A few amino acid substitutions are responsible for the higher thermostability of a novel NAD(+)-dependent bacillar alcohol dehydrogenase.
The gene adh-hT encoding a thermostable and thermophilic NAD(+)-dependent alcohol dehydrogenase (ADH) from the novel and more thermophilic Bacillus stearothermophilus LLD-R strain was cloned and its nucleotide sequence determined. The deduced protein sequence shows remarkable amino acid substitutions when compared to the sequence of the protein isolated from strain NCA1503 and significant similarity with the highly thermostable ADH from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The alignment of these sequences led to the identification of three amino acid replacements probably responsible for the higher thermostability of the novel bacillar ADH. Adh-hT gene expression in Escherichia coli, a fast purification procedure and the characterization of the recombinant enzyme are also described.
Dipartimento di Chimica Organica e Biologica, Università di Napoli, Italy.
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Cannio
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Rossi
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Bartolucci
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005798:Genes, Bacterial; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D016944:Sulfolobus / Q000201:enzymology
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase