Archaeal alcohol dehydrogenase active at increased temperatures and in the presence of organic solvents.
The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83 degrees C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD(+) as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t (1/2) = 42 min at 70 degrees C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.
Hess
Matthias
M
Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany.
Antranikian
Garabed
G
D019840:2-Propanol / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity; D019603:Thermoplasmales / Q000201:enzymology* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
D019843:Archaeal Proteins; D003067:Coenzymes; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012997:Solvents; D009243:NAD; D000431:Ethanol; D014508:Urea; D000426:Alcohol Dehydrogenase; D019840:2-Propanol