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1.
J Appl Microbiol ; 118(2): 356-67, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25376444

ABSTRACT

AIMS: To investigate the effect of culture conditions and medium components on exopolysaccharide (EPS) production by Pseudomonas stutzeri AS22 and to access the EPS performance as a metal-binding exopolysaccharide. METHODS AND RESULTS: The EPS production conditions of Ps. stutzeri AS22 in submerged culture were optimized using two approaches for EPS quantification, and its metal-binding capacity was evaluated using both single and mixed metal ions systems. Maximum EPS level was achieved after 24 h of incubation at 30°C with an initial pH of 8.0, 250 rev min(-1) stirring level and 10% inoculum size. 50 g l(-1) starch, 5 g l(-1) yeast extract, 0.5 g l(-1) NaCl, 1.4 g l(-1) K2 HPO4, 0.4 g l(-1) MgSO4, 0.4 g l(-1) CaCl2 and 1 g l(-1) mannose were found to be the most suitable carbon, nitrogen, mineral and additional carbohydrate sources, respectively. From metal-binding experiments, the crude EPS showed interesting metal adsorption capacity adopting the order Pb >> Co > Fe > Cu >> Cd. Lead was preferentially biosorbed with a maximal uptake of 460 mg g(-1) crude EPS. CONCLUSIONS: Under the optimal culture requirements, EPS level reached 10.2 g l(-1) after 24 h of fermentation, seven times more than the production under initial conditions. According to the metal-binding assay, the crude EPS has potential to be used as a novel biosorbent in the treatment of heavy metals-contaminated water. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results are interesting in terms of yield as well as efficiency for the potential use of the Ps. stutzeri exopolysaccharide as a metal-absorbent polymer in the bioremediation field.


Subject(s)
Metals, Heavy/metabolism , Polysaccharides, Bacterial/metabolism , Pseudomonas stutzeri/metabolism , Carbon/metabolism , Fermentation , Nitrogen/metabolism , Polysaccharides, Bacterial/biosynthesis , Pseudomonas stutzeri/isolation & purification
2.
J Appl Microbiol ; 108(2): 499-509, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19659698

ABSTRACT

AIMS: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine. METHODS AND RESULTS: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with beta-glucan formation and ropiness were grown on grape juice medium, dialysed MRS-derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol-sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf-). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. CONCLUSION: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than beta-glucan. SIGNIFICANCE AND IMPACT OF THE STUDY: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.


Subject(s)
Food Microbiology , Oenococcus/metabolism , Polysaccharides, Bacterial/biosynthesis , Wine/microbiology , Culture Media , Oenococcus/growth & development , beta-Glucans/metabolism , beta-Glucosidase/metabolism
3.
Oral Microbiol Immunol ; 23(5): 391-400, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18793362

ABSTRACT

INTRODUCTION: The development of therapeutic agents inhibiting the activity of glucosyltransferases (GTF) and their production of glucans is a potential strategy to reduce dental decay. The aim of this study was first to characterize a GTF preparation from Streptococcus sobrinus ATCC 33478 and then to evaluate the effects of select compounds and mouthrinses on insoluble glucan (ISG) formation by combined GTFs. METHODS: The purity of the crude GTF mixture was assessed by electrophoresis. The effects of pH, temperature, sucrose, and dextran T10 concentrations on GTF activity were analyzed and the chemical structure of the products was investigated. Finally, the inhibition of GTF by commercial mouthrinses used in oral hygiene and their active components (chlorhexidine, polyphenolic compounds, fluoride derivatives, polyols, cetylpyridinium chloride, and povidone iodine) was analyzed through the reductions in the overall reaction rate and the quantity of ISG synthesized. RESULTS: The S. sobrinus ATCC 33478 crude GTF preparation obtained contains a mixture of four different GTFs known for this species. For optimal adherent ISG formation, the reaction parameters were 37 degrees C, pH 6.5, sucrose 50 g/l, and dextran T10 2 g/l. Under these conditions, the most effective agents were chlorhexidine, cetylpyridinium chloride, and tannic acid. Eludril, Elmex, and Betadine were the most effective inhibitors of all the mouthrinses tested. CONCLUSION: As the formulation of commercial products considerably influences the efficiency of active components, the fast representative ISG inhibition test developed in this study should be of great interest.


