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PMID:22015539
| Citation |
Timpson, LM, Alsafadi, D, Mac Donnchadha, C, Liddell, S, Sharkey, MA and Paradisi, F (2012) Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea. Extremophiles 16:57-66 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1007/s00792-011-0405-0 |
| Keywords |
Alcohol Dehydrogenase/chemistry; Alcohol Dehydrogenase/isolation & purification; Alcohol Dehydrogenase/metabolism; Biocatalysis; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Haloarcula marismortui/enzymology; Mass Spectrometry; Seawater/microbiology; Tandem Mass Spectrometry; Water Microbiology |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
enables |
GO:0008106: alcohol dehydrogenase (NADP+) activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0008106: alcohol dehydrogenase (NADP+) activity |
ECO:0000314: |
F |
Table 2 depicts the HmADH12 gene followed Michaelis–Menten kinetics with the acetaldehyde substrate and NADP+ and NADPH cofactors in the reductive reaction. The enzyme was positively co-operative with ethanol as a substrate in the oxidative reaction. |
complete | ||||
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