13C-N.M.R.-spectroscopic investigation of agarose oligomers

Rochas, C.; Lahaye, Marc; Yaphe, W.; Viet, Minh Tan Phan

doi:10.1016/s0008-6215(00)90388-4

Cited by 68 publications

(19 citation statements)

References 16 publications

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“…The chemical shifts of the anomeric carbon signals in the spectrum of the spot (Fig. 4C) were identical to those reported previously (24). Typical resonance signals at the reducing end of neoagarobiose were found at chemical shifts of 92.21 and 96.19 ppm (Fig.…”

Section: P-nitrophenyl-␤-d-galactopyranoside and P-nitrophenyl-␣-d-supporting

confidence: 86%

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose

Temuujin

Chi

Chang

et al. 2011

Journal of Bacteriology

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Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating ␣-(1,3) and ␤-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by ␣/␤-agarases. In S. coelicolor, DagA was confirmed to be an endo-type ␤-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 ␤-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. ␤-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-␤-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K m and V max for agarose were 4.87 mg/ml (4 ؋ 10 ؊5 M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn 2؉ and Cu 2؉ was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo-and endo-type ␤-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.A gar, an important polysaccharide found in the cell walls of the Gracilariales and the Gelidiales red algae, is composed of 2 different components: agarose and agaropectin. Agarose consists of a linear chain of alternating residues of 3-O-linked ␤-Dgalactopyranose and 4-O-linked 3,6-anhydro-␣-L-galactose (7). Because agarose has excellent gelling ability, stabilizing properties, and high viscosity, it has been widely used for food, cosmetic, and pharmaceutical applications (2). Although agaropectin is presumably substituted by sulfate esters, pyruvate acetal, and methyl ethers on similar backbone units, its exact structure is still obscure (7). Recently, efforts have been focused on using terrestrial or marine plant biomasses, and the red alga is a good biomass resource candidate because of its easy cultivation in a broad range of oceans and rapid growth. A biomass must be efficiently degraded by chemical or enzymatic treatment for utilization, and the enzymatic process is preferred because it is more environmentally friendly and produces a smaller amount of toxic by-products than the chemical process.Agarases are classified into 2 groups on the basis of their mode of action: ␣-agarases (EC 3.2.1.158) and ␤-agarases (EC 3.2.1.81). ␤-Agarases hydrolyze ␤-1,4 linkages in agarose and produce neoagaro-oligosaccharides with D-galactose residues at t...

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Section: P-nitrophenyl-␤-d-galactopyranoside and P-nitrophenyl-␣-d-supporting

confidence: 86%

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose

Temuujin

Chi

Chang

et al. 2011

Journal of Bacteriology

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“…The method applied to the purification of calibrated oligo-agaroses was adapted from Rochas et al (4). 1 ml of 1.5% oligo-agarose mixture in deionized water was injected on a column (100 ϫ 5 cm) of Bio-Gel P2 (Bio-Rad) and was eluted with deionized water at the flow rate of 35 ml/min.…”

Section: Methodsmentioning

confidence: 99%

“…On the basis of x-ray fiber diffraction, optical rotation calculations, and solution gel transition, both a parallel double helix as well as a single helix structure for agarose have been proposed, where the individual polysaccharide chains have a left-handed 3-fold helix symmetry and a pitch of 1.90 nm (3,4). Agarose forms thermo-reversible gels structured by aggregates of agarose chains.…”

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confidence: 99%

The Three-dimensional Structures of Two β-Agarases

Allouch

Jam

Helbert

et al. 2003

Journal of Biological Chemistry

129

132

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Agars are important gelifying agents for biochemical use and the food industry. To cleave the ␤-1,4-linkages between ␤-D-galactose and ␣-L-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called ␤-agarases. ␤-Agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two ␤-agarases. The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 Å for ␤-agarases A and B, respectively. The structure of ␤-agarase A was solved by the multiple anomalous diffraction method, whereas ␤-agarase B was solved with molecular replacement using ␤-agarase A as model. Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/ base residues Glu-152 and Glu-189 for ␤-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a -carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity. The relationship between structure and enzymatic activity of the two ␤-agarases from Z. galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides. The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the ␤-agarases.Agarose is a hydrophilic polysaccharide found in the cell wall of marine red algae (Rhodophyceaea) (1), where it naturally occurs in the form of a pseudocrystalline matrix associated with cellulose (2). It consists of a linear backbone of galactopyranose residues linked by alternating ␣-1,3-and ␤-1,4-linkages. Whereas the ␤-linked residues are in the D configuration, the ␣-1,3-linked galactose units are in the rare L configuration and are further modified by a 3,6-anhydro bridge (2, 3) (Fig. 1a). On the basis of x-ray fiber diffraction, optical rotation calculations, and solution gel transition, both a parallel double helix as well as a single helix structure for agarose have been proposed, where the individual polysaccharide chains have a left-handed 3-fold helix symmetry and a pitch of 1.90 nm (3, 4). Agarose forms thermo-reversible gels structured by aggregates of agarose chains. These gels exhibit unique rheological properties and are widely used as texturing agents for various applications in the food industry or as a common laboratory medium for chromatographic separation or bacterial colony growth (5).Agarases are the glycoside hydrolases (GH) 1 that hydrolyze agarose. The ␤-agarases and the ␣-agarases cleave the internal ␤-1,4-and ␣-1,3-linkage of agarose, respectively. ␤-Agarases produce agaro-oligosaccharides in the series homologous to neoagarobiose (O-3,6-anhydro-␣-L-galact...

