Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on D-lyxose, suggesting that the enzyme is D-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²+. The apparent K(m) values for D-lyxose and D-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k(cat)/K(m)) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for D-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of D-lyxose and D-glucose from d-xylose and D-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM D-lyxose and D-mannose, 3.7 mM and 3.8 mM D-lyxose and D-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.