Structural characterisation and enzymic modification of exopolysaccharides from Lactococcus lactis (2000)

Since ancient times, lactic acid bacteria have been used for the preservation of food. Some of these bacteria are able to produce exopolysaccharides (EPSs), which may contribute to the peculiar rheology and texture of, for example, milk-derived products. Insight into the relationship between the chemical structure of EPSs and their physical properties can lead to tailor-made polysaccharides, which meet particular requirements in terms of structure and function. In this thesis, the elucidation of the chemical structures of three exopolysaccharides from Lactococcus lactis subsp. cremoris is described. Enzymes are used as a tool during the structural characterisation and modification of EPS and the action of three enzymes having activity towards (chemically modified) EPSs is investigated as well. Finally, a start has been made within this project to study the effect of structural changes of EPSs on the physical properties.In chapter 1, a brief general introduction into the research subject is given. Besides background information about the use of bacterial EPSs in food and the biosynthesis of EPS, attention is paid to common features in EPS structures from lactic acid bacteria. Different ways to obtain structurally related EPSs are presented and the use of enzymes in polysaccharide research is outlined.Chapter 2 describes the study of the chemical structure of EPS B40, explaining earlier reported analytical discrepancies.The EPS contains rhamnose:galactose:glucose:phosphate in a molar ratio of 1:1.3:2:1.1. 31 P NMR indicated that a single phosphate group is present as a phosphodiester. EPS B40 has chemically been modified using 0.3 M H 2 SO 4 , 28 M HF or 2 M NaOH. From these modifications it is concluded that during the hydrolysis step prior to sugar composition analysis the galactose 3-phosphate linkages are split only partially and that, consequently, the amount of galactose is underestimated. The backbone of HF-modified EPS B40 can be degraded by a crude cellulase preparation from Trichoderma viride . Purification and characterisation of the obtained oligomers (chapter 3), together with the characterisation of the polymer (chapter 2), has resulted in a chemical structure for EPS B40 identical to the repeating unit already described for EPS SBT 0495: In chapter 3, the enzyme activity responsible for the degradation of HF-modified EPS B40 is identified as an endoglucanase (endoV). Thus, after complete removal of galactosyl residues and phosphate and partial removal of rhamnosyl residues, endo glucanase is able to cleave the backbone consisting of glucosyl and galactosyl residues. Characterisation of the resulting homologous series of oligomers by MS and NMR unequivocally demonstrated that endoV is able to cleave theβ-(1→4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-modified EPS B40 is discussed on the basis of the subsite model for endoV, described in literature. The crude cellulase preparation from T. viride has also been shown to contain a phosphatase able to act on EPS B40 after removal of rhamnosyl and galactosyl residues by mild CF 3 CO 2 H treatment.In chapter 4, the structural elucidation of EPS B39 is outlined. This novel exopolysaccharide structure contains l-Rha, d-Gal and d-Glc in a molar ratio of 2:3:2. Enzymic modification, methylation analysis and 1D/2D NMR experiments (both 1 H- 1 H and 1 H- 13 C) revealed that EPS B39 consists of a branched heptasaccharide repeating unit with the following structure: Chapter 5 describes the chemical structure of EPS B891, which contains d-Gal and d-Glc in a molar ratio of 2:3. The polysaccharide is partially O -acetylated. By means of HF solvolysis, O -deacetylation, enzymic modification, methylation analysis and 1D/2D NMR studies the novel exopolysaccharide is shown to be composed of repeating units with the following structure: EPS B39 and O -deacetylated EPS B891 both contain lactosyl side chains and it is demonstrated that the terminally linked galactosyl residues can be removed by using a crude commercial enzyme preparation from Aspergillus aculeatus . The purification and characterisation of theβ-galactosidase responsible for this modification is described in chapter 6. The enzyme has a molecular mass of approximately 120 kDa, a pI between 5.3-5.7 and is optimally active at pH 5.4 and 55-60 o C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism is shown to be retaining and transglycosylation products are demonstrated using lactose as a substrate. Theβ-galactosidase is able to release terminally linked galactosyl residues from EPS B891 in presence of acetyl groups, but the hydrolysing rate after O -deacetylation is higher. Furthermore, O -deacetylated EPS B891 is degalactosylated faster than EPS B39.In chapter 7, the results of this thesis are discussed. Emphasis is placed on the approach of using enzymes in structure (-function) studies of exopolysaccharides. Furthermore, the use of (modified) exopolysaccharides for characterising enzyme activities is outlined. Finally, the influence of various structural modifications on the physical properties of EPSs is briefly discussed.