Kinetics of hydrolysis of chitin/chitosan oligomers in concentrated hydrochloric acid

The kinetics of hydrolysis in concentrated hydrochloric acid (12.07 M) of the fully N-acetylated chitin tetramer (GlcNAc(4)) and the fully N-deacetylated chitosan tetramer (GlcN(4)) were followed by determining the amounts of the lower DP oligomers as a function of time. A theoretical model was developed to simulate the kinetics of hydrolysis of the three different glycosidic linkages in the tetramers. The model uses two different rate constants for the hydrolysis of the glycosidic bonds in the oligomers, assuming that the glycosidic bond next to one of the end residues are hydrolysed faster than the two other glycosidic linkages. The two rate constants were estimated by fitting model data to experimental results. The results show that the hydrolysis of the tetramers is a nonrandom process as the glycosidic bonds next to one of the end residues are hydrolysed 2.5 and 2.0 times faster as compared to the other glycosidic linkages in the fully N-acetylated and fully N-deacetylated tetramer, respectively. From previous results on other oligomers and the reaction mechanism, it is likely that the glycosidic bond that is hydrolysed fastest is the one next to the nonreducing end. The absolute values for the rate constants for the hydrolysis of the glycosidic linkages in GlcNAc(4) were found to be 50 times higher as compared to the glycosidic linkages in GlcN(4), due to the catalytic role of the N-acetyl group and the presence of the positively charged amino-group on the N-deacetylated sugar residue.     

Catalytic properties and specificity of a recombinant, overexpressed D-mannuronate lyase

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.     

Structural characterization of Botryosphaeran: a (1-->3;1-->6)-beta-D-glucan produced by the ascomyceteous fungus,Botryosphaeria sp

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1-->3)-beta-D-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1-->6)-beta-linked glucosyl, and (1-->6)-beta-linked gentiobiosyl residues.     

Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2

A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40?°C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate.     

Isolation of Mutant Alginate Lyases with Cleavage Specificity for Di-guluronic Acid Linkages

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

Cell wall teichoic acids of Actinomadura viridis VKM Ac-1315T.

The cell walls of Actinomadura viridis contain poly(glycosylglycerol phosphate) chains of complex structure. On the basis of NMR spectroscopy of the polymer and glycosides thereof the following structural units were found: beta-D-Galp3Me-(1-->4)[beta-D-Glcp-(1-->6)]-beta-D-Galp-(1-->1)-++ +snGro (G1); beta-D-Galp-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2); beta-D-Galp3Me-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2a); beta-D-Galp-(1-->1)-snGro (G3); beta-D-Galp-(1-->1)[beta-D-Galp-(1-->2)]-snGro (G4); beta-D-Glcp-(1-->2)-snGro (G5). Glycosides G1, G2 and G3 were the predominant components of the teichoic acid: they formed the polymer chain via phosphodiester bonds involving C-3 of the glycerol residue and C-3 of the galactosyl residue which in turn glycosylates C-1 of the glycerol residue. Whether the different glycosides make up the one chain or whether there are several poly(glycosylglycerol phosphate) chains in the cell wall remains to be determined. It was suggested that the minor component G5 is located at the nonterminal end of the chains. Compound G4 which contains disubstituted glycerol residues (unusual for the teichoic acid) was also found as a minor component; this may be the glycoside of a new type of teichoic acid, or a glycoside on the terminal end of the above mentioned chains. In addition, small amounts of 1,3-poly(glycerol phosphate) chains were found in the cell wall.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

Determination of substituents distribution in carboxymethylpullulans by NMR spectroscopy

The distribution of carboxymethyl substituents in the alpha-(1 --> 6)-linked maltotriosyl repeating units of a carboxymethylpullulan (CMP) series was investigated by high resolution NMR spectroscopy on very short oligomers (DPn = 1.2-1.5) obtained by acid hydrolysis. A series of 2D NMR experiments on parent pullulan, hydrolysed pullulan and CMP was used to assign the proton and carbon chemical shifts of CMP acid hydrolysates. The degree of substitution (DS) and the relative distribution of -CH2COONa groups at OH-2, OH-3, OH-4 and OH-6 of glucose residues (DSi) were determined from 1H NMR measurements. From a set of CMP samples, widely different in degree of substitution, it was observed that the substitution at C-2 is predominant and decreases according to the order C-2 > C-3 > C-6 > C-4. Taking into account the availability of each OH group in the parent pullulan, an order of relative reactivity of hydroxyl groups is defined according to the relation: Ri = DSi/ni, where ni is the number of free OH groups in a maltotriose unit (MTU) for a given site C-i, the reactivity order was found to be OH-2 > OH-4 > OH-6 > OH-3.     

