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Abbas Mirshafiey - One of the best experts on this subject based on the ideXlab platform.

  • evaluation of the oral administration of α l Guluronic Acid on cox 1 and cox 2 gene expression profile in ankylosing spondylitis patients
    Drug Development Research, 2021
    Co-Authors: Arezoo Sadoughi, Sepideh Nazeri, Reza Mansouri, Abbas Mirshafiey
    Abstract:

    Ankylosing spondylitis (AS) is a chronic autoimmune arthritis disease with a genetic background, affecting the skeletal axis, sacroiliac, and peripheral joints. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for AS to alleviate the inflammation and pain. Despite the beneficial effect, their use is accompanied by a wide variety of possible side effects in the gastrointestinal and kidneys. The α-l-Guluronic Acid (G2013) is a new nonsteroidal anti-inflammatory patented (PCT/EP2017/067920) drug, which has shown its anti-inflammatory properties in the previous investigations. The present study revealed the oral administration effect of G2013 on COX-1 and COX-2 gene expression in AS patients. The blood samples of twelve 18-45 years old patients suffering AS and BASDAI >4, and BASFI >4, before and after 12 weeks of treatment with G2013 and 12 blood samples of healthy volunteers were collected and the effect of G2013 on the gene expression of COX-1 and COX-2 enzymes were assessed by Real-Time PCR. The results indicate that G2013 is able to reduce the gene expression level of COX-1 and COX-2 enzymes in treated AS patients compared to healthy control. Statistically significant differences were not observed between the treatment and the healthy control groups. According to the findings, G2013 might be categorized and introduced as a novel NSAID for the treatment of AS.

  • anti diabetic and angio protective effect of Guluronic Acid g2013 as a new nonsteroidal anti inflammatory drug in the experimental model of diabetes
    Endocrine‚ Metabolic & Immune Disorders-Drug Targets, 2020
    Co-Authors: Seyed Shahabeddin Mortazavijahromi, Shahab Alizadeh, Mohammad Hassan Javanbakht, Abbas Mirshafiey
    Abstract:

    BACKGROUND This study aimed to investigate the effects of Guluronic Acid (G2013) on blood sugar, insulin, and gene expression profile of oxLDL receptors (SR-A, CD36, LOX-1, and CD68) in the experimental model of diabetes. METHODS 18 Sprague Dawley rats were randomly assigned to three groups of healthy control, diabetic control, and G2013 group. Diabetes was induced through intraperitoneal (IP) injection of 60 mg/kg streptozotocin. The subjects were IP treated with 25 mg/kg of G2013 per day for 28 days. The body weight, food intake, fasting blood glucose and insulin were measured. In addition, the expression of mentioned genes was investigated through quantitative real-time PCR. RESULTS The data showed that the final weight increased significantly in the G2013-treated subjects compared to the diabetic control (p < 0.05). The results indicated that final food intake significantly reduced in the G2013-treated subjects compared to the diabetic control (p < 0.05). The study findings also suggested that the final fasting blood glucose significantly reduced in the G2013-treated group, whereas the final fasting serum insulin level significantly increased in this group compared to the diabetic control (p < 0.05). Moreover, the gene expression levels of SR-A, CD36, LOX-1, and CD68 in the G2013 group significantly reduced compared to the diabetic control (p < 0.05). CONCLUSION This study showed that G2013, could reduce blood glucose and increase insulin levels and reduce the gene expression level of oxLDL receptors. In addition, it may probably play an important role in reducing the severity of diabetes-induced inflammatory symptoms.

  • effect of Guluronic Acid g2013 as a new anti inflammatory drug on gene expression of pro inflammatory and anti inflammatory cytokines and their transcription factors in rheumatoid arthritis patients
    Iranian Journal of Allergy Asthma and Immunology, 2020
    Co-Authors: Tahereh Bakhtiari, Shahin Khadem Azarian, Afshin Ghaderi, Arman Ahmadzadeh, Abbas Mirshafiey
    Abstract:

    Rheumatoid arthritis (RA) as a long-term autoimmune disease is characterized by pain, swelling and joints destruction. The therapeutic efficacy of Guluronic Acid (G2013) (patented, DEU: 102016113017.6) was reported in phase I/II clinical trial in RA patients. In this study, we aimed to evaluate the effect of G2013 as a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property on genes expression of anti-inflammatory and pro-inflammatory cytokines and their transcription factors in the blood sample of RA patients. This study was performed on 12 patients with RA who had an inadequate response to conventional treatments which were disease-modifying anti-rheumatic drugs (DMARDs), NSAID, and biologics. G2013 was administered orally at a dose of 500 mg twice daily for 12 weeks. Before and after the treatment of patients with drug G2013, the peripheral blood mononuclear cells (PBMCs) were isolated for evaluating the gene expression level of interleukin 10 (IL10), interleukin 22 (IL22), interferon γ (IFNγ), and transcription factors specific to the T helper cell lineages, forkhead box P3 (Fox-P3), Aryl hydrocarbon receptor (AHR) and T-box-containing protein expressed in T cells (T-bet) using the real-time PCR method. Since these cytokines have a key role in the progression of RA and disease condition expected induction of IFNγ, AHR, IL22, T-bet, and reduction of IL10, Fox-P3. Results indicated a significant reduction in the level of IFNγ, AHR and a significant induction in IL10, Fox-P3 gene expression in comparison with the control group. In conclusion; the results of this investigation showed a part of the immunological mechanism of G2013 as a novel anti-inflammatory that could reduce pro-inflammatory cytokine and their transcription factors. Furthermore, it increased the anti-inflammatory cytokine and its transcription factor (clinical trial identifier: IRCT2016092813739N5).

