Preparation Of Oligosaccharides Research Articles

Elucidation of a specific alginate lyase Aly7Rm: The products demonstrated the strict recognition of G residue at subsites ±2

An alginate lyase Aly7Rm with novel subsite specificity was cloned, expressed and characterized in this article. Aly7Rm was a G-specific alginate lyase, exhibiting optimum reaction conditions at 30 °C and pH 5.0. UPSEC-VWD-MS analysis revealed that Aly7Rm degraded polysaccharides in a random endo-acting manner. The main degradation products of alginate were trisaccharide to octasaccharide and those of PG were disaccharide to hexasaccharide. The product distribution was related to the specificity of enzyme and the structure of substrates. By utilizing the HPAEC-PAD/MS method, the structure of products was identified and the specificities of subsite were defined. Aly7Rm accommodated both G and M at subsites −1 and strictly recognized G at subsite −2 and +2. This is the first report on the alginate lyase exhibiting G specificity at subsites ±2, which shed light on the diversity in subsite specificity of alginate lyases. The Aly7Rm with clear action pattern could serve as an efficient tool in the specific degradation of alginate and targeted preparation of oligosaccharides.

Structural Insights into the Substrate Recognition and Catalytic Mechanism of a GH16 βκ-Carrageenase from Wenyingzhuangia fucanilytica.

Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the βκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the βκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing βκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Sustainable Production of Ulva Oligosaccharides via Enzymatic Hydrolysis: A Review on Ulvan Lyase.

Ulvan is a water-soluble sulfated polysaccharide extracted from the green algae cell wall. Compared with polysaccharides, oligosaccharides have drawn increasing attention in various industries due to their enhanced biocompatibility and solubility. Ulvan lyase degrades polysaccharides into low molecular weight oligosaccharides through the β-elimination mechanism. The elucidation of the structure, catalytic mechanism, and molecular modification of ulvan lyase will be helpful to obtain high value-added products from marine biomass resources, as well as reduce environmental pollution caused by the eutrophication of green algae. This review summarizes the structure and bioactivity of ulvan, the microbial origin of ulvan lyase, as well as its sequence, three-dimensional structure, and enzymatic mechanism. In addition, the molecular modification of ulvan lyase, prospects and challenges in the application of enzymatic methods to prepare oligosaccharides are also discussed. It provides information for the preparation of bioactive Ulva oligosaccharides through enzymatic hydrolysis, the technological bottlenecks, and possible solutions to address these issues within the enzymatic process.

A chemo-enzymatic method for preparation of saturated oligosaccharides from alginate and other uronic acid-containing polysaccharides

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2–4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

Efficient preparation of alginate oligosaccharides by using alginate lyases and evaluation of the development promoting effects on Brassica napus L. in saline-alkali environment

Enzymatic degradation of alginate for the preparation of alginate oligosaccharides (AOS) is currently receiving significant attention in the field. AOS has been shown to promote crop growth and improve plant resistance to abiotic stresses. In this study, two PL6 family alginate lyases, AlyRmA and AlyRmB, were expressed and characterized. These enzymes demonstrate exceptional activity and stable thermophilicity compared to other known alginate lyases. AlyRmA (8855.34 U/mg) and AlyRmB (7879.44 U/mg) exhibited excellent degradation activity towards sodium alginate even at high temperatures (70 °C). The AlyRmA and AlyRmB were characterized and utilized to efficiently produce AOS. The study investigated the promotional effect of AOS on the growth of Brassica napus L. seedlings in a saline-alkaline environment. The results of this study demonstrate the high activity and thermal stability of AlyRmA and AlyRmB, highlighting their potential in the preparation of AOS. Moreover, the application of AOS prepared by AlyRmB could enhance the resistance of Brassica napus L. to saline-alkali environments, thereby broadening the potential applications of AOS.

Preparation of κ-carrageenan oligosaccharides by photocatalytic degradation: Structural characterization and antioxidant activity

To accurately, efficiently, and environmentally prepare carrageenan oligosaccharides, we have developed a method that uses H2O2 and TiO2 as catalysts for the photodegradation of κ-carrageenan (KC). The photodegradation of KC was monitored using various amounts of TiO2 and H2O2 and different concentrations of KC via HPLC and it could decrease the average molecular weight of KC into 1.6 kDa within 2 h. Further research under optimal conditions. As a control, the effects of UV, UV/H2O2, UV/TiO2, and H2O2/TiO2 treatments were studied. In contrast, UV/H2O2/TiO2 treatments showed a coordinated effect. The effect of degradation on the structure of KC was investigated by FT-IR, XRD, and there was no obvious remotion of sulfate groups. Furthermore, oral administration of KCO prolonged the healthy lifespan of nematodes induced by ultraviolet stress and significantly regulated oxidative stress. This study suggests that the precise preparation and application of KCO may be beneficial.

