A NADH-dependent mannitol dehydrogenase gene (mtlD) was cloned from Pseudomonas fluorescens, subcloned into an expression vector (pDEST110) and entered into different strains of E. coli to compare their protein expression and the enzyme specific activity. Purifications were accomplished by Ni(2+)-NTA affinity chromatography. Using this approach, the efficiency of purification process significantly increased (up to 90%) so that the purified enzyme gave a sharp single band (55 kDa) in SDS-PAGE. The results showed that among the strains, BL21 (DE3) plysS exhibited the maximum expression level of MDH(mannitol dehydrogenase) (11 mg L(-1)). Results from activity assay with fructose as substrate also showed that in this strain the specific activity of 63 U mg(-1) protein monitored for the enzyme, the record not reported before. Resazurin staining also indicated that the enzyme reduced fructose, whereas oxidized other substrates including mannitol, sorbitol and arabitol under optimal assay condition. From HPLC analysis it was showed for the first time that the enzyme could convert substrate isomaltulose to the specific products, GPM and GPS. Interestingly, because of the high specificity of the enzyme for substrate, the method can be used as an alternative approach to substitute nonspecific conventional method of isomalt production.