Structural analysis of bioengineered α-D-glucan produced by a triple mutant of the glucansucrase GTF180 enzyme from Lactobacillus reuteri strain 180:: Generation of (α1→4) linkages in a native (1→3)(1→6)-α-D-glucan

Site-directed mutagenesis of the glucansucrase gtf180 gene from Lactobacillus reuteri strain 180 was used to transform the active site region. The alpha-D-glucan (mEPS-PNNS) produced by the triple mutant V1027P:S1137N: A1139S differed in structure from that of the wild-type alpha-D-glucan (EPS180). Besides (alpha 1-->3) and (alpha 1-->6) linkages, as present in EPS180, mEPS-PNNS also contained (alpha 1-->4) linkages. Linkage analysis, periodate oxidation, and 1D/2D (1)H NMR spectroscopy of the intact mEPS-PNNS, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of mEPS-PNNS afforded a composite model, which includes all identified structural features.