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William Helbert - One of the best experts on this subject based on the ideXlab platform.
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Enzyme-Assisted Preparation of Furcellaran-Like κ-/β-Carrageenan
Marine Biotechnology, 2016Co-Authors: Aurelie Prechoux, Sabine Genicot, Hélène Rogniaux, William HelbertAbstract:Carrageenans are sulfated galactans that are widely used in industrial applications for their thickening and gelling properties, which vary according to the amount and distribution of ester sulfate groups along the galactan backbone. To determine and direct the sulfation of kappa-Carrageenan moieties, we purified an endo-kappa-Carrageenan sulfatase (Q15XH1 accession in UniprotKB) from Pseudoalteromonas atlantica T6c extracts. Based on sequence analyses and exploration of the genomic environment of Q15XH1, we discovered and characterized a second endo-kappa-Carrageenan sulfatase (Q15XG7 accession in UniprotKB). Both enzymes convert kappa-Carrageenan into a hybrid, furcellaran-like kappa-/beta-Carrageenan. We compared the protein sequences of these two new kappa-Carrageenan sulfatases and that of a previously reported tau-Carrageenan sulfatase with other predicted sulfatases in the P. atlantica genome, revealing the existence of additional new Carrageenan sulfatases.
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Discovery of a novel iota Carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora
Frontiers in Chemistry, 2014Co-Authors: Sabine Genicot, Tristan Barbeyron, William HelbertAbstract:Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for Carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of Carrageenans. As a result of the screening, an iota-Carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain Psc(T). It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc t-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH similar to 8.3 and at 40 +/- 5 degrees C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc t.-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-Carrageenan. The purified enzyme could transform iota-Carrageenan into hybrid iota-/alpha- or pure alpha-Carrageenan under controlled conditions. The gene encoding Psc t-CgsA, a protein of 1038 amino acids, was cloned into Escherichia colt, and the sequence analysis revealed that Psc t.-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes.
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Controlling Carrageenan Structure Using a Novel Formylglycine-Dependent Sulfatase, an Endo-4S-iota-Carrageenan Sulfatase
Marine Biotechnology, 2013Co-Authors: Aurelie Prechoux, Sabine Genicot, Hélène Rogniaux, William HelbertAbstract:Carrageenans are sulfated polysaccharides that are found in the cell walls of red algae. These polysaccharides have gelling and texturizing properties that are widely appreciated in industrial applications. However, these functional properties depend strongly on the sulfation of the moieties of the carrabiose repetition unit. Here we aimed to monitor the sulfate composition of gelling Carrageenan. To do so, we screened and purified from Pseudoalteromonas atlantica a 4S-iota Carrageenan sulfatase that converts ι-carrabiose into α-carrabiose units. The sequence of this protein matched the annotated Q15XH3 (Uniprot databank) formylglycine-dependent sulfatase found in the P. atlantica genome. With pure enzyme, ι-Carrageenan could be transformed into a hybrid ι-/α-Carrageenan or pure α-Carrageenan. Analysis of the distribution of the carrabiose moieties in hybrid Carrageenan chain using enzymatic degradation with Alteromonas fortis ι-carrageenase, coupled with chromatography and NMR spectroscopy experiments, showed that the sulfatase has an endo mode of action. The endo-character and the specificity of the sulfatase made it possible to prepare hybrid κ-/ι-/α-Carrageenan and κ-/α-Carrageenan starting from κ-/ι-Carrageenan.
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the cyclization of the 3 6 anhydro galactose ring of ι Carrageenan is catalyzed by two d galactose 2 6 sulfurylases in the red alga chondrus crispus
Plant Physiology, 2009Co-Authors: Sabine Genicotjoncour, Bernard Kloareg, Alexandra Poinas, Odile Richard, Philippe Potin, Brian Rudolph, William HelbertAbstract:Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the Carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in κ- and ι-Carrageenans through the enzymatic conversion of d-galactose-6-sulfate or d-galactose-2,6-disulfate occurring in μ- and ν-Carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of ν- into ι-Carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of Carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.
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The cyclization of the 3,6-anhydro-galactose ring of iota-Carrageenan is catalyzed by two D-galactose-2,6-sulfurylases in the red alga Chondrus crispus.