Structural characterization of the glycan part of glycoconjugate LbGp2 from Lycium barbarum L

A glycoconjugate with pronounced immunoactivity, designated as LbGp2, was isolated from the fruit of Lycium barbarum L. and purified to homogeneity by gel-filtration. Its carbohydrate content is up to 90.71% composed of Ara, Gal and amino acids. The molecular weight is 68.2 kDa as determined by size exclusive chromatography (SEC). The complete structure of the repeat unit of the glycan of LbGp2 was elucidated based on glycosidic linkage analysis, total acid hydrolysis, partial acid hydrolysis, 1H and 13C NMR spectroscopy. According to the experiments, the glycan possesses a backbone consisting of (1-->6)-beta-galactosyl residues, about fifty percent of which are substituted at C-3 by galactosyl or arabinosyl groups and the major nonreducing end being made of Ara (1 -->.     

Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.     

Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance

The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.     

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.     

Solid-state 13C NMR spectroscopy studies of xylans in the cell wall of Palmaria palmata (L. Kuntze,Rhodophyta)

The chemical structure and interactions of the cell wall polysaccharides from the red edible seaweed Palmaria palmata were studied by liquid-like magic-angle-spinning (MAS) and cross-polarization MAS (CPMAS) solid-state 13C NMR spectroscopy. The liquid-like MAS and CPMAS 13C NMR spectra of the rehydrated algal powder revealed the presence of beta-(1-->4)/beta-(1-->3)-linked D-xylan with chemical shifts close to those observed in the solution 13C NMR spectrum of the polysaccharide. Observation of mix-linked xylan in the liquid-like MAS 13C NMR spectrum indicated that part of this cell wall polysaccharide is loosely held in the alga. The CPMAS NMR spectrum of the dry algal powder alcohol insoluble residue (AIR) showed broad peaks most of which corresponded to the mix-linked xylan. Hydration of AIR induced a marked increase in the signal resolution also in the CPMAS NMR spectra together with a shift of the C-3 and C-4 signals of the (1-->3)- and (1-->4)-linked xylose, respectively. Such modifications were present in the spectrum of hydrated (1-->3)-linked xylan from the green seaweed Caulerpa taxifolia and absent in that of (1-->4)-linked xylan from P. palmata. This result emphasizes the important role of (1-->3) linkages on the mix-linked xylan hydration-induced conformational rearrangement. The mix-linked xylan signals were observed in the CPMAS NMR spectrum of hydrated residues obtained after extensive extractions by NaOH or strong chaotropic solutions indicating strong hydrogen bonds or covalent linkages. T(1 rho) relaxations were measured close or above 10 ms for the mix-linked xylan in the dry and hydrated state in AIR and indicated that the overall xylan chains likely remain rigid. Rehydration of the mix-linked xylan lead to a decrease in the motion of protons bounded to the C-1 and C-4 carbons of the (1-->4)-linked xylose supporting the re-organization of the xylan chains under hydration involving junction-zones held by hydrogen bonds between adjacent (1-->4)-linked xylose blocks. The CPMAS NMR spectrum of both dry and rehydrated residues obtained after NaOH and HCl extractions demonstrated the presence of cellulose and (1-->4)-linked xylans. The structures of the different polysaccharides are discussed in relation to their interactions and putative functions on the cell wall mechanical properties in P. palmata.     

