Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio

The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into a-d-glucans (labelled reuterans) with mainly a-(1fi4) glucosidic linkages (50% and 70%, respectively), plus a-(1fi6) 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 a-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 oligoand 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 a-(1fi4) and a-(1fi6) linkage ratios