L-Ribose isomerase (L-RI) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. L-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and L-ribose was utilized as a substrate. The recombinant L-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M-w) of 29.6 kDa. The measured M-w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer M 35 degrees C, pH 9.0, showing a specific activity of 54.96 U mg(-1). CrL-RIse displayed no major increase in activity with metal ions, excluding Mn2+. The estimated Km, K-cat K-cat/Km and V-max values of CrL-RIse were 37.8 mM, 10,416 min(-1), 275.43 min(-1) mM(-1), and 250 U mg(-1), respectively. The rate of L-ribulose production was 31 % (6.24, 12.11, and 20.89 g L-1) M equilibrium by utilizing 20, 40, and 70 g L-1 of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture L-ribulose from L-ribose.