The beneficial biological properties of L-fucose have extended its commercial application potential in pharmaceutical, cosmetic, and food industries. Enzymatic production of c-fucose with c-fucose isomerase (L-FucI) is considered a selective, green, and efficient strategy. Efficient sugar production requires thermophilic enzymes with increased reaction rate, reduced risk of microbial contamination, and high sugar solubility. No study has evaluated the applicability of thermophilic L-FucI for c-fucose production. In this study, we explored the biochemical properties of a thermostable L-FucI from Thermanaeromonas toyohensis (TtFucI) using L-fuculose as a substrate. The recombinant TtFucI exhibited thermophilicity and optimum activity at 70 degrees C. The specific activity, K-m, and k(cat) toward L-fuculose were 199.8 U/mg, 33.4 mM, and 901.7 s(-1), respectively. Mn2+ ions increased the activity of the enzyme by similar to 10 times and enhanced its thermal stability. Our study, on L-fucose synthesis by thermostable L-FucI, suggests the potential application of this enzyme for the industrial production of L-fucose.