Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the Lactobacillus reuteri wild-type strain and by mutant strains

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amou nts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masse s of 3,500 and 150 kDa, respectively. Methylation studies and C-13 or H-1 n uclear magnetic resonance analysis showed that the glucan has a unique stru cture consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubst ituted alpha-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was i dentified as a (2-->6)-beta-D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucans ucrase and levansucrase enzymes that occur in a cell-associated and a cell- free state after growth on sucrose, raffinose, or maltose but remain cell a ssociated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide g el electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of s pontaneous exopolysaccharide-negative mutants that had lost both glucansucr ase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan .