D-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. L-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable o-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn2+ with an optimal pH 7.5 and temperature 65 degrees C. The Michaelis-Menten constant (K-m), turnover number (k(cat)) and catalytic efficiency (k(cat/)K(m)) for L-rhamnose were 33.8 mM, 1189.8 min(-1) and 35.2 min(-1) mM(-1), respectively. At a higher temperattire, Mn2+ played a pivotal role in strengthening the thermostability of T. composti L-Rl. The differential scanning calorimetry (DSC) results showed the denaturing temperature (T-m) of T. composti L-RI was increased by 3 degrees C in presence of Mn2+. Although the T. composti L-RI displayed the optimum substrate as L-rhamnose, it could also effectively catalyze the isomerization between D-allulose and o-allose. When the reaction reached equilibrium, the sole product D-allose was produced from D-alluose by T. composti L-RI. (C) 2016 Elsevier Ltd. All rights reserved.