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    Characterization of a glucansucrase from Lactobacillus reuteri E81 and production of malto-oligosaccharides
    (Taylor & Francis Ltd, 2019) Ispirli, Humeyra; Yuzer, Mustafa Onur; Skory, Christopher; Colquhoun, Ian J.; Sagdic, Osman; Dertli, Enes
    Glucansucrases, which can be produced by different Lactic Acid Bacteria (LAB), catalyze the synthesis of alpha-glucans with different structures and properties using sucrose as substrate. In this study, a novel glucansucrase (GTFA) from Lactobacillus reuteri E81 was identified and heterologously expressed. Alignments of GTFA with other glucansucrases revealed its novelty and a putative 3D model structure was obtained. The biochemical properties of the truncated enzyme without the N-terminal variable region, GTFA-Delta N, was characterized. The K-m and V-max were found to be 7.5 mM and 1.49 IU/mg, respectively, and it showed optimum activities at pH 7 and at 50 degrees C. The GTFA-Delta N produced in vitro an alpha-glucan with (alpha 1 -> 3) and (alpha 1 -> 6) glycosidic linkages using sucrose as the substrate. Importantly, GTFA-Delta N synthesized DP = 9 oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively, as detected by TLC, HPLC, LC-MS and NMR analysis.
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    Preparation of gentiobiose-derived oligosaccharides by glucansucrase E81 and determination of prebiotic and immune-modulatory functions
    (Elsevier Sci Ltd, 2019) Ispirli, Humeyra; Colquhoun, Ian J.; Sahin, Engin; Sagdic, Osman; Dertli, Enes
    Gentiobiose-derived oligosaccharides were synthesized by the acceptor reaction of glucansucrase E81 obtained from Lactobacillus reuteri E81 with sucrose and gentiobiose as donor-acceptor sugars, respectively. The reaction products were monitored by TLC analysis and gentiobiose-derived oligosaccharides up to DP 8 were formed during the acceptor reaction as determined by ESI-MS/MS analysis. The glycosylation of the gentiobiose with alpha-(1 -> 6) linkages and alpha-(1 -> 3) linkages was shown by H-1 and C-13 NMR analysis confirming the structure of these gentiobiose-derived oligosaccharides. The in vitro prebiotic function of the oligosaccharides was determined in which probiotic strains were stimulated whereas no growth was observed in pathogen strains. Gentiobiosederived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-alpha cytokines in HT29 cells in a dose dependent manner. This study showed the production and functional characterisation of gentiobiose-derived oligosaccharides establishing a promising avenue for future applications.