RESUMEN
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).
Asunto(s)
Alcohol Deshidrogenasa/fisiología , Bacterias Anaerobias/efectos de los fármacos , Farmacorresistencia Bacteriana , Etanol/farmacología , Ácidos Grasos/fisiología , Bacterias Anaerobias/genética , Bacterias Anaerobias/crecimiento & desarrollo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Medios de Cultivo , Etanol/metabolismo , FermentaciónRESUMEN
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
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Bacterias Anaerobias/enzimología , Bacilos Grampositivos Asporogénicos Irregulares/enzimología , Alcohol Deshidrogenasa/análisis , Alcohol Deshidrogenasa/genética , Análisis Mutacional de ADN , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
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).
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Bacterias Anaerobias/enzimología , Bacterias Anaerobias/genética , Genes Bacterianos , Bacilos Grampositivos Asporogénicos Irregulares/enzimología , Bacilos Grampositivos Asporogénicos Irregulares/genética , Alcohol Deshidrogenasa/biosíntesis , Alcohol Deshidrogenasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Estabilidad de Enzimas , Escherichia coli , Calor , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , TermodinámicaRESUMEN
Enzymes synthesized by thermophiles (organisms with optimal growth temperatures > 60 degrees C) and hyperthermophiles (optimal growth temperatures > 80 degrees C) are typically thermostable (resistant to irreversible inactivation at high temperatures) and thermophilic (optimally active at high temperatures, i.e., > 60 degrees C). These enzymes, called thermozymes, share catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, thermozymes usually retain their thermal properties, suggesting that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, and crystal structure comparisons indicate that thermozymes are, indeed, very similar to mesophilic enzymes. No obvious sequence or structural features account for enzyme thermostability and thermophilicity. Thermostability and thermophilicity molecular mechanisms are varied, differing from enzyme to enzyme. Thermostability and thermophilicity are usually caused by the accumulation of numerous subtle sequence differences. This review concentrates on the mechanisms involved in enzyme thermostability and thermophilicity. Their relationships with protein rigidity and flexibility and with protein folding and unfolding are discussed. Intrinsic stabilizing forces (e.g., salt bridges, hydrogen bonds, hydrophobic interactions) and extrinsic stabilizing factors are examined. Finally, thermozymes' potential as catalysts for industrial processes and specialty uses are discussed, and lines of development (through new applications, and protein engineering) are also proposed.