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 (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C 30 fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).Microorganisms such as Saccharomyces or Zymomonas strains that are used for industrial ethanol production from glucose or sucrose have high alcohol tolerance for growth (i.e., Ͼ6% [vol/vol]). Other species that produce ethanol from cheaper substrates such as cellulose or starch, like Clostridium thermocellum or Thermoanaerobacter ethanolicus, generally have a low alcohol tolerance for growth (Ͻ2% [vol/vol]). In general, alcohol-producing microbes respond to increasing solvent concentrations by increasing the percentage of unsaturated versus saturated fatty acids, long-chain fatty acids, and hopanes into their cytoplasmic membranes (2,8,9). These structural changes prevent the loss of membrane function from fluidization caused by a high solvent concentration.Thermophilic ethanol fermentations offer the potential of direct degradation of cellulose or starch and direct recovery of ethanol at fermentation temperatures under reduced pressure (5,16,17,18,21,23). This potential has not been demonstrated because of low-end product concentrations caused by bacterial ethanol inhibition. Thermophilic bacteria employ two different pathways for ethanol production, using either a primary alcohol dehydrogenase (ADH), as in C. thermocellum, or primary and secondary ADHs, as in T. ethanolicus (13,15). Herrero and coworkers (3, 4) studied ethanol tolerance in C. thermocellum and concluded that the low tolerance to ethanol (Ͻ2% [vol/vol]) was a combined result of general solvent effects on membrane fluidity and a specific inhibition of enzymes involved in sugar metabolism. Work in the labs of Ljundahl, Wiegel, Demain, Zeikus, and others has showed that thermophilic anaerobic bacteria can adapt their tolerance to about 4% (vol/vol) ethanol (for a review, see reference 16).We previously demonstrated (15) that moderate ethanol tolerance (Ͻ4% [vol/vol]) of a T. ethanolicus mutant strain was related to enzymatic prevention of metabolic inhibition caused by ethanol overreducing the pyridine nucleotide pool and inhibiting glycolysis. The ethanol-tolerant mutant 39EA lacked primary ADH...

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The substrate specificity of wild-type and Ser39 → Thr (S39T) secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus was examined. The S39T mutation increases activity for 2-propanol without any significant effect on NADP+ binding. There is no significant effect of the mutation on the primary and secondary alcohol specificity of SADH. However, an effect on the enantiospecificity of SADH by the S39T mutation is demonstrated. Throughout the temperature range from 15 to 55 °C, wild-type SADH exhibits a preference for (S)-2-pentanol. In contrast, a temperature-dependent reversal of enantiospecificity is observed for 2-butanol, with a racemic temperature of 297 K. Throughout the same range of temperatures, S39T SADH exhibits higher enantiospecificity for the (R)-enantiomers of both 2-butanol and 2-pentanol. Examination of individual k cat/K m values for each enantiomer of the chiral alcohols reveals that the effect of the mutation is to decrease (S)-2-butanol specificity, and to preferentially enhance (R)-2-pentanol specificity relative to (S)-2-pentanol. These results are the first step toward expanding the synthetic utility of SADH to allow efficient preparation of a range of (R)-alcohols.

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 enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 degrees C when the enzymes were incubated at 55 degrees C before assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 degrees C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change. Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).

The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had

The amino acid sequences of cyclomaltodextrinase (CDase) from Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosuIfuricum 39E) and other amylolytic enzymes were compared by using linear alignment and hydrophobic cluster analysis. Two Asp and one Glu residue, which were considered to be the catalytic residues of the compared enzymes according to crystallographic or protein engineering experiments, were also conserved in CDase. ASPIRE, Asp4*' and GIu'~~ of the CDase were individually replaced by means of site-directed mutagenesis. The mutant enzymes completely lost activity, suggesting that these residues play an important role in catalysis.

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