Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.
Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.
Centre for Synthesis and Chemical Biology, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland.
Leanne M
LM
Timpson
Diya
D
Alsafadi
Cillín
C
Mac Donnchadha
Susan
S
Liddell
Michael A
MA
Sharkey
Francesca
F
Paradisi
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019614:Haloarcula marismortui / Q000201:enzymology*; D013058:Mass Spectrometry; D012623:Seawater / Q000382:microbiology*; D053719:Tandem Mass Spectrometry; D014871:Water Microbiology
D000426:Alcohol Dehydrogenase