'Protaminobacter rubrum' sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a 325RLDRD329 motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other -glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg325 and Arg328 in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R325Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter–1 and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter–1) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g–1 with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.