Abstract
The genome of Natronomonas pharaonis encodes genes annotated as alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3), enzymes involved in alcohol metabolism. These genes (adh and aldH2) occur in a single copy on the chromosome. We have studied the role of these genes in ethanol metabolism in N. pharaonis. Reverse transcription-PCR analysis showed that the aldH2 gene was inducible by ethanol, but the adh gene was transcribed both in the presence and absence of ethanol. The gene encoding for ALDH of N. pharaonis (NpALDH) was cloned into a pET41a vector containing a glutathione S-transferase tag, expressed in Escherichia coli and purified by glutathione sepharose affinity chromatography. The GST-NpALDH fusion protein was cleaved by bovine enterokinase and the target enzyme showed a molecular mass of approximately 60 kDa by SDS-PAGE. The enzyme was thermophilic and alkaliphilic, the optimal temperature and pH being 60°C and 8.0, respectively. NpALDH was salt independent, being most active at 0.25 M NaCl or KCl.
| Original language | English |
|---|---|
| Pages (from-to) | 849-854 |
| Number of pages | 6 |
| Journal | Extremophiles |
| Volume | 12 |
| Issue number | 6 |
| DOIs | |
| State | Published - Nov 2008 |
Bibliographical note
Funding Information:Acknowledgments This work was supported by the grants from the Major State Basic Research Development Program of China (973 Program) (Grant No. 2004CB719604-3) and the National Natural Science Foundation of China (Grant No. 30670048).
Keywords
- Aldehyde dehydrogenase
- Haloalkaliphilic
- Molecular cloning
- Natronomonas pharaonis
- Reverse transcription-PCR