The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Alain Heyraud - One of the best experts on this subject based on the ideXlab platform.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units
Carbohydrate Research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Abstract Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxM B (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp∼30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp>20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units.
Carbohydrate research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxMB (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp approximately 30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp > 20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
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Catalytic properties and specificity of a recombinant, overexpressed D-Mannuronate lyase.
Carbohydrate research, 1998Co-Authors: F Chavagnat, Alain Heyraud, P Colin-morel, M Guinand, J WallachAbstract:Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true Mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polyMannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligoMannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.
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hplc analysis of saturated or unsaturated oligoguluronates and oligoMannuronates application to the determination of the action pattern of haliotis tuberculata alginate lyase
Carbohydrate Research, 1996Co-Authors: Alain Heyraud, Philippe Colinmorel, Sylvie Girond, Christophe Richard, Bernard KloaregAbstract:Abstract The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-β- d -Mannuronate blocks, leading to the release of O -(4-deoxy-α- l - erythro -hex-4-enopyranosyluronic acid)-(1 → 4)- O -(β- d -mannopyranosyluronic acid)-(1 → 4)- O -β- d -mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligoMannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligoMannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G—M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G—G nor the M—G diads. This lyase should therefore be referred to as a Mannuronate β-eliminase, indicating that the enzyme performs β-elimination on Mannuronate residues only, from both the M—M and G—M diads of alginates.
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HPLC analysis of saturated or unsaturated oligoguluronates and oligoMannuronates. Application to the determination of the action pattern of Haliotis tuberculata alginate lyase
Carbohydrate research, 1996Co-Authors: Alain Heyraud, Sylvie Girond, Christophe Richard, P Colin-morel, Bernard KloaregAbstract:The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-Mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligoMannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligoMannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a Mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on Mannuronate residues only, from both the M-M and G-M diads of alginates.
Tsuyoshi Muramatsu - One of the best experts on this subject based on the ideXlab platform.
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Structure–activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells
FEBS letters, 2005Co-Authors: Mami Iwamoto, Yoshiko Iwamoto, Tatsuya Oda, Maki Kurachi, Takuji Nakashima, Daekyung Kim, Kenichi Yamaguchi, Tsuyoshi MuramatsuAbstract:Guluronate and Mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polyMannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and Mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.
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Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells.
Bioscience biotechnology and biochemistry, 2003Co-Authors: Yoshiko Iwamoto, Tadashi Tamura, Tatsuya Oda, Tsuyoshi MuramatsuAbstract:Enzymatically depolymerized guluronate and Mannuronate oligomers were prepared from polyuronates with an alginate lyase from a Pseudoalteromonas sp., and their effects on mononuclear cells from human peripheral blood were examined. Conditioned medium prepared by the incubation of cells with an untreated polyuronate had little effect on growth of human leukemic U937 cells, but a medium prepared with depolymerized uronate oligomers inhibited their growth. Inhibition was greater in a medium prepared with guluronate oligomer than one prepared with Mannuronate oligomer. The cytotoxic activity of the medium was heat-labile and nondialyzable. Apoptotic nuclear morphological changes and increased caspase-3-like activity were found in U937 cells treated with a medium prepared with depolymerized uronates. The medium prepared with purified tetra-guluronate and tetra-Mannuronate also was cytotoxic; these effects were inhibited by antibodies to tumor necrosis factor-α. Our results suggested that enzymatically depolyme...
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purification and characterization of bifunctional alginate lyase from alteromonas sp strain no 272 and its action on saturated oligomeric substrates
Bioscience Biotechnology and Biochemistry, 2001Co-Authors: Yoshiko Iwamoto, Tatsuya Oda, Ryoko Araki, Kenichi Iriyama, Hisataka Fukuda, Shinziro Hayashida, Tsuyoshi MuramatsuAbstract:A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-Mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-Mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and Mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the Mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).
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Primary structure of Mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability
Journal of Protein Chemistry, 1996Co-Authors: Tsuyoshi Muramatsu, Kenji Komori, Narumi Sakurai, Koji Yamada, Yasuyuki Awasaki, Kazumasa Fukuda, Tatsuya OdaAbstract:The complete amino acid sequences of two isoforms, SP1 and SP2, of Mannuronate lyase from a wreath shell, Turbo cornutus , were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of Mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.
