Characterization of a Thermophilic L-Rhamnose Isomerase from Thermoanaerobacterium saccharolyticum NTOU1

L-Rhamnose isomerase (EC 5.3.1.14, L-Rhl) catalyzes the reversible aldose ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-Rhl was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-Rhl, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-Rhl was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-Rhl 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 degrees C. L-Rhl from T. saccharolyticum NTOU1 is the most thermostable L-Rhl 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.