Structural characterization of the polysaccharide produced by Lactobacillus helveticus CNRZ32 and assignment of the function of genes involved in its biosynthesis

Abstract

Strain CNRZ32 of Lactobacillus helveticus was grown in chemically defined medium or milk, and its exopolysaccharide (EPS) was isolated from the supernatant or the whole culture and purified by TCA and acetone precipitations and solvent extractions. Purity was checked by gel permeation chromatography. The molecular mass determined by size-exclusion chromatography was 6.4 × 10⁵ g/mol. Structural elucidation was performed by chemical, chromatographic, and spectroscopic methods. Monosaccharide and configuration analyses gave the following sugar composition: d-Gal, 2; d-Glc, 1; d-GlcNAc, 1; l-Rha, 3. Methylation analysis indicated the presence of terminal and 3,6-disubstituted Gal, 4-substituted Glc, 3,4,6-trisubstituted GlcNAc, as well as terminal and 2,4-disubstituted Rha. A phosphoethanolamine substituent was detected by NMR spectroscopy. On the basis of one- and two-dimensional homo- and heteronuclear NMR data, the structure of the EPS was consistent with a multibranched heptasaccharide repeating unit with the following sequence: {4[Rhaα-3]GlcNAc6PEtNβ-3[Galβ-6]Galα-4[Rhaα-2]Rhaβ-4Glcβ-}_n. Electrospray MS and MS/MS data are given for oligosaccharides produced after deacetylation, deamination, and reduction. A correlation between the EPS sequence and genes of the L. helveticus CNRZ32 eps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit is proposed. The structure is compared to that of EPSs produced by other L. helveticus strains.

Keywords: Gene function; Heptasaccharide repeating unit; High-resolution NMR spectroscopy; Phosphoethanolamine substituent; Repeating unit biosynthesis; Structure determination.