Subject(s)
Dental Plaque/prevention & control , Glucans/antagonists & inhibitors , Glucosyltransferases/antagonists & inhibitors , Mouthwashes/pharmacology , Pharmaceutical Preparations, Dental/pharmacology , Polysaccharides, Bacterial/drug effects , Streptococcus sobrinus/enzymology , Anti-Infective Agents, Local/pharmacology , Cariostatic Agents/pharmacology , Cetylpyridinium/pharmacology , Chlorhexidine/analogs & derivatives , Chlorhexidine/pharmacology , Dextrans/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Fluorides/pharmacology , Glucans/chemistry , Glucosyltransferases/drug effects , Humans , Hydrogen-Ion Concentration , Materials Testing , Phenols/pharmacology , Polymers/pharmacology , Polyphenols , Polysaccharides, Bacterial/chemistry , Povidone-Iodine/pharmacology , Solubility , Streptococcus sobrinus/drug effects , Sucrose/pharmacology , Tannins/pharmacology , Temperature
4.
Carbohydr Res ; 339(6): 1119-27, 2004 Apr 28.
Article in English | MEDLINE | ID: mdl-15063200

ABSTRACT

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.


Subject(s)
Pectins/chemistry , Polysaccharides/chemistry , Anions , Carbohydrate Conformation , Carbohydrate Sequence , Cell Wall/metabolism , Chromatography , Chromatography, Ion Exchange , Edetic Acid/chemistry , Galactans/chemistry , Hexuronic Acids/chemistry , Magnetic Resonance Spectroscopy , Microscopy, Electron, Scanning , Molecular Sequence Data , Opuntia , Temperature , Time Factors , Uronic Acids/chemistry
5.
Appl Environ Microbiol ; 67(11): 5197-203, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11679345

ABSTRACT

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.


Subject(s)
Polysaccharide-Lyases/isolation & purification , Polysaccharide-Lyases/metabolism , Sinorhizobium meliloti/enzymology , Amino Acid Sequence , Molecular Sequence Data , Polysaccharide-Lyases/chemistry , Sinorhizobium meliloti/growth & development , Substrate Specificity
6.
Carbohydr Res ; 334(2): 127-33, 2001 Aug 23.
Article in English | MEDLINE | ID: mdl-11502268

ABSTRACT

Burkholderia caribensis strain MWAP71 was isolated from rhizosphere soil microaggregates in Martinique. The extracellular polysaccharide produced by this strain was found to be composed of D-glucose (D-Glc), 6-deoxy-L-talose (L-6dTal), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and an O-acetyl group in a molar ratio of 2:1:1:1. The primary structure of the polysaccharide was shown by sugar analysis, electrospray mass spectrometry, partial acid hydrolysis and 1-D and 2-D NMR spectroscopy to consist of a tetrasaccharide repeating unit having the following structure: [structure in text].


Subject(s)
Burkholderia/chemistry , Deoxy Sugars/chemistry , Glucose/chemistry , Hexoses/chemistry , Polysaccharides, Bacterial/chemistry , Sugar Acids/chemistry , Magnetic Resonance Spectroscopy , Polysaccharides, Bacterial/analysis , Soil Microbiology , Species Specificity , Spectrometry, Mass, Electrospray Ionization
7.
Carbohydr Res ; 332(2): 167-73, 2001 May 18.
Article in English | MEDLINE | ID: mdl-11434374

ABSTRACT

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.


Subject(s)
Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/isolation & purification , Rhizobium/chemistry , Acetylation , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Ion Exchange , Gas Chromatography-Mass Spectrometry , Glucose/analysis , Medicago sativa/microbiology , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Rhamnose/analysis , Rhizobium/isolation & purification
8.
Glycoconj J ; 18(6): 465-74, 2001 Jun.
Article in English | MEDLINE | ID: mdl-12084982

ABSTRACT

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, lacto-N-neodifucohexaose Galbeta1-4(Fucalpha1-3)Glc-NAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, and lacto-N-neodifucooctaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an alpha-1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon. To prevent fucosylation at the glucosyl residue, lactulose Galbeta1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.


