Abstract
Microbial exopolysaccharides (EPSs) are useful for the food industry as viscosifying, stabilising, emulsifying, and gelling agents. The improvement of texture in fermented dairy products by generally recognised as safe (GRAS) lactic acid bacteria, is caused in part by the in situ production of EPSs. Most studied are heteropolysaccharides (HePSs) produced
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by LAB, and many studies have been performed into their structure-function relationships. Many parts of the biosynthesis of these EPSs have been elucidated in recent years. In case of homopolysaccharides (HoPSs) only initial structural studies have been performed. The research described in this thesis was part of the EET (Economy, Ecology, Technology) programme "Bioprimer" (EETK01129), researching LAB α-D-glucan EPSs as anti-corrosive additives for use in heavy-duty coatings. The aim of the study was to perform complete structural characterisations of α-D-glucans, produced by glucansucrase (GTF) enzymes from Lactobacilli. Some α-D-glucans have been indicated as promising anti-corrosives, whereas others showed no anti-corrosive activity in a pilot study. To understand the mechanism of anti-corrosive activity, detailed structural knowledge of these glucans is required. In order to perform the challenging analysis of α-D-glucans, an NMR structural-reporter-group concept was established (Chapter 2), based on known di- and trisaccharide standards. Making use of the structural-reporter-group concept, the primary structure of EPSs produced in vitro by the glucansucrase (GTF180) enzyme from Lb. reuteri strain 180 (Chapter 3; EPS180) and the glucansucrase (GTFA) enzyme from Lb. reuteri strain 35-5 (Chapter 4; EPS35-5), using sucrose as a substrate, were analysed. Based on all data gathered visual presentations, that include all identified structural features, were formulated. During the analysis of each EPS, new structural-reporter-group signals were added to the concept. Using the enhanced structural-reporter-group concept the EPS product produced from sucrose by a triple mutant of the glucansucrase (GTF180;V1027P:S1137N:A1139S) enzyme from Lb. reuteri strain 180 (mEPS-PNNS) was analysed (Chapter 5). Again a picture was formulated, that includes all structural elements indentified, and the structural-reporter-group concept was complemented. In Chapter 6, structural characterisations were performed on α-D-glucans produced from sucrose by 12 mutant glucansucrase GTF180 enzymes of Lb. reuteri strain 180. Mutations were selected based on sequences observed in other glucansucrase enzymes, i.e. reuteransucrase (NNS), mutansucrase (NNV), alternansucrase (YDA), and dextransucrase (SEV) enzymes, as well as single mutant variants of these sequences. Making use of the structural-reporter-group concept the 1H NMR spectroscopic data were interpreted, and combined with methylation analysis data to formulate visual representations, depicting all structural elements identified for the 12 mutant EPSs.
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