Structural studies of the Vibrio cholerae o-antigen

The dominant part of the O-antigen of Vibrio cholerae is a homopolysaccharide composed of (1→2)-linked 4-amino-4,6-dideoxy-α-d-mannopyranosyl (perosaminyl) residues, the amino groups of which are acylated by 3-deoxy-l-glycero-tetronic acid. Most of the amino sugar is decomposed during acid hydrolysis. Treatment of the polymer with anhydrous hydrogen fluoride, which cleaves the glycosidic linkages but does not cause N-deacylation, followed by acid hydrolysis under mild conditions, produced the monomer in good yield. Treatment of the N-deacylated polysaccharide with nitrous acid caused deamination with concomitant rearrangements, typical of 4-amino-4-deoxyhexopyranosyl residues in which the amino group occupies an equatorial position.     

Structure determination of the glycolipid sulfate from the extreme halophile Halobacterium cutirubrum

A sulfur-containing glycolipid, accounting for ca. 25% of the total polar lipids, has been isolated from the extreme halophile Halobacterium cutirubrum. The ammonium salt of the lipid was found to have the molecular formula C(61)H(117)O(21)S.NH(4), and on strong acid hydrolysis it yielded 2,3-di-O-phytanyl-sn-glycerol, glucose, mannose, galactose, and sulfate in equimolar proportions. Infrared and NMR spectra indicated the presence of a secondary sulfate group. Solvolysis of the lipid in 0.004 m HCl in tetrahydrofuran resulted in rapid release of inorganic sulfate and formation of galactosyl-mannosyl-glucosyl diphytanyl glycerol ether. With higher acid concentration (0.25 m methanolic HCl), stepwise hydrolysis of monosaccharide units occurred, giving mannosyl-glucosyl glycerol diphytanyl ether and monoglucosyl glycerol diphytanyl ether. The position of attachment of the sugars and of the sulfate group was determined by methylation of the free acid form of the glycolipid sulfate, followed by acid hydrolysis and gas-liquid chromatographic analysis of the partially methylated sugars as the alditol acetates. The configuration of the glycosidic linkages was established both by optical rotation measurements and by specific enzymatic hydrolysis. The results obtained established the structure as 2,3-di-O-phytanyl-1-O-[beta-d-galactopyranosyl-3'-sulfate-(1' -->6')-O-alpha-d-mannopyranosyl-(1' --> 2')-O-alpha-d-glucopyranosyl]-sn-glycerol.     

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.     

Enzymatic synthesis of a library of beta-(1-->4) hetero- D-glucose and D-xylose-based oligosaccharides employing cellodextrin phosphorylase

Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1-->4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.49) as the enzyme catalyst, with alpha-D-glucose 1-phosphate (1) or alpha-D-xylose 1-phosphate (2) as the donor substrates, and cellobiose (3), xylobiose (4), betaGlc-(1-->4)-Xyl (5), or betaXyl-(1-->4)-Glc (6) as the acceptor substrates. All enzymatic reactions were performed at pH 7.0 and the products purified by gel-filtration chromatography. We successfully synthesized all six hetero-trisaccharides and 10 of the 14 possible hetero-tetrasaccharides. It was not found possible to synthesize the four tetrasaccharides with a Xyl-->Glc sequence at their non-reducing ends employing this method. The stereochemistries of the isolated products were assessed by analysis of their 2D NMR spectra (DQF-COSY, TOCSY, HSQC, HMBC), confirming that all of the glycosidic bonds in the products were beta-(1-->4) linkages.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

Effect of (1-->3)- and (1-->4)-linkages of fully sulfated polysaccharides on their anticoagulant activity

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.     

Complete assignment of 1H and 13C NMR spectra of Gigartina skottsbergii lambda-carrageenan using carrabiose oligosaccharides prepared by enzymatic hydrolysis

lambda-Carrageenan extracted from Gigartina skottsbergii tetrasporophyte was completely digested by a purified Pseudoalteromonas carrageenovora lambda-carrageenase. The main digestion products were fractionated and analysed by (1)H and (13)C NMR spectroscopy. All the oligosaccharides observed belong to the neo-carrabiose oligosaccharide series indicating that the lambda-carrageenase cleaves the beta-(1-->4) glycosidic bonds. (1)H and (13)C NMR spectra recorded on oligomers from DP 2 to DP 8 were fully interpreted allowing unambiguous assignment of the lambda-carrageenan spectra. Besides the typical oligo-lambda-carrageenans, we have also characterised a heptasulfated tetrasaccharide which demonstrates the random over-sulfation along the chain of G. skottsbergii lambda-carrageenan.