Subject(s)
Escherichia coli/genetics , Fucose/metabolism , Fucosyltransferases/metabolism , Genetic Engineering , Helicobacter pylori/enzymology , Oligosaccharides/metabolism , Chromatography, Gel , Chromatography, Thin Layer , Escherichia coli/cytology , Fermentation , Fucosyltransferases/genetics , Helicobacter pylori/genetics , Kinetics , Lactose/metabolism , Lactulose/metabolism , Magnetic Resonance Spectroscopy , Methylation , Oligosaccharides/chemistry , Oligosaccharides/isolation & purification , Spectrometry, Mass, Fast Atom Bombardment
9.
Int J Biol Macromol ; 27(4): 269-77, 2000 Jul 12.
Article in English | MEDLINE | ID: mdl-10921853

ABSTRACT

The Rhizobium sp. T1 strain. which induces nodule formation on alfalfa and clover roots, produces, during growth, an extracellular polysaccharide composed of D-glucose and D-glucuronic acid noted glucoglucuronan. During the bacterial growth, the pH of the medium decreases slightly. The control of pH in the growth medium slightly reduces the glucoglucuronan production. Under the conditions tested in the present work, the weight-average molecular weight of the polymers produced with or without pH control are similar: Mw approximately 2 x 10(6); the repeating unit determined by chemical and NMR analyses corresponds to the disaccharide: --> 3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1 -->.


Subject(s)
Biopolymers/chemistry , Medicago sativa/microbiology , Polysaccharides, Bacterial/chemistry , Rhizobium/metabolism , Biopolymers/biosynthesis , Fermentation , Glucose/analysis , Glucuronic Acid/analysis , Hydrogen-Ion Concentration , Nuclear Magnetic Resonance, Biomolecular , Polysaccharides, Bacterial/biosynthesis , Rhizobium/chemistry
10.
Glycoconj J ; 16(3): 205-12, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10596895

ABSTRACT

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.


Subject(s)
Amino Sugars/chemistry , Escherichia coli/metabolism , Oligosaccharides/biosynthesis , Carbohydrate Sequence , Chromatography , Cloning, Molecular , Magnetic Resonance Spectroscopy , Molecular Sequence Data , N-Acetyllactosamine Synthase/biosynthesis , Oligosaccharides/chemistry
11.
Carbohydr Res ; 308(3-4): 417-22, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9711832

ABSTRACT

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.


Subject(s)
Alginates/metabolism , Polysaccharide-Lyases/metabolism , Pseudomonas aeruginosa/chemistry , Glucuronic Acid , Hexuronic Acids , Nuclear Magnetic Resonance, Biomolecular
12.
Cell Mol Biol (Noisy-le-grand) ; 44(3): 447-54, 1998 May.
Article in English | MEDLINE | ID: mdl-9620440

ABSTRACT

Structural studies were performed in two atypical polysaccharides, PS-1 and PS-2 isolated from the broth of a Tn5 mutant strain of Xanthomonas campestris. Sugar composition, methylation and nuclear magnetic resonance analyses were determined. PS-1 is composed by repeating trisaccharide units containing D-glucose, D-mannose and having the structure. carbohydrate sequence [see text]. Preliminary studies on the PS-2 show a polymer composed in a large extent of rhamnose. Unexpectedly, this polysaccharide is soluble in alcoholic solutions.


Subject(s)
Cellulose/chemistry , Mannose/metabolism , Xanthomonas campestris/metabolism , Carbohydrate Sequence , Cellulose/biosynthesis , Cellulose/isolation & purification , Cellulose/metabolism , Methylation , Molecular Sequence Data , Mutation , Polysaccharides/biosynthesis , Polysaccharides/isolation & purification , Xanthomonas campestris/genetics
13.
Plant Physiol ; 116(3): 1013-21, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9501133

ABSTRACT

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-beta-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2, 4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an alpha-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced alpha-l-fucosidase.

14.
Carbohydr Res ; 308(3-4): 409-15, 1998 Jun.
Article in English | MEDLINE | ID: mdl-11001631

ABSTRACT

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.