  • effects of Guluronic Acid g2013 on gene expression of tlr2 tlr4 myd88 tnf α and cd52 in multiple sclerosis under in vitro conditions
    Immunopharmacology and Immunotoxicology, 2019
    Co-Authors: Seyedeh Masoomeh Noorbakhsh, Zahra Aghazadeh, Mona Oraei, Alireza Razavi, Nahid Beladi Moghadam, Payam Saadat, Mostafa Hoseini, Maryam Mobini, Abbas Mirshafiey
    Abstract:

    AbstractContext: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-Guluronic Acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive...

  • evaluation of the acute and 28 day sub acute intravenous toxicity of α l Guluronic Acid alg g2013 in mice
    Drug and Chemical Toxicology, 2019
    Co-Authors: Ahmad Mahdianshakib, Mohammad Sadegh Hashemzadeh, Ali Anissian, Mona Oraei, Abbas Mirshafiey
    Abstract:

    α-l-Guluronic Acid (ALG; G2013) has been previously introduced as a new anti-inflammatory agent with promising therapeutic effects. Thus, in the present study, we aimed to evaluate the acute and su...

Gudmund Skjakbraek - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis and function of long Guluronic Acid blocks in alginate produced by azotobacter vinelandii
    Biomacromolecules, 2019
    Co-Authors: Olav Andreas Aarstad, Finn Lillelund Aachmann, Anne Tondervik, Havard Sletta, Annalucia Stanisci, Gerd Inger Sætrom, Gudmund Skjakbraek
    Abstract:

    With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown ...

  • heparin like properties of sulfated alginates with defined sequences and sulfation degrees
    Biomacromolecules, 2014
    Co-Authors: Oystein Arlov, Finn Lillelund Aachmann, Anders Sundan, Terje Espevik, Gudmund Skjakbraek
    Abstract:

    Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-d-mannuronic Acid (M) and α-l-Guluronic Acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar mo...

  • alginate sequencing an analysis of block distribution in alginates using specific alginate degrading enzymes
    Biomacromolecules, 2012
    Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund Skjakbraek
    Abstract:

    Distribution and proportion of β-d-mannuronic and α-l-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diGuluronic Acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with 1H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (FG = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerase...

  • isolation of mutant alginate lyases with cleavage specificity for di Guluronic Acid linkages
    Journal of Biological Chemistry, 2010
    Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard Sletta
    Abstract:

    Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-l-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.

  • the pseudomonas fluorescens algg protein but not its mannuronan c 5 epimerase activity is needed for alginate polymer formation
    Journal of Bacteriology, 2003
    Co-Authors: Martin Gimmestad, Gudmund Skjakbraek, Helga Ertesvag, Trond E Ellingsen, Havard Sletta, Karianne Bakkevig, Sumita Jain, Sangjin Suh, Dennis E Ohman, Svein Valla
    Abstract:

    Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic Acid residues to alpha-L-Guluronic Acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino Acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic Acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of Guluronic Acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Svein Valla - One of the best experts on this subject based on the ideXlab platform.

  • isolation of mutant alginate lyases with cleavage specificity for di Guluronic Acid linkages
    Journal of Biological Chemistry, 2010
    Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard Sletta
    Abstract:

    Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-l-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.

  • characterization of three new azotobacter vinelandii alginate lyases one of which is involved in cyst germination
    Journal of Bacteriology, 2009
    Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren Glaerum Svanem, Svein Valla
    Abstract:

    Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic Acid and α-l-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between Guluronic Acid and mannuronic Acid, resulting in a Guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

  • Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination
    2009
    Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren, Glærum Svanem, Svein Valla
    Abstract:

    Alginates are polysaccharides composed of 1-4-linked -D-mannuronic Acid and -L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a -elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2. All three enzymes preferably cleave the bond between Guluronic Acid and mannuronic Acid, resulting in a Guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinat

  • structural and mutational characterization of the catalytic a module of the mannuronan c 5 epimerase alge4 from azotobacter vinelandii
    Journal of Biological Chemistry, 2008
    Co-Authors: Henriette J Rozeboom, Svein Valla, Tonje M Bjerkan, Kor H Kalk, Synnove Holtan, Finn Lillelund Aachmann, Bauke W Dijkstra
    Abstract:

    Abstract Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic Acid and its C-5 epimer α-l-Guluronic Acid. The polymer is first produced as polymannuronic Acid and the Guluronic Acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino Acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction.

