Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of d-mannose to d-glucose, whereas they did not act on beta-(1 -> 4)-mannobiose, N-acetyl-d-glucosamine, and d-fructose, which are substrates of known AGE superfamily members. The k(cat)/K-m values of Runsl_4512 and Dfer_5652 for d-mannose epimerization were 3.89 and 3.51min(-1)mM(-1), respectively. Monitoring the Runsl_4512 reaction through H-1-NMR showed the formation of beta-d-glucose and beta-d-mannose from d-mannose and d-glucose, respectively. In the reaction with beta-d-glucose, beta-d-mannose was produced at the initial stage of the reaction, but not in the reaction with alpha-d-glucose. These results indicate that Runsl_4512 catalyzed the 2-epimerization of the beta-anomer substrate with a net retention of the anomeric configuration. Since H-2 was obviously detected at the 2-C position of d-mannose and d-glucose in the equilibrated reaction mixture produced by Runsl_4512 in (H2O)-H-2, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500g/L d-glucose at 50 degrees C and pH8.0, Runsl_4512 and Dfer_5652 produced d-mannose with a 24.4 and 22.8% yield, respectively. These d-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing d-mannose.