D-Mannose is the aldose isomer of D-fructose and displays unique physiological functions and health applications. As a result, it has attracted increasing interest from the public. Because of its wide substrate specificity, D-lyxose isomerase (D-LI) has been applied to D-mannose bioproduction. In this article, the Thermoflavimicrobium dichotomicum D-LI encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). The novel protein T. dichotomicum D-LI was identified and characterized, and its maximum enzyme activity was exhibited at pH 7.5 (phosphate buffer) and 60 degrees C in the presence of Mn2+. Similar to other D-LIs, T. dichotomicum D-LI formed a homodimeric structure and showed strict conservation of the metal coordination and substrate binding sites. The D-LI half-life (t(1/2)) was 9.91 h and 3.05 h at 55 and 60 degrees C, respectively, and its melting temperature (T-m) was 72.85 degrees C. T. dichotomicum D-LI displayed the highest specific activity towards D-lyxose among the substrates tested, followed by D-mannose. It also efficiently converted D-fructose to D-mannose, and the equilibrium ratio between D-mannose and D-fructose during T. dichotomicum D-LI activity was 25:75. From 500 g/L D-fructose, 110.5 g/L D-mannose was produced after 6 h, suggesting that T. dichotomicum D-LI might provide an alternative for D-mannose production.