Structural Characterization of Bioengineered α-D-Glucans Produced by Mutant Glucansucrase GTF180 Enzymes of Lactobacillus reuteri Strain 180

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradation analysis, and 1D/2D H-1 NMR spectroscopy. Comparison of the results with structural data of the previously described wild type EPS180 and triple mutant mEPS-PNNS revealed a broad variation of structural elements between mEPS molecules. The amount of (alpha 1 -> 3) linkages varied from 14-43%, the amount of (alpha 1 -> 4) linkages (not present in the wild type) from 0-12%, and the amount of (alpha 1 -> 6) linkages from 51-86%. The average molecular weight (M-w) ranged from 9.4 to 32.3 MDa and the degree of branching varied from 8-20%. Using a previously established H-1 NMR structural-reporter-group concept, composite models, that include all identified structural features, were formulated for all mEPS molecules. Variations in the mEPS structures strongly affected the physical properties of the mEPSs.