Sunday, May 4, 2008
2-30

Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG

Young Jae Jeon1, Jiunn C. N. Fong2, Eny Riyanti1, Brett A. Neilan1, Peter L. Rogers1, and Charles Svenson1. (1) School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2052, Sydney, Australia, (2) Environmental Toxicology, University of California, Santa Cruz, 453 Physical Sciences Building, Santa Cruz, CA 95064

A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterized. 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 1,020-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 E. coli DH5 (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 modelling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases.

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