Efficient Expression and Characterization of an Endo-Type Lyase HCLase_M28 and Its Gradual Scale-Up Fermentation for the Preparation of Chondroitin Sulfate Oligosaccharides.

Glycosaminoglycan (GAG) lyases have been critical in structural and functional studies of GAGs. HCLase_M28, a lyase identified from the genome of Microbacterium sp. M28 was heterologously expressed, enzymatically characterized, and prepared in large-scale fermentation for the production of chondroitin sulfate (CS) oligosaccharides. Results showed that the expression of HCLase_M28 in Escherichia coli BL21 (DE3)-pET24a-HCLase_M28opt1 and Bacillus subtilis W800-pSTOP1622-HCLase_M28opt2 were 108-fold and 25-fold that of wide strain. The optimal lytic reaction of HCLase_M28 happened in 20mM Tris-HCl (pH 7.2) at 50°C with a specific activity of 190.9U/mg toward CS-A. The degrading activity was slightly simulated in presence of 1mM Ca2+ and Mn2+ while severely inhibited by Hg+, Cu2+, Fe3+, and SDS. TLC and ESI-MS analysis proved HCLase_M28 was an endolytic lyase and degraded CS and hyaluronic acid into unsaturated disaccharides. Through a gradual scale-up of fermentation in 5L, 100L, and 1000L, a highly efficient intracellular expression of HCLase_M28 with an activity of 3.88 × 105U/L achieved within a 34h of cultivation. Through ultrafiltration, CS oligosaccharides with DP of 2 to 8 as the main components could be controllably prepared. The successful large-scale fermentation made HCLase_M28 a promising enzyme for industrial production of CS oligosaccharides.

Preparation, Structural Analysis, and Intestinal Probiotic Properties of a Novel Oligosaccharide from Enzymatic Degradation of Huangshui Polysaccharide.

Huangshui polysaccharide (HSP) has attracted more and more interest due to its potential health benefits. Despite being an excellent source for the preparation of oligosaccharides, there are currently no relevant research reports on HSP. In the present study, a novel oligosaccharide (HSO) with a molecular weight of 1791 Da and a degree of polymerization of 11 was prepared through enzymatic degradation of crude HSP (cHSP). Methylation and NMR analyses revealed that the main chain of HSO was (1 → 4)-α-d-glucose with two O-6-linked branched chains. Morphological observations indicated that HSO exhibited smooth surface with lamellar and filamentary structure, and the glycan size ranged from 0.03 to 0.20 μm. Notably, HSO significantly promoted the proliferation of Bifidobacterium, Bacteroides, and Phascolarctobacterium, thereby making positive alterations in intestinal microbiota composition. Moreover, HSO markedly increased the content of short-chain fatty acids during in vitro fermentation. Metabolomics analysis illustrated the important metabolic pathways primarily involving glucose metabolism, amino acid metabolism, and fatty acid metabolism.

Action Pattern of a Novel G-Specific Alginate Lyase: Determination of Subsite Specificity by HPAEC-PAD/MS.

G-specific alginate lyases are important tools for alginate fragment biodegradation and oligosaccharide production, which have great potential in alginate refining research. In this research, a novel G-specific alginate lyase Aly7Ce was cloned, expressed, and characterized, with the optimal reaction conditions at 30 °C and pH 8.0. By employing the UPSEC-VWD-MS method, Aly7Ce was confirmed as a random endoacting alginate lyase. Its minimum substrate was tetrasaccharide, and the final product majorly consisted of disaccharide to tetrasaccharide. HPAEC-PAD/MS method was employed to investigate the structurally different unsaturated alginate oligosaccharides. The substrate recognition and subsite specificity of Aly7Ce were revealed by detecting the oligosaccharide pattern in the enzymatic products with oligosaccharides or polysaccharides as substrates. Aly7Ce mainly attacked the second glycosidic linkage from the nonreducing end of oligosaccharide substrates. The subsite specificity of Aly7Ce was revealed as -2 (M/G), - 1 (G), + 1 (M/G), and +2 (M/G). The regular oligosaccharide products of Aly7Ce could be applied for the efficient preparation of ΔG, ΔGG, and ΔGGG with high purity. The G-specific alginate lyase Aly7Ce with a well-defined product composition and action pattern provided a novel tool for the modification and structural elucidation of alginate, as well as for the targeted preparation of oligosaccharides.

