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

Angela Pennacchio, Biagio Pucci, Francesco Secundo, Francesco La Cara, Mosè Rossi, Carlo A. Raia

Research output: Contribution to journalArticle

Abstract

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adhTt) was heterologously overexpressed in Escherichia coli, and the protein (ADH Tt) was purified to homogeneity and characterized. ADHTt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ∼73°C and a 30-min half-inactivation temperature of ∼90°C, as well as good tolerance to common organic solvents. ADHTt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and α-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, α-tetralone, and α-methyl and α-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-α-(trifluoromethyl)benzyl alcohol (93% ee), (S)-α-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 the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

Original languageEnglish
Pages (from-to)3949-3958
Number of pages10
JournalApplied and Environmental Microbiology
Volume74
Issue number13
DOIs
Publication statusPublished - Jul 2008

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

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