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- MakaleAçık Erişim2025Scopus SJRQ1Characterization and immune-modulatory roles of branched glucan and acetylated gluco-oligosaccharides produced by glucansucrase 40 from Leuconostoc mesenteroides YTU 40
Dere, Sevda; İspirli, Hümeyra; Korkmaz, Kader; Tuncil, Yunus Emre; Sagdic, Osman; Narbad, Arjan; Ayyash, Mutamed; Dertli, Enes
Glucansucrases (GS) are functional enzymes for the production of distinct glucan and gluco-oligosaccharide structures with potential bioactive characteristics. In this study, an active glucansucrase (GS40) was identified within the genome of Leuconostoc mesenteroides YTU 40 that was further cloned, heterologously expressed and biochemically characterised. GS40 was capable of production of a branched dextran with ~ 20% (1→3)-α-D-Glc residues detected by1H and13C NMR analysis. Acceptor reactions of GS40 with maltose and cellobiose were performed and distinct maltose/cellobiose derivative acetylated gluco-oligosaccharides were produced detected by TLC, NMR and LC-MS analysis. The bioactive characteristics of branched dextran and gluco-oligosaccharides were tested by determination of their immune-modulatory roles in terms of triggering IL-12, IL-10, IL-4 and TNF-α cytokine levels. These findings can be important to unveil the role of glucansucrases for the production of gluco-oligosaccharides that can be used as functional ingredients for food and other industries.
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İncele - MakaleYalnız künye2023Scopus SJRQ1Utilisation of an active branching sucrase from Lactobacillus kunkeei AP-37 to produce techno-functional poly-oligosaccharides
İspirli, Hümeyra; Korkmaz, Kader; Arioglu-Tuncil, Seda; Bozkurt, Fatih; Sağdıç, Osman; Tunçil, Yunus Emre; Narbad, Arjan; Dertli, Enes
Glucansucrase AP-37 was extracted from the culture supernatant of Lactobacillus kunkeei AP-37 and characteristics of the glucan produced by the active glucansucrase in terms of structural and functional roles were determined in this study. A molecular weight around 300 kDa was observed for glucansucrase AP-37 and its acceptor reactions with maltose, melibiose and mannose were also conducted to unveil the prebiotic potential of the poly-oligosaccharides formed via these reactions. The core structure of glucan AP-37 was determined by 1H and 13C NMR and GC/MS analysis which revealed that glucan AP-37 was a highly branched dextran composing of high levels of (1 → 3)-linked α-D-glucose units with low levels of (1 → 2)-linked α-D-glucose units. The structural features of the glucan formed, demonstrated that glucansucrase AP-37 was an α-(1 → 3) branching sucrase. Dextran AP-37 was further characterised by FTIR analysis and XRD analysis demonstrated its amorphous nature. A fibrous compact morphology was observed for dextran AP-37 with SEM analysis whereas TGA and DSC analysis revealed its high stability as no degradation was observed up to 312 °C. Finally, the prebiotic potential of the dextran AP-37 and the gluco-oligosaccharides produced with the acceptor reaction of α-(1 → 3) branching sucrase AP-37 were determined and promising results were found for the gluco-oligosaccharides to act as prebiotics.
AtıfScopus13WoS12OpenAlex13İncele - MakaleAçık Erişim2022Scopus SJRQ1Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1
Cebeci, Fatma; Mayer, Melinda J.; Rossiter, John T.; Mithen, Richard; Narbad, Arjan
Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.
AtıfScopus21WoS14OpenAlex16İncele - 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 Erişim2015Scopus SJRQ1Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785
Dertli, Enes; Mayer, Melinda J.; Narbad, Arjan
Background: The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens. Results: Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses. Conclusions: This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.
AtıfScopus201WoS179OpenAlex211İncele - MakaleAçık Erişim2013Scopus SJRQ1Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics
Horn, Nikki; Wegmann, Udo; Dertli, Enes; Mulholland, Francis; Collins, Samuel R. A.; Waldron, Keith W.; Bongaerts, Roy J.; Mayer, Melinda J.; Narbad, Arjan
As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.
AtıfScopus71WoS66OpenAlex72İncele - 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.
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