Subject(s)
Polysaccharide-Lyases/metabolism , Alginates/metabolism , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/metabolism , Substrate Specificity
15.
Carbohydr Res ; 314(3-4): 211-20, 1998 Dec 31.
Article in English | MEDLINE | ID: mdl-10335590

ABSTRACT

The extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83 was found to be composed of D-glucose and D-galactose in a molar ratio of 2:3. The primary structure of the polysaccharide was shown by sugar analysis, methylation analysis, FABMS, partial acid hydrolysis and nuclear magnetic resonance (NMR) spectroscopy to consist of a pentasaccharide repeating unit having the following structure: -->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6 )-alpha-D -Glcp-(1-->3)-beta-D-Galf-(1-->


Subject(s)
Lactobacillus/chemistry , Polysaccharides/chemistry , Carbohydrate Sequence , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Methylation , Molecular Sequence Data , Molecular Structure , Polysaccharides/isolation & purification
16.
Int J Biol Macromol ; 21(1-2): 3-9, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9283009

ABSTRACT

The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated. During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected. A glucuronan lyase was identified, this new bacterial lyase produces d.p. 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity.


Subject(s)
Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/metabolism , Sinorhizobium meliloti/enzymology , Sinorhizobium meliloti/genetics , Acetylation , Carbohydrate Metabolism , Carbohydrates/chemistry , Chromatography/methods , Magnetic Resonance Spectroscopy , Mutation , Polysaccharides/chemistry , Polysaccharides/metabolism , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/metabolism
17.
Carbohydr Res ; 302(1-2): 35-42, 1997 Jul 11.
Article in English | MEDLINE | ID: mdl-9249951

ABSTRACT

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.


Subject(s)
Escherichia coli/metabolism , Oligosaccharides/biosynthesis , Acetylation , Amidohydrolases/genetics , Amidohydrolases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Magnetic Resonance Spectroscopy , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Oligosaccharides/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping
18.
Carbohydr Res ; 291: 115-26, 1996 Sep 23.
Article in English | MEDLINE | ID: mdl-8864226

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-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.


Subject(s)
Alginates/metabolism , Mollusca/enzymology , Oligosaccharides/chemistry , Polysaccharide-Lyases/metabolism , Uronic Acids/chemistry , Alginates/isolation & purification , Animals , Binding Sites , Chromatography, Gel , Chromatography, High Pressure Liquid , Hexuronic Acids/chemistry , Magnetic Resonance Spectroscopy , Mannose/analogs & derivatives , Oligosaccharides/analysis , Oligosaccharides/metabolism , Osmolar Concentration , Uronic Acids/analysis
19.
Carbohydr Res ; 289: 11-23, 1996 Aug 19.
Article in English | MEDLINE | ID: mdl-8805773

ABSTRACT

The 1H and 13C NMR chemicals shifts of the various saturated and unsaturated timers obtained by acid or enzymatic depolymerisation of homopolymeric blocks of alginates are reported. In addition, 13C NMR chemical shifts are assigned for several saturated oligomers of higher polymerisation degrees. Breakdown of alginate and of homopolymeric alginate blocks by Haliotis tuberculata alginate lyase was monitored with 1H NMR spectroscopy and the signals relevant to the identification of the lyase products are pointed out. The enzymes performs beta-elimination on the mannuronic acid residues, independently of their immediately surrounding neighbours. Application of this approach to the analysis of the substrate specificity of alginate lyases is discussed.


Subject(s)
Alginates/chemistry , Mollusca/enzymology , Polysaccharide-Lyases/metabolism , Animals , Eukaryota/chemistry , Glucuronic Acid , Hexuronic Acids/chemistry , Hexuronic Acids/isolation & purification , Hydrolysis , Klebsiella pneumoniae/enzymology , Magnetic Resonance Spectroscopy , Substrate Specificity , Uronic Acids/chemistry , Uronic Acids/isolation & purification
20.
Carbohydr Res ; 285: 69-79, 1996 May 14.
Article in English | MEDLINE | ID: mdl-9011378

ABSTRACT

The exocellular polysaccharide from Sphingomonas paucimobilis strain I-886 has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy, and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [formula: see text] The absolute configuration of the uronic acid was deduced from 1H NMR chemical shifts and is most likely D. Some preparations of the polysaccharide also contain phosphate and O-acyl groups which have not been identified or localised.


Subject(s)
Gram-Negative Aerobic Bacteria/chemistry , Polysaccharides, Bacterial/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Glucose/analysis , Hexuronic Acids/analysis , Hydrolysis , Magnetic Resonance Spectroscopy , Methylation , Molecular Sequence Data , Rhamnose/analysis
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