Original Paper

Archives of Microbiology

, Volume 187, Issue 6, pp 499-510

First online:

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea

  • Xiangxian YingAffiliated withDepartment of Biology, University of Waterloo
  • , Ying WangAffiliated withDepartment of Biology, University of WaterlooXinjiang Agricultural University
  • , Hamid R. BadieiAffiliated withDepartment of Chemistry, University of Waterloo
  • , Vassili KaranassiosAffiliated withDepartment of Chemistry, University of Waterloo
  • , Kesen MaAffiliated withDepartment of Biology, University of Waterloo Email author 

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Abstract

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO2, H2, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent Km values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

Keywords

Thermotoga hypogea Alcohol dehydrogenase Iron Extreme thermophile Anaerobe Thermostability