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“…The procedure of Dutton & Lim (1985) was followed with lysozyme-extracted polymer and with Ultra-Pure Agarose (BRL) as a control. This latter material is known to have alternating a-and P-glycosidic bonds in its structure (Rochas et al, 1986).…”

Section: Separation Techniques (A)mentioning

confidence: 99%

Composition of an unusual accessory polymer containing N-lactyl-3-amino-3,6-dideoxyhexose from walls of Bacillus megaterium NCIB 7581

Bishop

White

1993

Journal of General Microbiology

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A polysaccharide of molecular mass at least 300000 Da was isolated from walls of BaciZZus megateriurn NCIB 7581. Three sugars, N-lactyl-3-~0-3,6dideoxyhexose, N-acetylglucosamine and glucose were present in equimolar proportions, and accounted for 90 % of the carbon of the polysaccharide. The remainder of the polymer was an unidentified acidic molecule, with a hydrophobic nature. IntroductionAn acidic accessory polymer containing glucose and glucosamine was recognized in walls of Bacillus megaterium NCIB 758 1 (preceding paper : Bishop et al., 1993). The present paper describes a further study of this polymer which has led to the identification of N-lactyl-3-amino-3,6-dideoxyhexose as a major component. MethodsOrganisms. Bacillus megaterium NCIB 758 1 and the double mutant GW-1 which requires diaminopimelate plus lysine for growth are described by Bishop et al. (1993).Growth of bacteria, isolation of walls and of the acidic polymer. All of these procedures were as described before (Bishop et al., 1993).Tests with lectins. The lysozyme-extracted polymer was tested against concanavalin A Type V (from Sigma) by the agar diffusion method of Goldstein & So (1965) with Type I1 glycogen (Sigma) as positive control, and against Solanum tuberosum lectin with a positive control of peptidoglycan (B. megaterium) from which the accessory polymer had been removed by extraction with HC1.Hydrolysis of the polymer. Routinely the polymer (irrespective of the method of isolation) was hydrolysed as a solution (3 mg ml-' or less) in 2 M-HCl in a sealed tube for 2 h in boiling water. When diaminopimelate was to be measured colorirnetrically, hydrolysis was at 105 "C in 6 M-HCl for 18 h. Hydrolysates were dried repeatedly in a desiccator over silica gel and solid NaOH, or else were freeze-dried. Hydrolysis for only 20 min in 1 M-HC~ in boiling water liberated N-lactylaminodideoxyhexose optimally. Isolation of oligosaccharides from partial hydrolysates of the polymer.Lysozyme-extracted polymer (20 mg) was kept at 100 "C in 0 1 M-HCl (15 ml) for 2 h. The hydrolysate was rotary evaporated, taken up in water (1-5 ml) and eluted with water through a column of Bio-Gel P2. Samples from fractions were assayed, and two discrete peaks of anthrone-positive material were found to emerge between the void volume and the elution volume of monosaccharides. These two impure oligosaccharide isolates were freeze-dried, and each was separately recovered from Dowex-50 with O~M -H C~, then eluted once more through the Bio-Gel P2 column.Treatment of polymer with HF. Lysozyme-extracted polymer (6 mg) or walls of Lactobacillus plantarum (6 rng) were separately added to 300 pl HF (50 %, w/v) at 0 O C in a plastic tube. The mixtures were left at 0 "C for 1 h with occasional shaking, then the acid was carefully neutralized with 10 M-NaOH while the temperature was kept at 0 "C. Fine adjustment to pH 7 was done with 1 M-NaOH and 1 M-HCl, using indicator paper. The wall suspension was centrifuged and the supernatant liquid was kept. Both neutralized solutions were...

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13C-N.M.R.-spectroscopic investigation of agarose oligomers

Cited by 68 publications

References 16 publications

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose

The Three-dimensional Structures of Two β-Agarases

Composition of an unusual accessory polymer containing N-lactyl-3-amino-3,6-dideoxyhexose from walls of Bacillus megaterium NCIB 7581

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