The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into alpha-d-glucans (labelled reuterans) with mainly alpha-(1→4) glucosidic linkages (50% and 70%, respectively), plus alpha-(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 alpha-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 alpha-(1→4) and alpha-(1→6) linkage ratios. © 2008 The Authors. Chemicals/CAS: 1,4 alpha glucan branching enzyme, 9001-97-2; glucoside, 50986-29-3; isomaltose, 499-40-1; maltose, 16984-36-4, 69-79-4; sucrase, 37288-39-4; sucrose, 122880-25-5, 57-50-1; Bacterial Proteins; Disaccharides; Glucans; Glucosyltransferases, EC 2.4.1.-; Isomaltose, 499-40-1; leucrose, 7158-70-5; Maltose, 69-79-4; Oligosaccharides; Recombinant Fusion Proteins; Sucrose, 57-50-1