An alanine racemase (EC 18.104.22.168) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 amino
acid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.