Extremophiles

, Volume 13, Issue 2, pp 299–311

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

  • Xiangxian Ying
  • Amy M. Grunden
  • Lin Nie
  • Michael W. W. Adams
  • Kesen Ma
Original Paper

Abstract

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg−1, which was very similar to that of the native enzyme (110 ± 3 U mg−1). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower Km-values for acetaldehyde and NADPH and higher Km-values for ethanol and NADP+. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.

Keywords

Iron-containing alcohol dehydrogenase Heterologous expression Hyperthermophile Archaea Thermococcus 

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Copyright information

© Springer 2008

Authors and Affiliations

  • Xiangxian Ying
    • 1
    • 4
  • Amy M. Grunden
    • 2
  • Lin Nie
    • 1
  • Michael W. W. Adams
    • 3
  • Kesen Ma
    • 1
  1. 1.Department of BiologyUniversity of WaterlooWaterlooCanada
  2. 2.Department of MicrobiologyNorth Carolina State UniversityRaleighUSA
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of GeorgiaAthensUSA
  4. 4.Zhejiang University of TechnologyZhejiangChina

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