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 or1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted α-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2→6)-β-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.

Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11

“…It was therefore concluded that all the L. fermentum strains tested express one or more glycosyltranferases. Expression of GTF activity by wildtype L. fermentum BR11 is not unexpected as it has been found that several strains of the closely related species Lactobacillus reuteri express both GTFs and fructosyltransferases [19].…”

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

“…reuteri levansucrase a¤nity for sucrose (K m = 7 mM) is comparable to values reported for FTF of S. salivarius (K m = 5 mM; [10]) and SacB of B. subtilis (K m = Table 2 Comparison of 13 C NMR chemical shifts of fructan produced by the puri¢ed levansucrase and of fructan present in culture supernatants of Lb. reuteri strain 121 grown on MRSr [8] 4 mM; [11]). The strong inhibitory e¡ects of Cu 2 and Fe 3 ions on Lb.…”

“…Sucrase enzyme activity in Lb. reuteri strain 121 occurs both in a cell-associated form and as a free, extracellular enzyme [8]. In MRSm cultures sucrase enzyme activity is largely cell-associated, which may explain the lower sucrase activity observed.…”

“…Further con¢rmation was obtained from the identical 1 H NMR spectra of the FTF fructan (results not shown) and the fructan produced by MRSr grown Lb. reuteri strain 121 [8]. The puri¢ed FTF enzyme thus is a levansucrase.…”

“…All other positive strains synthesized polymers consisting of glucose or other sugars [7]. The fructan, a linear polymer consisting of only L (2,6) linked fructosyl residues (a levan), had an estimated size of 150 kDa [8].…”