Plant Physiology, 2009Co-Authors: Sabine Genicot-joncour, Bernard Kloareg, Alexandra Poinas, Odile Richard, Philippe Potin, Brian Rudolph, William HelbertAbstract:Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the Carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in kappa- and iota-Carrageenans through the enzymatic conversion of d-galactose-6-sulfate or d-galactose-2,6-disulfate occurring in mu- and nu-Carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of nu- into iota-Carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of Carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.
Camille Michon - One of the best experts on this subject based on the ideXlab platform.
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Starch/Carrageenan mixed systems: Penetration in, adsorption on or exclusion of Carrageenan chains by granules?
Food Hydrocolloids, 2014Co-Authors: A. Matignon, P. Barey, M. Desprairies, S. Mauduit, J.m. Sieffermann, Camille MichonAbstract:Abstract This study focused on the understanding of starch/Carrageenan interactions. Seven Carrageenan samples and a modified waxy maize starch were used. Two types of blends were made: starch was pasted in Carrageenan solutions or swelled starch granules were mixed with Carrageenan solutions. For each blend, the Carrageenan concentration in the continuous phase was determined by indirect titration that permitted to evaluate the percentages of Carrageenan trapped by starch granules. The presence of Carrageenan in the continuous phase was shown to impact the swollen starch granules' sizes and characteristics. The level of viscosity in the continuous phase was shown to depend on the starch/Carrageenan interactions but also on the released amylopectin concentration. Starch pasting was shown to be unnecessary to promote interactions as the same percentage of trapped Carrageenan was found when put into contact with starch granules either before or after swelling. Carrageenans were found to be both excluded and adsorbed on starch granules. The level of adsorption of Carrageenan on starch granules seemed to be promoted by low-negatively-charged Carrageenan and by low-molecular-weight Carrageenan.
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Starch/Carrageenan mixed systems: Penetration in, adsorption on or exclusion of Carrageenan chains by granules?
Food Hydrocolloids, 2014Co-Authors: A. Matignon, P. Barey, M. Desprairies, S. Mauduit, J.m. Sieffermann, Camille MichonAbstract:This study focused on the understanding of starch/Carrageenan interactions. Seven Carrageenan samples and a modified waxy maize starch were used. Two types of blends were made: starch was pasted in Carrageenan solutions or swelled starch granules were mixed with Carrageenan solutions. For each blend, the Carrageenan concentration in the continuous phase was determined by indirect titration that permitted to evaluate the percentages of Carrageenan trapped by starch granules. The presence of Carrageenan in the continuous phase was shown to impact the swollen starch granules' sizes and characteristics. The level of viscosity in the continuous phase was shown to depend on the starch/Carrageenan interactions but also on the released amylopectin concentration. Starch pasting was shown to be unnecessary to promote interactions as the same percentage of trapped Carrageenan was found when put into contact with starch granules either before or after swelling. Carrageenans were found to be both excluded and adsorbed on starch granules. The level of adsorption of Carrageenan on starch granules seemed to be promoted by low-negatively-charged Carrageenan and by low-molecular-weight Carrageenan. (C) 2013 Elsevier Ltd. All rights reserved.
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Starch gelatinization in Carrageenan solutions: exclusions, adsorption or absoprption of Carrageenan chains by starch granules?
2012Co-Authors: A. Matignon, Delphine Huc-mathis, Philippe Barey, Marc Desprairies, Stéphane Mauduit, Camille MichonAbstract:Starch-hydrocolloid combinations are widely used in food products. Hydrocolloids influence the texture of starch-containing media, indicating possible interactions. However, the mechanisms are not yet elucidated. One of the proposed (but not established) mechanism suggests exclusion, at least partly, of the hydrocolloid by starch granules during gelatinization and thus an increase of hydrocolloid concentration in the continuous phase [1]. The objective of this work was to investigate the behavior of κ (Ck) and ι (Ci) Carrageenans during gelatinization of modified waxy maize starch for different Carrageenan/starch ratios. Two experiments were done: one with increasing starch concentration (from 0.5 to 4 %)/Carrageenan (0.2 %), and the other with starch (2 %)/increasing Carrageenan concentration (0.1 to 0.4 % Ck and Ci). The starch dispersed in Carrageenan solutions was gelatinized in non destroying conditions (up to 85◦C, 10◦C/min) and separated from the suspensions by centrifugation (10000 g/5 min). The Carrageenan concentration in the supernatant was evaluated with the help of Ck and Ci reference curves connecting the apparent viscosity measured at 20◦C to the concentration of Carrageenan. This was compared to values calculated using two extreme assumptions: (H1) Carrageenan penetrates into the starch granules driven by solvent and (H2) Carrageenan does not penetrate at all into them (total exclusion). A percentage of Carrageenan that is trapped by starch granules (% Ctrapped) was then evaluated. The % Ctrapped was higher for Ck (∼ 80 %), than for Ci (∼ 30 %). However, neither the increase of starch nor Carrageenan concentration in the mixtures affected the % Ctrapped (Ck and Ci). In order to understand the mechanism of trapping of Carrageenan by swelling starch granules, the influence of the contact moment between starch and Carrageenan (before and after gelatinization) was studied. Similar % Ctrapped was obtained, independent of whether the contact moment was before or after gelatinization. Confocal laser scanning microscopy visualizations of the blends clearly showed Carrageenan adsorbed on starch granules. As a conclusion, Carrageenan chains were found to be partly excluded, and partly adsorbed by starch granules. The proportion of each phenomenon depended on Carrageenan types, but not on the mixture ratios.
