Cloning and Sequencing of the Alcohol Dehydrogenase Gene from Bacillus stearothermophilus and Alteration of the Optimum pH of the Enzyme by Protein Engineering

Abstract

Thermophilic bacteria produce various useful enzymes. Some of them have been purified and characterized, and their structural genes have already been cloned and sequenced [1–3]. Bacillus stearothermophilus NCA 1503 was found to produce a thermostable alcohol dehydrogenase (ADH-T) amounting to 1–2% of soluble cell protein. By using this strain, ethanol was produced from sucrose or glucose as a carbon source under anaerobic condition at high temperatures [4, 5]. Alcohol dehydrogenases (ADH) were also isolated from both eucaryotes [6–9] and procaryotes [10, 11], and they exhibited various features. The ADH reaction mechanism was originally studied using horse liver ADH based on X- ray crystallographic analysis and kinetic studies [6, 12, 13]. The catalysis with horse liver ADH is performed by the proton release system consisting of a zinc atom, water molecule, and serine and histidine residues. By a series of intensive studies, the system including the 2’-hydroxyl group of the nicotinamide ribose was proposed for horse liver and human liver ADH [14–16]. Threonine and histidine of human liver ADH corresponded to serine and histidine of horse liver ADH, respectively, both of which functioned as catalytic sites [16, 17]. The amino acid residues responsible for substrate specificity in human liver ADH and yeast ADH were also proposed based on three-dimensional information on the horse liver ADH [18–20].