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Primary structure of Mannuronate lyases SP1 and SP2 from Turbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability.
Journal of protein chemistry, 1996Co-Authors: Tsuyoshi Muramatsu, Kenji Komori, Narumi Sakurai, Koji Yamada, Yasuyuki Awasaki, Kazumasa Fukuda, Tatsuya OdaAbstract:The complete amino acid sequences of two isoforms, SP1 and SP2, of Mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of Mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.
J Wallach - One of the best experts on this subject based on the ideXlab platform.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units
Carbohydrate Research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Abstract Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxM B (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp∼30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp>20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units.
Carbohydrate research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxMB (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp approximately 30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp > 20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
Bernard Kloareg - One of the best experts on this subject based on the ideXlab platform.
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hplc analysis of saturated or unsaturated oligoguluronates and oligoMannuronates application to the determination of the action pattern of haliotis tuberculata alginate lyase
Carbohydrate Research, 1996Co-Authors: Alain Heyraud, Philippe Colinmorel, Sylvie Girond, Christophe Richard, Bernard KloaregAbstract:Abstract The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-β- d -Mannuronate blocks, leading to the release of O -(4-deoxy-α- l - erythro -hex-4-enopyranosyluronic acid)-(1 → 4)- O -(β- d -mannopyranosyluronic acid)-(1 → 4)- O -β- d -mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligoMannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligoMannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G—M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G—G nor the M—G diads. This lyase should therefore be referred to as a Mannuronate β-eliminase, indicating that the enzyme performs β-elimination on Mannuronate residues only, from both the M—M and G—M diads of alginates.
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HPLC analysis of saturated or unsaturated oligoguluronates and oligoMannuronates. Application to the determination of the action pattern of Haliotis tuberculata alginate lyase
Carbohydrate research, 1996Co-Authors: Alain Heyraud, Sylvie Girond, Christophe Richard, P Colin-morel, Bernard KloaregAbstract:The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-Mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligoMannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligoMannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a Mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on Mannuronate residues only, from both the M-M and G-M diads of alginates.
P Colin-morel - One of the best experts on this subject based on the ideXlab platform.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units
Carbohydrate Research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Abstract Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxM B (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp∼30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp>20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
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An enzymatic method for preparation of homopolyMannuronate blocks and strictly alternating sequences of mannuronic and guluronic units.
Carbohydrate research, 1998Co-Authors: Alain Heyraud, P Colin-morel, C Gey, F Chavagnat, M Guinand, J WallachAbstract:Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polyMannuronate lyase AlxMB (only acting on two or more consecutive, nonacetylated Mannuronate units) to prepare either Mannuronate blocks (poly-M blocks) with dp approximately 30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp > 20. The poly-M blocks were obtained by lysis of a P. aeruginosa polyMannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both Mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the Mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.
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HPLC analysis of saturated or unsaturated oligoguluronates and oligoMannuronates. Application to the determination of the action pattern of Haliotis tuberculata alginate lyase
Carbohydrate research, 1996Co-Authors: Alain Heyraud, Sylvie Girond, Christophe Richard, P Colin-morel, Bernard KloaregAbstract:The chromatographic behaviour of various saturated and unsaturated oligouronates obtained by acid or enzymatic degradation of homopolymeric blocks of alginates was investigated by isocratic anion exchange liquid chromatography. This approach was then applied to the determination of the catalytic properties of Haliotis tuberculata alginate lyase. This enzyme presents a high affinity for poly-beta-D-Mannuronate blocks, leading to the release of O-(4-deoxy-alpha-L-erythro-hex-4-enopyranosyluronic acid)-(1-->4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranuronic acid as the main end reaction product. Kinetic analysis with oligoMannuronates of various sizes indicate that the catalytic site of Haliotis tuberculata lyase (abalone) best accommodates an oligoMannuronate pentamer. The abalone lyase, however, is also capable of cleaving the G-M linkages of alginate heteropolymeric sequences. In contrast, it does not degrade the G-G nor the M-G diads. This lyase should therefore be referred to as a Mannuronate beta-eliminase, indicating that the enzyme performs beta-elimination on Mannuronate residues only, from both the M-M and G-M diads of alginates.