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- MakaleAçık Erişim2019Scopus SJRQ3Characterization of a glucansucrase from Lactobacillus reuteri E81 and production of malto-oligosaccharides
İspirli, Hümeyra; Yüzer, Mustafa Onur; Skory, Christopher; Colquhoun, Ian J.; Sağdıç, Osman; Dertli, Enes
Glucansucrases, which can be produced by different Lactic Acid Bacteria (LAB), catalyze the synthesis of α-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-ΔN, was characterized. The Km and Vmax were found to be 7.5 mM and 1.49 IU/mg, respectively, and it showed optimum activities at pH 7 and at 50 °C. The GTFA-ΔN produced in vitro an α-glucan with (α1 → 3) and (α1 → 6) glycosidic linkages using sucrose as the substrate. Importantly, GTFA-ΔN 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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İncele - MakaleAçık Erişim2019Scopus SJRQ2Preparation of gentiobiose-derived oligosaccharides by glucansucrase E81 and determination of prebiotic and immune-modulatory functions
İspirli, Hümeyra; Colquhoun, Ian J.; Şahin, 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 α-(1 → 6) linkages and α-(1 → 3) linkages was shown by 1H and 13C 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. Gentiobiose-derived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-α 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.
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İncele - MakaleAçık Erişim2018Scopus SJRQ1Structural analysis of the α-D-glucan produced by the sourdough isolate Lactobacillus brevis E25
Dertli, Enes; Colquhoun, Ian J.; Côté, Gregory L.; Le Gall, Gwénaëlle; Narbad, Arjan
Cereal-associated Lactic Acid Bacteria (LAB) are well known for homopolymeric exopolysaccharide (EPS) production. Herein, the structure of an EPS isolated from sourdough isolate Lactobacillus brevis E25 was determined. A modified BHI medium was used for production of EPS-E25 in order to eliminate potential contaminants. Analysis of sugar monomers in EPS revealed that glucose was the only sugar present. Structural characterisation of EPS by NMR and methylation analysis revealed that E25 produced a highly branched α-glucan with (α1 → 3) and (α1 → 6) glycosidic linkages, and was similar in structure to a previously reported EPS from Lactobacillus reuteri 180. The 1H and 13C NMR data were contrasted with newly recorded data for known polysaccharides (alternan, commercial dextran) which also contain α-(1,3,6)Glc branch points. It was found in both E25 EPS and alternan that NMR parameters could be used to distinguish glucose residues that had the same substitution pattern but occupied different positions in the structure.
AtıfScopus78WoS73OpenAlex80İncele - MakaleAçık Erişim2016Scopus SJRQ1EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785
Dertli, Enes; Mayer, Melinda J.; Colquhoun, Ian J.; Narbad, Arjan
Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.
AtıfScopus43WoS38OpenAlex39İncele - MakaleAçık Erişim2013Scopus SJRQ1Structure and biosynthesis of two exopolysaccharides produced by lactobacillus johnsonii FI9785
Dertli, Enes; Colquhoun, Ian J.; Gunning, A. Patrick; Bongaerts, Roy J.; Le Gall, Gwénaëlle; Bonev, Boyan B.; Mayer, Melinda J.; Narbad, Arjan
Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4) -β-Gal p-(1-4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (δepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the δepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.
AtıfScopus131WoS120OpenAlex134İncele