Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (20 Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioesterase
نویسنده
چکیده
The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (20 Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 20 Adh had a lower catalytic efficiency for the oxidation of 1P alcohols, including ethanol, than for the oxidation of secondary (20) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined byNADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (10 Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE)
منابع مشابه
Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme.
The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 degrees C with a half-life of approx. 1.7 h at 90 degrees C. An NADP(H)-dependent enzym...
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Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent M(r)s of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetram...
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A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM ...
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