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- MakaleAçık Erişim2026Scopus SJRQ2Tailoring Physicochemical and Technological Properties of α-Glucans Through Mutations of GTFA from Lactobacillus Reuteri E81
Gökcan, Hande; İspirli, Hümeyra; Dertli, Enes
Glucansucrases catalyze the production of α-glucans using sucrose as substrate with distinct physicochemical characteristics originates from mainly the type of linkages, degree of branching and molecular mass. New forms of α-glucans can be produced by applying specific mutations to glucansucrases. In this study, a single and double mutation at Leu933 and Leu935 sites of glucansucrase E81 was applied and L933I and L933I-L935F sites modified glucansucrase was obtained. Structural characterisation of bioengineered α-glucans revealed that ratio of (1 → 3):(1 → 6) linked α-Glc units altered at L933I whereas no (1 → 3)-linked α-Glc units were presented for L933I-L935F modified glucansucrase detected by NMR analysis. Both bioengineered α-glucans had lower molecular weights in comparison to glucan E81 detected by GPC analysis. TGA and DSC analysis were applied to detect the thermal profiles of bioengineered α-glucans whereas FTIR and XRD analysis were applied to determine the alterations in the structural and physical nature of the bioengineered α-glucans, respectively. Finally, SEM and AFM analysis were applied to explore the morphological modifications of the bioengineered α-glucans. This study provides a better understanding for the potential role of final glucan structures on their physicochemical characteristics. Keywords: glucansucrase; carbohydrate engineering; mutation.
AtıfScopus2WoS2OpenAlex2
İncele - MakaleYalnız künye2021Scopus SJRQ1Production of lactose derivative hetero-oligosaccharides from whey by glucansucrase E81 and determination of prebiotic functions
İspirli, Hümeyra; Dertli, Enes
Bioactive oligosaccharides are important compounds for development of functional foods and ingredients. In this study, lactose derivative hetero-oligosaccharides were produced by acceptor reaction of glucansucrase E81 with whey. The synthesis of oligosaccharides was monitored by Thin Layer Chromatography (TLC) analysis followed by 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy analysis which demonstrated the glycosylation of lactose with α-(1 → 2), α-(1 → 3) and α-(1 → 4) linkages. Liquid chromatography–mass spectrometry (LC-MS) analysis demonstrated the formation of DP 7 oligosaccharides from whey. The synthesized oligosaccharides were not digested under in vitro human gastric juice and α-amylase conditions. The prebiotic effect of lactose derivative hetero-oligosaccharides was tested and no growth in pathogenic strains was observed whereas certain probiotic strains utilised lactose derivative hetero-oligosaccharides as Carbon source and their growth was promoted. These findings can be important to reveal the potential of glucansucrases to produce functional oligosaccharides from different sources including waste materials.
AtıfScopus17WoS16OpenAlex16İncele - MakaleAçık Erişim2021Scopus SJRQ1Synthesis and characterization of cellobiose-derived oligosaccharides with Bifidogenic activity by glucansucrase E81
İspirli, Hümeyra; Bowman, Michael J.; Skory, Christopher D.; Dertli, Enes
Cellobiose-derived oligosaccharides were produced from acceptor reactions with glucansucrase E81 and their prebiotic potentials were determined in this study. The major product was a mixture of trisaccharides formed by the addition of an α-linked glucose to cellobiose at either the 2-O position of the reducing end (86%) glucose or the 6-O position of the non-reducing end glucose (11%) demonstrated by NMR spectroscopy and LC-MS-LTQ analysis. The purified DP 3 cellobiose-derived oligosaccharides were then tested for in vitro human gastric juice and α-amylase digestion and no digestion was observed during the 6 h digestion period. Importantly, DP 3 cellobiose-derived oligosaccharides promoted the growth of Bifidobacteria at high levels demonstrating a Bifidogenic effect and a mild level of proliferation was observed for Lactobacilli suggesting the prebiotic roles of cellobiose-derived oligosaccharides.
AtıfScopus7WoS5OpenAlex6İncele - MakaleYalnız künye2020Scopus SJRQ3Production of mannose-containing oligosaccharides by glucansucrase E81 and determination of their functional characteristics
İspirli, Hümeyra; Dertli, Enes
Oligosaccharides are one of the functional ingredients to be used in food technology. In this study, by using an active glucansucrase GTFA-ΔN E81 in the acceptor reaction of mannose, mannose-containing oligosaccharides were produced and their functionality was tested. The formation of the oligosaccharides were visualised by TLC analysis and mannose-containing oligosaccharides up to DP 7 were determined by LC-MS analysis. The presence of the (1,6)Glc and (1,3)Glc units within the oligosaccharides were determined by NMR analysis. The in vitro immune-modulatory functions of the mannose-containing oligosaccharides were determined but no induction in IL-4, IL-10, IL-12 and TNF-α cytokine levels were detected. Importantly this oligosaccharide mixture showed prebiotic effect by triggering the growth of tested probiotics and not affecting the growth of pathogen strains. Our findings reveals the potential of the role of glucansucrases for the production of functional oligosaccharides.
AtıfScopus8WoS7OpenAlex8İncele - MakaleYalnız künye2020Scopus SJRQ1Impact of glucan type exopolysaccharide (EPS) production on technological characteristics of sourdough bread
İspirli, Hümeyra; Özmen, D.; Yılmaz, Mustafa Tahsin; Sağdıç, O.; Dertli, Enes
In this study, technological role of α-glucan with (α1-3) and (α1-6) glycosidic linkages produced by Lactobacillus reuteri E81 on sourdough rheology and bread texture was investigated. To achieve this, a glucan deficient ΔgtfA mutant was produced by the deletion of the glucansucrase gene from strain E81. By comparison of glucan-positive and glucan-negative strains for rheological properties of sourdough revealed that in situ α-glucan production resulted in higher elasticity in sourdough. However, no additional effect of in situ glucan on textural characteristics of bread was observed although addition of glucan ex situ at 0.09% level significantly decreased bread hardness. Scanning Electron Microscopy (SEM) images of lyophilized sourdoughs confirmed association of glucan and starch granules forming a film like structure in sourdough.
AtıfScopus71WoS58OpenAlex67İncele - MakaleAçık Erişim2020Scopus SJRQ2Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81
İspirli, Hümeyra; Kaya, Yasemin; Dertli, Enes
Glucansucrases are responsible for the production of α-glucans using sucrose as the substrate. Glucansucrases may also produce different oligosaccharides by transferring the glucose moiety from sucrose to a variety carbohydrates acting as acceptor nucleophile. In this study, melibiose-derived oligosaccharides were produced by the glucansucrase from Lactobacillus reuteri E81 expressed without the N-terminal region (GtfA-ΔN). The reaction products were characterized by TLC, LC-MS and NMR analysis and it was found that GtfA-ΔN synthesized melibiose-derived oligosaccharides (DP3 and DP4) by adding glucose units through alpha 1->3 or 1->6 glycosidic bond. The functional characteristics of these melibiose-derived oligosaccharides were determined by testing the immune-modulatory functions in HT-29 cells and testing their growth promoting effects for important probiotic and pathogenic strains. The melibiose-derived oligosaccharides triggered the production of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine TNF-α depending on their concentrations. Finally, melibiose-derived oligosaccharides showed bifidogenic effect as potential prebiotics.
AtıfScopus15WoS14OpenAlex15İncele - 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.
AtıfScopus40WoS40OpenAlex40İncele - 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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