C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 μM) with this stable NAD synthetase was possible.<br>

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