A Recombinant Alginate Lyase Algt1 with Potential in Preparing Alginate Oligosaccharides at High-Concentration Substrate.

Alginate lyase has been demonstrated as an efficient tool in the preparation of functional oligosaccharides (AOS) from alginate. The high viscosity resulting from the high concentration of alginate poses a limiting factor affecting enzymatic hydrolysis, particularly in the preparation of the fragments with low degrees of polymerization (DP). Herein, a PL7 family alginate lyase Algt from Microbulbifer thermotolerans DSM 19189 was developed and expressed in Pichia pastoris. The recombinant alginate lyase Algt1 was constructed by adopting the structural domain truncation strategy, and the enzymatic activity towards the alginate was improved from 53.9 U/mg to 212.86 U/mg compared to Algt. Algt1 was stable when incubated at 40 °C for 90 min, remaining with approximately 80.9% of initial activity. The analyses of thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC), and electrospray ionization mass spectrometry (ESI-MS) demonstrated that the DP of the minimum identifiable substrate of Algt1 was five, and the main hydrolysis products were AOS with DP 1-4. Additionally, 1-L the enzymatic hydrolysis system demonstrated that Algt1 exhibited an effective degradation at alginate concentrations of up to 20%, with the resulting products of monosaccharides (14.02%), disaccharides (21.10%), trisaccharides (37.08%), and tetrasaccharides (27.80%). These superior properties of Algt1 make it possible to efficiently generate functional AOS with low DP in industrial processing.

RETRACTED: Biochemical characterization of a cold-adapted κ-carrageenase from Colwellia echini: A biotechnological tool for efficient preparation of carrageenan oligosaccharides

κ-Carrageenase (EC 3.2.1.83) is a glycoside hydrolase that efficiently breaks down κ-carrageenan into κ-carrageenan oligosaccharides (κ-COS). In this study, a novel GH16 family κ-carrageenase called CeCgkB was cloned from the genome of Colwellia echini. Escherichia coli BL21 (DE3) was used to express CeCgkB. CeCgkB has a complete length of 987 bp, encoding 329 amino acid residues, and a molecular weight of 37.44 kDa. Biochemical characterization indicated that CeCgkB exhibits cold-adapted properties and was able to reach a maximum activity of 1530 U/mg at pH 8.0 and 35 °C. Kinetic parameters showed that CeCgkB has a low Km value (2 mg/mL), indicating high substrate affinity. As an endo-type κ-carrageenase, CeCgkB can degrade κ-carrageenan into products of varying DP, with a preference for DP4, DP6, and DP10. ESI-MS analysis revealed that the major products of CeCgkB consist of κ-carratetraose, κ-carrahexaose, and κ-carradecaose. As a result of CeCgkB's high activity, cold-adapted, and principal products, it is a promising biotechnological tool for the efficient preparation of κ-carrageenan oligosaccharides.

Preparation and potential antitumor activity of alginate oligosaccharides degraded by alginate lyase from Cobetia marina

It is of great significance to develop marine resources and study its potential biological activity by using alginate lyase produced by marine psychrophilic bacteria. In the previous study, a new marine psychrophilic bacterium (Cobetia marina HQZ08) was screened from the growth area of Laminaria japonica, and it was found that the strain could efficiently produce alginate-degrading enzyme (Aly30). In this paper, the ability of Aly30 to degrade alginate was optimized and the optimal degradation conditions were obtained. It was found that the main degradation product of alginate oligosaccharides was trisaccharide. In vitro cell experiments showed that the antitumor activity of low molecular weight alginate oligosaccharides was better than that of high molecular weight alginate oligosaccharides. In summary, Aly30 had the potential to produce alginate oligosaccharides with low degree of polymerization and antitumor activity, which provided a reference for the enzymatic preparation and application of alginate oligosaccharides.

Biochemical characterization of a novel bifunctional alginate lyase from Microbulbifer arenaceous

