The novel isolated Rhizobium sp. S10 was identified as n-glucoside 3-dehydrogenase (G3DH) producing microbe. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme complex. The recombinant G3DH (rG3DH) was purified with relatively high specific activity of 38.54 U/mg compared to the previously characterized and cloned G3DHs. The purified rG3DH showed the highest activity at pH 7.0, 40 degrees C toward cellobiose. It can also oxidize a broad range of mono-disaccharides including saccharide derivatives. The glycosides oxidizing activity combined with chemical reaction, could produce o-gulose from lactitol via 3-ketolactitol.