Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis.
Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70 degrees C, respectively. The optimal pH was 9.0. Zn2+ significantly inhibited activity. The Km value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.
Cao
Yi
Y
College of Life Sciences, Zhejiang University, Hangzhou 310058, People's Republic of China.
Liao
Li
L
Xu
Xue-wei
XW
Oren
Aharon
A
Wang
Ce
C
Zhu
Xu-feng
XF
Wu
Min
M
D000079:Acetaldehyde / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism*; D006216:Halobacteriaceae / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010802:Phylogeny; D011189:Potassium Chloride / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000378:metabolism
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000431:Ethanol; D012965:Sodium Chloride; D011189:Potassium Chloride; D000426:Alcohol Dehydrogenase; D000079:Acetaldehyde; D015032:Zinc