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Structure evolution of Carrageenan/milk gels: effect of shearing, Carrageenan concentration and nu fraction on rheological behavior
Food Hydrocolloids, 2005Co-Authors: Camille Michon, V Langendorff, Celine Chapuis, P Boulenguer, Gérard CuvelierAbstract:Abstract Carrageenans, sulphated polysaccharides extracted from red seaweeds, are widely added to milk systems because of the gelation they promote and for their ability to recover a structure at rest after shearing. The gelation of iota-Carrageenan is closely related to the helix-coil transition they undergo at approximately 48 °C in milk. Carrageenan gels (e.g. iota with and without nu precursors) were formed in milk or permeate by decreasing the temperature from 60 to 10 °C following various mechanical treatments, e.g. gelation and ageing of the gel at rest, gelation under shearing and ageing at rest. Gels were characterized rheologically during cooling and ageing, performed in the dynamic mode, to follow the mechanical recovery of the mixed systems at rest. In many cases, a strain-hardening behavior was observed using strain sweep measurements: above the upper limit of the linear domain, stress increased more than proportionally to the strain. Two types of iota-Carrageenan were compared: iota with and without nu precursors. The content of the precursor affects the ability to form helical structures. Samples without precursor gave the highest level of helical structures. The Carrageenan concentration effect was tested in the range 0.1–0.5 wt%. Differences in sheared and unsheared gel properties, particularly in large strain conditions, are discussed in terms of gelation process, phase separation, Carrageenan/milk proportion and Carrageenan type. The industrial interest in using either type of Carrageenan is discussed.
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Structure evolution of Carrageenan/milk gels: effect of shearing, Carrageenan concentration and nu fraction on rheological behavior
Food Hydrocolloids, 2005Co-Authors: Camille Michon, V Langendorff, Celine Chapuis, P Boulenguer, Gérard CuvelierAbstract:Carrageenans, sulphated polysaccharides extracted from red seaweeds, are widely added to milk systems because of the gelation they promote and for their ability to recover a structure at rest after shearing. The gelation of iota-Carrageenan is closely related to the helix-coil transition they undergo at approximately 48 degreesC in milk. Carrageenan gels (e.g. iota with and without nu precursors) were formed in milk or permeate by decreasing the temperature from 60 to 10 degreesC following various mechanical treatments, e.g. gelation and ageing of the gel at rest, gelation under shearing and ageing at rest. Gels were characterized rheologically during cooling and ageing, performed in the dynamic mode, to follow the mechanical recovery of the mixed systems at rest. In many cases, a strain-hardening behavior was observed using strain sweep measurements: above the upper limit of the linear domain, stress increased more than proportionally to the strain. Two types of iota-Carrageenan were compared: iota with and without nu precursors. The content of the precursor affects the ability to form helical structures. Samples without precursor gave the highest level of helical structures. The Carrageenan concentration effect was tested in the range 0.1-0.5 wt%. Differences in sheared and unsheared gel properties, particularly in large strain conditions, are discussed in terms of gelation process, phase separation, Carrageenan/milk proportion and Carrageenan type. The industrial interest in using either type of Carrageenan is discussed
