Get access

Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD+ and NADP+

Authors

  • Xi Wu,

    1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; telephone: +81-45-924-5766; fax: +81-45-924-5766
    2. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China; telephone: +86-10-62774771; fax: +86-10-62770304
    Search for more papers by this author
  • Hiroki Kobori,

    1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; telephone: +81-45-924-5766; fax: +81-45-924-5766
    Search for more papers by this author
  • Izumi Orita,

    1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; telephone: +81-45-924-5766; fax: +81-45-924-5766
    Search for more papers by this author
  • Chong Zhang,

    1. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China; telephone: +86-10-62774771; fax: +86-10-62770304
    Search for more papers by this author
  • Tadayuki Imanaka,

    1. Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Shiga, Japan
    Search for more papers by this author
  • Xin-Hui Xing,

    Corresponding author
    1. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China; telephone: +86-10-62774771; fax: +86-10-62770304
    • Department of Chemical Engineering, Tsinghua University, Beijing 100084, China; telephone: +86-10-62774771; fax: +86-10-62770304.
    Search for more papers by this author
  • Toshiaki Fukui

    Corresponding author
    1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; telephone: +81-45-924-5766; fax: +81-45-924-5766
    • Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; telephone: +81-45-924-5766; fax: +81-45-924-5766.
    Search for more papers by this author

Abstract

A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP+ and NAD+ in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP+ regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD+ regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45–60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air. Biotechnol. Bioeng. 2012;109: 53–62. © 2011 Wiley Periodicals, Inc.

Ancillary