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Applied and Environmental Microbiology, August 2008, p. 5183-5194, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00181-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from "Protaminobacter rubrum"{triangledown} ,{dagger}

Hyeon Cheol Lee,1* Jin Ha Kim,1 Sang Yong Kim,1 and Jung Kul Lee2

BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521,1 Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea2

Received 20 January 2008/ Accepted 7 June 2008

"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 {alpha}-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.

* Corresponding author. Mailing address: BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521, Republic of Korea. Phone: 82-2-747-9796. Fax: 82-2-747-0750. E-mail: churry{at}biongene.com

{triangledown} Published ahead of print on 13 June 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2008, p. 5183-5194, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00181-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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