Now showing 1 - 2 of 2
  • Publication
    Heterologous overexpression, purification and characterisation of an alcohol dehydrogenase (ADH2) from Halobacterium sp. NRC-1
    Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterization of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography (IMAC). His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.
      382Scopus© Citations 8
  • Publication
    A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii
    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.
      511Scopus© Citations 37