The Experts below are selected from a list of 1080 Experts worldwide ranked by ideXlab platform

Tiina Alamae - One of the best experts on this subject based on the ideXlab platform.

  • a highly active endo levanase bt1760 of a dominant mammalian gut commensal bacteroides thetaiotaomicron cleaves not only various bacterial levans but also levan of timothy grass
    PLOS ONE, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  • A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass
    PloS one, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (

  • Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
    2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    * Synthesized by Pseudomonas syringae pv. tomato Levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.

  • Levansucrases of a pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo and polysaccharides
    New Biotechnology, 2015
    Co-Authors: Triinu Visnapuu, Karin Mardo, Tiina Alamae
    Abstract:

    Gut microbiota influences more physiological and developmental processes of humans and animals than earlier expected. Therefore, the possibility to shape the composition and activity of this bacterial population by prebiotics becomes especially important. Inulin, a β-2,1 linked fructan polymer, from plants and fructooligosaccharides (FOS) derived from it are recognized and already widely used as prebiotics while β-2,6 linked fructans have received much less attention from scientific community. In this mini-review, we will address β-2,6 linked fructans: levan and levan-type FOS as novel potential prebiotics and summarize the literature data on Levansucrases of Pseudomonas bacteria which are producing these fructans. The major attention is drawn to stable and highly efficient Levansucrases of Pseudomonas syringae pv. tomato, among which the Lsc3 protein has been most thoroughly studied using biochemical methods as well as extensive mutagenesis of the protein.

  • mutational analysis of conserved regions harboring catalytic triad residues of the Levansucrase protein encoded by the lsc 3 gene lsc3 of pseudomonas syringae pv tomato dc3000
    Biotechnology and Applied Biochemistry, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi, Tiina Alamae
    Abstract:

    Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for Levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.

Triinu Visnapuu - One of the best experts on this subject based on the ideXlab platform.

  • a highly active endo levanase bt1760 of a dominant mammalian gut commensal bacteroides thetaiotaomicron cleaves not only various bacterial levans but also levan of timothy grass
    PLOS ONE, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  • A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass
    PloS one, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (

  • Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
    2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    * Synthesized by Pseudomonas syringae pv. tomato Levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.

  • Levansucrases of a pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo and polysaccharides
    New Biotechnology, 2015
    Co-Authors: Triinu Visnapuu, Karin Mardo, Tiina Alamae
    Abstract:

    Gut microbiota influences more physiological and developmental processes of humans and animals than earlier expected. Therefore, the possibility to shape the composition and activity of this bacterial population by prebiotics becomes especially important. Inulin, a β-2,1 linked fructan polymer, from plants and fructooligosaccharides (FOS) derived from it are recognized and already widely used as prebiotics while β-2,6 linked fructans have received much less attention from scientific community. In this mini-review, we will address β-2,6 linked fructans: levan and levan-type FOS as novel potential prebiotics and summarize the literature data on Levansucrases of Pseudomonas bacteria which are producing these fructans. The major attention is drawn to stable and highly efficient Levansucrases of Pseudomonas syringae pv. tomato, among which the Lsc3 protein has been most thoroughly studied using biochemical methods as well as extensive mutagenesis of the protein.

  • mutational analysis of conserved regions harboring catalytic triad residues of the Levansucrase protein encoded by the lsc 3 gene lsc3 of pseudomonas syringae pv tomato dc3000
    Biotechnology and Applied Biochemistry, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi, Tiina Alamae
    Abstract:

    Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for Levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.

Karin Mardo - One of the best experts on this subject based on the ideXlab platform.

  • a highly active endo levanase bt1760 of a dominant mammalian gut commensal bacteroides thetaiotaomicron cleaves not only various bacterial levans but also levan of timothy grass
    PLOS ONE, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  • A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass
    PloS one, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (

  • Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
    2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    * Synthesized by Pseudomonas syringae pv. tomato Levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.

  • Levansucrases of a pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo and polysaccharides
    New Biotechnology, 2015
    Co-Authors: Triinu Visnapuu, Karin Mardo, Tiina Alamae
    Abstract:

    Gut microbiota influences more physiological and developmental processes of humans and animals than earlier expected. Therefore, the possibility to shape the composition and activity of this bacterial population by prebiotics becomes especially important. Inulin, a β-2,1 linked fructan polymer, from plants and fructooligosaccharides (FOS) derived from it are recognized and already widely used as prebiotics while β-2,6 linked fructans have received much less attention from scientific community. In this mini-review, we will address β-2,6 linked fructans: levan and levan-type FOS as novel potential prebiotics and summarize the literature data on Levansucrases of Pseudomonas bacteria which are producing these fructans. The major attention is drawn to stable and highly efficient Levansucrases of Pseudomonas syringae pv. tomato, among which the Lsc3 protein has been most thoroughly studied using biochemical methods as well as extensive mutagenesis of the protein.

  • mutational analysis of conserved regions harboring catalytic triad residues of the Levansucrase protein encoded by the lsc 3 gene lsc3 of pseudomonas syringae pv tomato dc3000
    Biotechnology and Applied Biochemistry, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi, Tiina Alamae
    Abstract:

    Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for Levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.

Heiki Vija - One of the best experts on this subject based on the ideXlab platform.

  • a highly active endo levanase bt1760 of a dominant mammalian gut commensal bacteroides thetaiotaomicron cleaves not only various bacterial levans but also levan of timothy grass
    PLOS ONE, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  • A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass
    PloS one, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (

  • Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
    2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    * Synthesized by Pseudomonas syringae pv. tomato Levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.

  • mutational analysis of conserved regions harboring catalytic triad residues of the Levansucrase protein encoded by the lsc 3 gene lsc3 of pseudomonas syringae pv tomato dc3000
    Biotechnology and Applied Biochemistry, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi, Tiina Alamae
    Abstract:

    Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for Levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.

  • High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan
    MDPI AG, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Maria Gromkova, Tiina Alamae
    Abstract:

    Bacterial Levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of Levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged Levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized Levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the Levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes

Anneli Aasamets - One of the best experts on this subject based on the ideXlab platform.

  • a highly active endo levanase bt1760 of a dominant mammalian gut commensal bacteroides thetaiotaomicron cleaves not only various bacterial levans but also levan of timothy grass
    PLOS ONE, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

  • A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass
    PloS one, 2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the Levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, Levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (

  • Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.
    2017
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Tiina Alamae
    Abstract:

    * Synthesized by Pseudomonas syringae pv. tomato Levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see Table 1.

  • High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan
    MDPI AG, 2014
    Co-Authors: Karin Mardo, Triinu Visnapuu, Heiki Vija, Anneli Aasamets, Katrin Viigand, Maria Gromkova, Tiina Alamae
    Abstract:

    Bacterial Levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of Levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged Levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized Levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the Levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes