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

Abstract

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (D-glucose) and a fructan (D-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or 1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted -glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (26)--D-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel 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 spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase 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.