A gene (ORF TM0392) encoding a putative trehalose synthase (TmTreT) in Thermotoga maritima was cloned and expressed in Escherichia coil. The recombinant enzyme was purified to homogeneity by heat treatment and a glutathione-sepharose affinity column chromatography. The purified enzyme existed exclusively as a monomer in a native state. The optimum pH and temperature for this enzyme were 6.0 and 65 degrees C. The glutathione-S-transferase (GST)-fusion enzyme had greater thermostability than thrombin-treated free enzyme. TmTreT had diverse substrate specificities. The enzyme effectively created a free trehalose from several nucleoside diphosphate (NDP)-glucoses as a donor and glucose as an acceptor. Inversely, the enzyme was also capable of employing several NDPs such as UDP, ADP, GDP, and CDP with trehalose to produce corresponding NDP-glucoses. The enzyme was able to employ other monosaccharides, such as mannose and fructose, as acceptors to synthesize disaccharide analogues of trehalose. The mannose-containing analogue was not hydrolyzed by trehalase and the rat intestinal enzymes. Furthermore, the analogue showed a competitive inhibition to the intestinal disaccharidases with K-i values of approximately 0.8-1.6 mM. The results suggest that the enzyme is an useful trehalose synthase that can regenerate NDP-glucoses from NDPs and produce the inhibitory trehalose analogues of indigestible disaccharides. (C) 2010 Elsevier Inc. All rights reserved.