Skip to main content

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Applied Microbiology and Biotechnology volume 97, pages 195–203 (2013)Cite this article

Abstract

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD+-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Adams MWW, Perler FB, Kelly RM (1995) Extremozymes: expanding the limits of biocatalysis. Nat Biotechnol 13:662–668

    Article  CAS  Google Scholar 

  • Allers T, Ngo HP, Mevarech M, Lloyd RG (2004) Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol 70:943–953

    Article  CAS  Google Scholar 

  • Allers T, Barak S, Liddell S, Wardell K, Mevarech M (2010) Improved strains and plasmid vectors for conditional overexpression of his-tagged proteins in Haloferax volcanii. Appl Environ Microbiol 76:1759–1769

    Article  CAS  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  CAS  Google Scholar 

  • Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408

    Article  CAS  Google Scholar 

  • Bitan-Banin G, Ortenberg R, Mevarech M (2003) Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene. J Bacteriol 185:772–778

    Article  CAS  Google Scholar 

  • Camacho M, Rodriguez-Arnedo A, Bonete M (2002) NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli. FEMS Microbiol Lett 209:155–160

    Article  CAS  Google Scholar 

  • Cao Y, Liao L, Xu X, Oren A, Wang C, Zhu X, Wu M (2008a) Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis. Extremophiles 12:471–476

    Article  CAS  Google Scholar 

  • Cao Y, Liao L, Xu X, Oren A, Wu M (2008b) Aldehyde dehydrogenase of the haloalkaliphilic archaeon Natronomonas pharaonis and its function in ethanol metabolism. Extremophiles 12:849–854

    Article  CAS  Google Scholar 

  • Cendrin F, Chroboczek J, Zaccai G, Eisenberg H, Mevarech M (1993) Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui. Biochemistry 32:4308–4313

    Article  CAS  Google Scholar 

  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500

    Article  CAS  Google Scholar 

  • Connaris H, Chaudhuri JB, Danson MJ, Hough DW (1999) Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli. Biotechnol Bioeng 64:38–45

    Article  CAS  Google Scholar 

  • Coquelle N, Talon R, Juers DH, Girard E, Kahn R, Madern D (2010) Gradual adaptive changes of a protein facing high salt concentrations. J Mol Biol 404:493–505

    Article  CAS  Google Scholar 

  • Danson MJ, Hough DW (1998) Structure, function and stability of enzymes from the Archaea. Trends Microbiol Rev 6:307–314

    Article  CAS  Google Scholar 

  • Dym O, Mevarech M, Sussman JL (1995) Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 267:1344–1346

    Article  CAS  Google Scholar 

  • Eichler J (2001) Biotechnological uses of archaeal extremozymes. Biotechnol Adv 19:261–278

    Article  CAS  Google Scholar 

  • Flam F (1994) The chemistry of life at the margins. Science 265:471–472

    Article  CAS  Google Scholar 

  • Friest JA, Maezato Y, Broussy S, Blum P, Berkowitz DB (2010) Use of a robust dehydrogenase from an archael hyperthermophile in asymmetric catalysis-dynamic reductive kinetic resolution entry into (S)-profens. J Am Chem Soc Commun 132:5930–5931

    Article  CAS  Google Scholar 

  • Giacomini D, Galletti P, Quintavalla A, Gucciardo G, Paradisi F (2007) Highly efficient asymmetric reduction of arylpropionic aldehydes by horse liver alcohol dehydrogenase through dynamic kinetic resolution. Chem Commun 21:4038–4040

    Article  Google Scholar 

  • Goldberg K, Schroer K, Lütz S, Liese A (2007) Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes. Appl Microbiol Biotechnol 76:237–248

    Article  CAS  Google Scholar 

  • Gray CJ (1988) Additives and enzyme stability. Biocatal Biotrans 1:187–196

    Article  CAS  Google Scholar 

  • Guy CP, Haldenby S, Brindley A, Walsh DA, Briggs GS, Warren MJ, Allers T, Bolt EL (2006) Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP. J Mol Biol 358:46–56

    Article  CAS  Google Scholar 

  • Hartman AL, Norais C, Badger JH, Delmas S, Haldenby S, Madupu R, Robinson J, Khouri H, Ren Q, Lowe TM, Maupin-Furlow J, Pohlschroder M, Daniels C, Pfeiffer F, Allers T, Eisen JA (2010) The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS One 5:e9605

