Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG.
A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5alpha (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(+/-0.3) U mg(-1) protein, compared to 0.1(+/-0.01) U mg(-1) protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases.
Jeon
Young Jae
YJ
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia.
Fong
Jiunn C N
JC
Riyanti
Eny I
EI
Neilan
Brett A
BA
Rogers
Peter L
PL
Svenson
Charles J
CJ
D020001:1-Butanol / Q000378:metabolism; D020003:1-Octanol / Q000378:metabolism; D019840:2-Propanol / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000235:genetics; D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D019850:Heptanol / Q000378:metabolism; D000441:Hexanols / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000439:Pentanols / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000441:Hexanols; D000439:Pentanols; D000431:Ethanol; C036260:1-hexanol; D019850:Heptanol; D020001:1-Butanol; D000426:Alcohol Dehydrogenase; D000446:Aldehyde-Lyases; C021355:hexose phosphate synthetase; D019747:Aldose-Ketose Isomerases; C023918:phospho-3-hexuloisomerase; C024999:n-pentanol; D019840:2-Propanol; D020003:1-Octanol