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- Title
Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio.
- Authors
Kralj, Slavko; van Leeuwen, Sander S.; Valk, Vincent; Eeuwema, Wieger; Kamerling, Johannis P.; Dijkhuizen, Lubbert
- Abstract
The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into α-d-glucans (labelled reuterans) with mainly α-(1→4) glucosidic linkages (50% and 70%, respectively), plus α-(1→6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1–3; O1–3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize α-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in α-(1→4) and α-(1→6) linkage ratios.
- Subjects
LACTOBACILLUS; HYDROLYSIS; IMMUNOSPECIFICITY; ENZYME analysis; GLUCANS; SUCROSE; HYBRID enzymes
- Publication
FEBS Journal, 2008, Vol 275, Issue 23, p6002
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2008.06729.x