N. Cayot - One of the best experts on this subject based on the ideXlab platform.
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Impact of the composition of polysaccharide composite gels on small molecules diffusion: A rheological and NMR study
Food Research International, 2010Co-Authors: Géraldine Savary, C. Moreau, N. CayotAbstract:The gelation mechanism of Carrageenan depends on the amount and nature of the polysaccharide, and is cation sensitive. From a rheological approach, this specificity leads to different textural properties. In composite gels with Carrageenans, starch and sucrose, the presence of κ/κ2-Carrageenan, even at low levels, has an impact on textural and structural properties. In this study, rheological and diffusion NMR measurements were performed on composite gels to probe gel structure at the macro- and micro-scale. Variations were made in the gel composition by varying the Carrageenan content and the nature of the polysaccharide and cations. We showed that all the factors that increased the rigidity of the composite gels – polysaccharide content and specific cations – decreased significantly the diffusion of small molecules such as sucrose and ethyl butyrate.
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Impact of the composition of polysaccharide composite gels on small molecules diffusion: A rheological and NMR study
Food Research International, 2010Co-Authors: Géraldine Savary, C. Moreau, N. CayotAbstract:The gelation mechanism of Carrageenan depends on the amount and nature of the polysaccharide, and is cation sensitive. From a theological approach, this specificity leads to different textural properties. In composite gels with Carrageenans, starch and sucrose, the presence Of kappa/kappa 2-Carrageenan, even at low levels, has an impact on textural and structural properties. In this study, theological and diffusion NMR measurements were performed on composite gels to probe gel structure at the macro- and micro-scale. Variations were made in the gel composition by varying the Carrageenan content and the nature of the polysaccharide and cations. We showed that all the factors that increased the rigidity of the composite gels - polysaccharide content and specific cations - decreased significantly the diffusion of small molecules such as sucrose and ethyl butyrate. (C) 2009 Elsevier Ltd. All rights reserved.
Géraldine Savary - One of the best experts on this subject based on the ideXlab platform.
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Impact of the composition of polysaccharide composite gels on small molecules diffusion: A rheological and NMR study
Food Research International, 2010Co-Authors: Géraldine Savary, C. Moreau, N. CayotAbstract:The gelation mechanism of Carrageenan depends on the amount and nature of the polysaccharide, and is cation sensitive. From a rheological approach, this specificity leads to different textural properties. In composite gels with Carrageenans, starch and sucrose, the presence of κ/κ2-Carrageenan, even at low levels, has an impact on textural and structural properties. In this study, rheological and diffusion NMR measurements were performed on composite gels to probe gel structure at the macro- and micro-scale. Variations were made in the gel composition by varying the Carrageenan content and the nature of the polysaccharide and cations. We showed that all the factors that increased the rigidity of the composite gels – polysaccharide content and specific cations – decreased significantly the diffusion of small molecules such as sucrose and ethyl butyrate.
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Impact of the composition of polysaccharide composite gels on small molecules diffusion: A rheological and NMR study
Food Research International, 2010Co-Authors: Géraldine Savary, C. Moreau, N. CayotAbstract:The gelation mechanism of Carrageenan depends on the amount and nature of the polysaccharide, and is cation sensitive. From a theological approach, this specificity leads to different textural properties. In composite gels with Carrageenans, starch and sucrose, the presence Of kappa/kappa 2-Carrageenan, even at low levels, has an impact on textural and structural properties. In this study, theological and diffusion NMR measurements were performed on composite gels to probe gel structure at the macro- and micro-scale. Variations were made in the gel composition by varying the Carrageenan content and the nature of the polysaccharide and cations. We showed that all the factors that increased the rigidity of the composite gels - polysaccharide content and specific cations - decreased significantly the diffusion of small molecules such as sucrose and ethyl butyrate. (C) 2009 Elsevier Ltd. All rights reserved.
Sabine Genicot - One of the best experts on this subject based on the ideXlab platform.
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Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria
Nature Communications, 2017Co-Authors: Elizabeth Ficko-blean, Sabine Genicot, Aurelie Prechoux, François Thomas, Tatiana Rochat, Robert Larocque, Yongtao Zhu, Mark Stam, Murielle Jam, Alexandra CalteauAbstract:Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for Carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted Carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The Carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.
