Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l -RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be similar to 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70A degrees C, respectively, with a k (cat) of 8,971 min(-1) and a k (cat)/K (m) of 17 min(-1) mM(-1) for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans l-RhI is distinguished from other l-RhIs by its high temperature optimum (70A degrees C) with high thermal stability of showing 100% activity for 10 h at 60A degrees C. The half-life of the enzyme was more than 900 min and similar to 25 min at 60A degrees C and 70A degrees C, respectively, making B. halodurans l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.