    Article  Google Scholar 

  • Hough DW, Danson MJ (1999) Extremozymes. Curr Opin Chem Biol 3:39–46

    Article  CAS  Google Scholar 

  • Huisman GW, Liang J, Krebber A (2010) Practical chiral alcohol manufacture using ketoreductases. Curr Opin Chem Biol 14:122–129

    Article  CAS  Google Scholar 

  • Lesk AM (1995) NAD-binding domains of dehydrogenases. Curr Opin Struct Biol 5:775–783

    Article  CAS  Google Scholar 

  • McKinley-McKee J (1964) The mechanism of action and the active centre of the alcohol dehydrogenases. Progr Biophys Mol Biol 14:225–262

    Article  Google Scholar 

  • Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H, Allers T (2007) Genetic and physical mapping of DNA replication origins in Haloferax volcanii. PLoS Genet 3:e77

    Article  Google Scholar 

  • Oren A (2002) Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J Ind Microbiol Biotechnol 28:56–63

    CAS  Google Scholar 

  • Parkot J, Gröger H, Hummel W (2010) Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters. Appl Microbiol Biotechnol 86:1813–1820

    Article  CAS  Google Scholar 

  • Pire C, Esclapez J, Diaz S, Perez-Pomares F, Ferrer J, Bonete MJ (2009) Alteration of coenzyme specificity in halophilic NAD(P)+ glucose dehydrogenase by site-directed mutagenesis. J Mol Catal B: Enzym 59:261–265

    Article  CAS  Google Scholar 

  • Schmid RD (1979) Stabilized soluble enzymes. Adv Biochem Eng Biotechnol 12:41–118

    CAS  Google Scholar 

  • Sellek GA, Chaudhuri JB (1999) Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 25:471–482

    Article  CAS  Google Scholar 

  • Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310

    Article  CAS  Google Scholar 

  • Timpson LM, Alsafadi D, Mac Donnchadha C, Liddell S, Sharkey MA, Paradisi F (2012) Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea. Extremophiles 16:57–66

    Article  CAS  Google Scholar 

  • Vallee BL, Hoch FL (1957) Zinc in horse liver alcohol dehydrogenase. J Biol Chem 225:185–195

    CAS  Google Scholar 

  • Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60

    Article  CAS  Google Scholar 

  • Wierenga RK, De Maeyer MCH, Hol WGJ (1985) Interaction of pyrophosphate moieties with alpha-helixes in dinucleotide-binding proteins. Biochemistry 24:1346–1357

    Article  CAS  Google Scholar 

  • Wilkinson GN (1961) Statistical estimations in enzyme kinetics. Biochem J 80:324–332

    CAS  Google Scholar 

  • Yakushi T, Matsushita K (2010) Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology. Appl Microbiol Biotechnol 86:1257–1265

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a research grant awarded to F.P. by Science Foundation Ireland (SFI), a University Research Fellowship awarded to T.A. by the Royal Society, and funding provided by the Islamic Development Bank (IsDB).

Author information

Authors and Affiliations

  1. Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland

    Leanne M. Timpson, Ann-Kathrin Liliensiek, Diya Alsafadi, Jennifer Cassidy & Francesca Paradisi

  2. UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

    Michael A. Sharkey

  3. Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

    Susan Liddell

  4. Institute of Genetics, School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK

    Thorsten Allers

Authors
  1. Leanne M. Timpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ann-Kathrin Liliensiek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Diya Alsafadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jennifer Cassidy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael A. Sharkey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Susan Liddell
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thorsten Allers
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Francesca Paradisi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Thorsten Allers or Francesca Paradisi.

Additional information

L. M. Timpson and A.-K. Liliensiek contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Timpson, L.M., Liliensiek, AK., Alsafadi, D. et al. A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii . Appl Microbiol Biotechnol 97, 195–203 (2013). https://doi.org/10.1007/s00253-012-4074-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4074-4

Keywords

  • Alcohol dehydrogenase
  • Biocatalyst discovery
  • Protein characterization
  • Extremophile
  • Haloferax volcanii
  • Organic solvents