Abstract:l-Rhamnose isomerase (EC 22.214.171.124, l-RhI) catalyzes the reversible aldose−ketose isomerization between l-rhamnose and l-rhamnulose. In this study, the l-rhi gene encoding l-RhI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active l-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. l-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified l-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40−70 °C. l-RhI from T. saccharolyticum NTOU1 is the most thermostable l-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.