Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus (Articolo in rivista)

Type
Label
  • Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus (Articolo in rivista) (literal)
Anno
  • 2008-01-01T00:00:00+01:00 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#doi
  • 10.1128/AEM.00217-08 (literal)
Alternative label
  • Angela Pennacchio; Biagio Pucci; Francesco Secundo; Francesco La Cara; Mosè Rossi; Carlo A. Raia. (2008)
    Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus
    in Applied and environmental microbiology (Print)
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Angela Pennacchio; Biagio Pucci; Francesco Secundo; Francesco La Cara; Mosè Rossi; Carlo A. Raia. (literal)
Pagina inizio
  • 3949 (literal)
Pagina fine
  • 3958 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroVolume
  • 74 (literal)
Rivista
Note
  • ISI Web of Science (WOS) (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni
  • Ist Biochim Prot, CNR, I-80131 Naples, Italy Ist Chim & Riconoscimento Mol, CNR, I-20131 Milan, Italy (literal)
Titolo
  • Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus (literal)
Abstract
  • The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily has been identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The ttadh gene was heterologously overexpressed in Escherichia coli and the protein (TtADH) was purified to homogeneity and characterized. TtADH is a tetrameric enzyme of identical 26,961 Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity up to approximately 73 degrees C, a 30 min half-inactivation temperature of approximately 90 degrees C, and good tolerance towards common organic solvents. TtADH shows a strict requirement for NAD(H) as the coenzyme, preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. The thermophilic enzyme catalyses the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, alpha-methyl and alpha-ethyl benzoylformate to (S)-(-)-1-phenylethanol (>99% enantiomeric excess, ee), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-1-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee) and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of D37 in discriminating NAD(H) from NADP(H) in members of the SDR superfamily. (literal)
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