Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “ Protaminobacter rubrum ”

Abstract

"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 325 RLDRD 329 motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional activesite 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 Arg 325 and Arg 328 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 R 325 Q 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.Isomaltose (6-O-␣-D-glucopyranosyl-D-glucose) is a disaccharide that comprises an ␣-1,6-glycosidic bond between two glucose molecules. Its caloric value is less than 50% that of sucrose, and its viscosity is lower than that of maltose (8). Further, its properties are similar to those of other well-known low-cariogenic sugar alcohols or sucrose isomers, since much less acid and glucan are formed by Streptococcus mutans than sucrose. Isomaltose is a member of the isomalto-oligosaccharide group, whose members contain at least one ␣-1,6-glycosidic linkage, such as pannose, isomaltotriose, isomaltotetraose, nigerose, and isopanose. Isomalto-oligosaccharides are known to improve the general well-being of humans and animals when ingested orally on a daily basis. Isomaltose has potential applications, not only in the food industry, such as in confections, processed fruits and vegetables, and canned and bottled food, but also as an ingredient in animal feed, cosmetics, and medicines. It is generated as a by-product during chemical and enzymatic reactions that use liquefied starch or a glucose-containing solution as a reactant. However, it has not yet been possible to obtain a disaccharide-enriched product ...

Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from “ Pseudomonas mesoacidophila ” MX-45

“…Surprisingly, Aroonnual et al (3) reported that the replacement of RLDRD by RYDRA in PalI from K. pneumoniae NK33 had no effect on the ratio of isomaltulose to trehalulose formation. Recently, the importance of the Arg residues of this motif has been demonstrated in an SI from P. rubrum, and mutations at these sites led to enzymes with altered product compositions but with decreased sucrose conversion rates and some synthesis potential for isomaltose (19). Interestingly, the hydrolytic mutant MutB R311C has its amino acid alteration at one of these conserved Arg residues within the so-called "isomerization" motif.…”

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

“…Based on the product preference of sucrose isomers, SIases can be divided into two groups, isomaltulose synthases and trehalulose synthases. The protein sequences of isomaltulose synthases from Klebsiella sp. LX3 [14], Protaminobacter rubrum [15], Serratia plymuthica [16,17], Pantoea dispersa UQ68J [18], and Enterobacter sp. FMB1 [19] share high homology with those of trehalulose synthases from Agrobacterium radiobacter [20] and Pseudomonas mesoacidophila (also known as Rhizobium sp. by Goulter et al) [21-23].…”

“…In 2003, Zhang et al identified a unique catalytic 325 RLDRD 329 motif in PalI affecting the product specificity of sucrose isomerization [26]. Mutations on any charged residues in this motif dramatically increased the production ratio of trehalulose over isomaltulose [15,26]. Interestingly, the amino acid sequences of this motif are well conserved in isomaltulose synthases but vary between isomaltulose synthases and trehalulose synthases.…”

Structure/Function Relationships of Sucrose Isomerases with Different Product Specificity

“…The mutations of Arg298 resulted in a substantial decrease of the isomaltulose production, and a slight increase in the production of trehalulose and isomaltose as in the quantity of formed monosaccharides. 16) When the arginine was replaced by a lysine, the decrease in isomaltulose production was significantly less than for the other three in other mutations. As concerns the mutation of the second arginine of the isomerisation motif, Arg301, when it was mutated to one of the four above mentioned residues, the amount of produced isomaltulose was lower than for the wild type, and the formed products seemed to be independent on the nature of the mutation.…”

“…9) As concerns the P. rubrum sucrose isomerase, it was proposed based on modeling studies that this motif and especially the two arginines may be involved in fructose binding. 16) Among the five amino acid residues forming the isomerization motif in SmuA, Lee and colleagues carried out a series of mutations on Arg298 and Arg301, which were replaced by Asp, Ala, Gln and Lys. The mutations of Arg298 resulted in a substantial decrease of the isomaltulose production, and a slight increase in the production of trehalulose and isomaltose as in the quantity of formed monosaccharides.…”