The L-rhamnose isomerase gene ( L-rhi) encoding for L-rhamnose isomerase ( L-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6 x His sequence at a C-terminal of the protein. The open reading frame of L-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced L-RhI was achieved in a large quantity ( 470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant L-RhI exhibited maximum activity at 65 C ( pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60 C for 60 min. The apparent affinity ( Km) and catalytic efficiency ( k(cat)/K-m) for L-rhamnose ( at 65 degrees C) were 4.89 mM and 8.36 x 10(5) M-1 min(-1) 1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity ( 42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50 C, for D-allose, L-mannose, D-ribulose, and L-talose from D-psicose, L-fructose, D-ribose and L-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant L-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.