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Enzyme-Assisted Preparation of Furcellaran-Like κ-/β-Carrageenan
Marine Biotechnology, 2016Co-Authors: Aurelie Prechoux, Sabine Genicot, Hélène Rogniaux, William HelbertAbstract:Carrageenans are sulfated galactans that are widely used in industrial applications for their thickening and gelling properties, which vary according to the amount and distribution of ester sulfate groups along the galactan backbone. To determine and direct the sulfation of kappa-Carrageenan moieties, we purified an endo-kappa-Carrageenan sulfatase (Q15XH1 accession in UniprotKB) from Pseudoalteromonas atlantica T6c extracts. Based on sequence analyses and exploration of the genomic environment of Q15XH1, we discovered and characterized a second endo-kappa-Carrageenan sulfatase (Q15XG7 accession in UniprotKB). Both enzymes convert kappa-Carrageenan into a hybrid, furcellaran-like kappa-/beta-Carrageenan. We compared the protein sequences of these two new kappa-Carrageenan sulfatases and that of a previously reported tau-Carrageenan sulfatase with other predicted sulfatases in the P. atlantica genome, revealing the existence of additional new Carrageenan sulfatases.
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Discovery of a novel iota Carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora
Frontiers in Chemistry, 2014Co-Authors: Sabine Genicot, Tristan Barbeyron, William HelbertAbstract:Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for Carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of Carrageenans. As a result of the screening, an iota-Carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain Psc(T). It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc t-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH similar to 8.3 and at 40 +/- 5 degrees C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc t.-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-Carrageenan. The purified enzyme could transform iota-Carrageenan into hybrid iota-/alpha- or pure alpha-Carrageenan under controlled conditions. The gene encoding Psc t-CgsA, a protein of 1038 amino acids, was cloned into Escherichia colt, and the sequence analysis revealed that Psc t.-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes.
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Controlling Carrageenan Structure Using a Novel Formylglycine-Dependent Sulfatase, an Endo-4S-iota-Carrageenan Sulfatase
Marine Biotechnology, 2013Co-Authors: Aurelie Prechoux, Sabine Genicot, Hélène Rogniaux, William HelbertAbstract:Carrageenans are sulfated polysaccharides that are found in the cell walls of red algae. These polysaccharides have gelling and texturizing properties that are widely appreciated in industrial applications. However, these functional properties depend strongly on the sulfation of the moieties of the carrabiose repetition unit. Here we aimed to monitor the sulfate composition of gelling Carrageenan. To do so, we screened and purified from Pseudoalteromonas atlantica a 4S-iota Carrageenan sulfatase that converts ι-carrabiose into α-carrabiose units. The sequence of this protein matched the annotated Q15XH3 (Uniprot databank) formylglycine-dependent sulfatase found in the P. atlantica genome. With pure enzyme, ι-Carrageenan could be transformed into a hybrid ι-/α-Carrageenan or pure α-Carrageenan. Analysis of the distribution of the carrabiose moieties in hybrid Carrageenan chain using enzymatic degradation with Alteromonas fortis ι-carrageenase, coupled with chromatography and NMR spectroscopy experiments, showed that the sulfatase has an endo mode of action. The endo-character and the specificity of the sulfatase made it possible to prepare hybrid κ-/ι-/α-Carrageenan and κ-/α-Carrageenan starting from κ-/ι-Carrageenan.
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degradation of λ Carrageenan by pseudoalteromonas carrageenovora λ carrageenase a new family of glycoside hydrolases unrelated to κ and ι carrageenases
Biochemical Journal, 2007Co-Authors: Marion Guibet, Sébastien Colin, Gurvan Michel, Bernard Kloareg, Sabine Genicot, Tristan Barbeyron, William HelbertAbstract:Carrageenans are sulfated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulfate ester groups. λ-Carrageenan is the most sulfated Carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of λ-Carrageenan, the λ-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the λ-carrageenase, referred to as CglA, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains κ-carrageenases, and GH82, that contains ι-carrageenases. This large enzyme (105 kDa) features a low-complexity region, suggesting the presence of a linker connecting at least two independent modules. The N-terminal region is predicted to fold as a β-propeller. The main degradation products have been purified and characterized as neo-λ-carratetraose [DP (degree of polymerization) 4] and neo-λ-carrahexaose (DP6), indicating that CglA hydrolyses the β-(1→4) linkage of λ-Carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and 1H-NMR monitoring of the enzymatic degradation of λ-Carrageenan indicate that CglA proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-λ-carrabiose oligosaccharides, in the present study we demonstrate that the active site of CglA comprises at least 8 subsites (−4 to +4) and that a DP6 oligosaccharide binds in the subsites −4 to +2 and can be hydrolysed into DP4 and DP2.