Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum by. viciae 3841 (R1GDH) was cloned and overexpressed in Escherichia coil. The R1GDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114 kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35 degrees C, respectively. The purified recombinant RIGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RIGDH with a K-m of 8.8 mM, k(cat) of 835 min(-1) and a k(cat)/K-m of 94.9 min(-1) mM(-1). Although GDHs have been characterized from a few other sources, RIGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RIGDH a good choice for practical applications. (C) 2014 Elsevier Inc. All rights reserved.