  • the pseudomonas fluorescens algg protein but not its mannuronan c 5 epimerase activity is needed for alginate polymer formation
    Journal of Bacteriology, 2003
    Co-Authors: Martin Gimmestad, Gudmund Skjakbraek, Helga Ertesvag, Trond E Ellingsen, Havard Sletta, Karianne Bakkevig, Sumita Jain, Sangjin Suh, Dennis E Ohman, Svein Valla
    Abstract:

    Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic Acid residues to alpha-L-Guluronic Acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino Acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic Acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of Guluronic Acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Helga Ertesvag - One of the best experts on this subject based on the ideXlab platform.

  • Guluronic Acid content as a factor affecting turbidity removal potential of alginate
    Environmental Science and Pollution Research, 2016
    Co-Authors: Helga Ertesvag, Cigdem Kivilcimdan Moral, Dilek F Sanin
    Abstract:

    Alginates are natural polymers composed of mannuronic and Guluronic Acid residues. They are currently extracted from brown algae; however, alginate can also be synthesized by some species of Azotobacter and Pseudomonas. Alginates with different proportion of mannuronic and Guluronic Acids are known to have different characteristics and form gels at different extents in the presence of calcium ions. The aim of this work was to investigate the usefulness of alginate as a non-toxic coagulant used in purification of drinking water. This study utilized alginates from Azotobacter vinelandii having different Guluronic Acid levels. These were obtained partly by changing the cultivation parameters, partly by epimerizing a purified alginate sample in vitro using the A. vinelandii mannuronan C-5 epimerase AlgE1. The different alginates were then used for coagulation together with calcium. The results showed that turbidity removal capability was dependent on the content of Guluronic Acid residues. For the best performing samples, the turbidity decreased from 10 NTU to 1 NTU by the use of only 2 mg/L of alginate and 1.5 mM of calcium chloride.

  • isolation of mutant alginate lyases with cleavage specificity for di Guluronic Acid linkages
    Journal of Biological Chemistry, 2010
    Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard Sletta
    Abstract:

    Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-l-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.

  • characterization of three new azotobacter vinelandii alginate lyases one of which is involved in cyst germination
    Journal of Bacteriology, 2009
    Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren Glaerum Svanem, Svein Valla
    Abstract:

    Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic Acid and α-l-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between Guluronic Acid and mannuronic Acid, resulting in a Guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

  • Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination
    2009
    Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren, Glærum Svanem, Svein Valla
    Abstract:

    Alginates are polysaccharides composed of 1-4-linked -D-mannuronic Acid and -L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a -elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2. All three enzymes preferably cleave the bond between Guluronic Acid and mannuronic Acid, resulting in a Guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinat

  • the pseudomonas fluorescens algg protein but not its mannuronan c 5 epimerase activity is needed for alginate polymer formation
    Journal of Bacteriology, 2003
    Co-Authors: Martin Gimmestad, Gudmund Skjakbraek, Helga Ertesvag, Trond E Ellingsen, Havard Sletta, Karianne Bakkevig, Sumita Jain, Sangjin Suh, Dennis E Ohman, Svein Valla
    Abstract:

    Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic Acid residues to alpha-L-Guluronic Acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino Acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic Acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of Guluronic Acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Olav Andreas Aarstad - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis and function of long Guluronic Acid blocks in alginate produced by azotobacter vinelandii
    Biomacromolecules, 2019
    Co-Authors: Olav Andreas Aarstad, Finn Lillelund Aachmann, Anne Tondervik, Havard Sletta, Annalucia Stanisci, Gerd Inger Sætrom, Gudmund Skjakbraek
    Abstract:

    With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown ...

  • Biosynthesis and Function of Long Guluronic Acid-Blocks in Alginate Produced by Azotobacter vinelandii
    2019
    Co-Authors: Olav Andreas Aarstad, Finn Lillelund Aachmann, Anne Tondervik, Havard Sletta, Annalucia Stanisci, Gerd Inger Sætrom, Gudmund Skjåk-bræk
    Abstract:

    With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of Guluronic Acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic Acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young’s modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources

  • alginate sequencing an analysis of block distribution in alginates using specific alginate degrading enzymes
    Biomacromolecules, 2012
    Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund Skjakbraek
    Abstract:

    Distribution and proportion of β-d-mannuronic and α-l-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diGuluronic Acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with 1H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (FG = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerase...

  • Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.
    Biomacromolecules, 2011
    Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund Skjåk-bræk
    Abstract:

    Distribution and proportion of β-d-mannuronic and α-l-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, ...

  • isolation of mutant alginate lyases with cleavage specificity for di Guluronic Acid linkages
    Journal of Biological Chemistry, 2010
    Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard Sletta
    Abstract:

    Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-l-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.