Bio‒production of alginate oligosaccharides (AOSs), a type of functional food additive, is a promising way for green utilization of algae, in which alginate hydrolyzing enzymes play a key role. A novel alginate lyase gene (MiAly17A) from a marine bacterium Microbulbifer arenaceous was heterologously expressed in E. coli. The coding sequence of the gene shared the highest identity of 86% with a polysaccharide lyase (PL) family 17 alginate lyase (AlgL17) from Microbulbifer sp. ALW1. The recombinant enzyme (MiAly17A) was purified and biochemically characterized. MiAly17A showed maximal enzyme activity at 40 °C and pH 7.5, respectively. It was stable at the temperatures below 35 °C and within pH 5.0–8.0. The enzyme activities were increased by 5.3 and 5.6 folds in the presence of 100 mM of K+ and Na+, respectively. MiAly17A was bifunctional and could hydrolyze sodium alginate to release unsaturated monosaccharides and oligosaccharides with degrees of polymerization (DP) 2–7. The enzyme catalyzed the cleavage of glycosidic bonds from the non-reducing ends and the backbone of the tested oligosaccharides (DP ≥ 4), exhibiting both exolytic and endo-lytic activities. Moreover, MiAly17A was used for the production of alginate oligosaccharides from sodium alginate, and the highest conversion ratio of 68% was obtained. The unique properties may possess the enzyme great potential for preparation of alginate oligosaccharides.

Preparation of Dendrobium officinale flower oligosaccharides and the value-added effects on three types of probiotics

The present study prepared Dendrobium officinale flower oligosaccharides and investigated their added value in vitro. The crude oligosaccharides obtained by water extraction and alcohol precipitation were purified by ultrafiltration, and the value-added effects of different concentrations of D. officinale flower oligosaccharides on Saccharomycetes, Lactobacillus bulgaricus, and Streptococcus thermophilus were examined. The in vitro growth kinetic models for the three types of probiotics were established according to the logistic equation. Our results showed that a concentration of 8 g/L D. officinale flower oligosaccharides was most suitable for promoting the growth of the three types of probiotics, and the growth rates were significantly improved compared to the control group. Nonlinear fitting was performed for the in vitro growth kinetic models of the three bacteria according to the logistic equation, and the degrees of fit reached 0.9895, 0.9899, and 0.99, respectively, indicating a good fit. These kinetic models suitably depicted the in vitro growth processes of the three bacteria. D. officinale flower oligosaccharides promote the growth of Saccharomycetes, L. bulgaricus and S. thermophilus and improve the physiological activities of these microorganisms. This study provides a theoretical basis for the application of D. officinale flower oligosaccharides in the development of probiotic products.

Production, Characterization and Application of a Novel Chitosanase from Marine Bacterium Bacillus paramycoides BP-N07.

Chitooligosaccharides (COS), a high-value chitosan derivative, have many applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitosanase, which catalyzes the hydrolysis of chitosan, can cleave β-1,4 linkages to produce COS. In this study, a chitosanase-producing Bacillus paramycoides BP-N07 was isolated from marine mud samples. The chitosanase enzyme (BpCSN) activity was 2648.66 ± 20.45 U/mL at 52 h and was able to effectively degrade chitosan. The molecular weight of purified BpCSN was approximately 37 kDa. The yield and enzyme activity of BpCSN were 0.41 mg/mL and 8133.17 ± 47.83 U/mg, respectively. The optimum temperature and pH of BpCSN were 50 °C and 6.0, respectively. The results of the high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of chitosan treated with BpCSN for 3 h showed that it is an endo-chitosanase, and the main degradation products were chitobiose, chitotriose and chitotetraose. BpCSN was used for the preparation of oligosaccharides: 1.0 mg enzyme converted 10.0 g chitosan with 2% acetic acid into oligosaccharides in 3 h at 50 °C. In summary, this paper reports that BpCSN has wide adaptability to temperature and pH and high activity for hydrolyzing chitosan substrates. Thus, BpCSN is a chitosan decomposer that can be used for producing chitooligosaccharides industrially.

Preparation of water-soluble chitosan oligosaccharides by oxidative hydrolysis of chitosan powder with hydrogen peroxide

Chitosan (CS) is only soluble in weak acid medium, thereby limiting its wide utilisation in the field of biomedicine, food, and agriculture. In this report, we present a method for preparing water-soluble CS oligosaccharides (COSs) at high concentration (∼10%, w/v) via the oxidative hydrolysis of CS powder with molecular weight (Mw) ∼90,000 g/mol) in 2% H2O2 solution at ambient temperature by a two-step process, namely, the heterogeneous hydrolysis step and homogeneous hydrolysis step. The resultant COSs were characterised by gel permeation chromatography (GPC), fourier transforms infrared spectroscopy (FT-IR), ultraviolet–visible spectroscopy (UV–Vis), proton nuclear magnetic resonance spectroscopy (1H NMR) and X-ray diffraction (XRD) spectroscopy. The resulting products were composed of COSs (Mw of 2000−6600 g/mol) that were completely soluble in water. The results also indicated that the structure of COSs was almost unchanged compared with the original CS unless Mw was low. Accordingly, COSs with low Mw (∼2000 g/mol) and high concentration (10%, w/v) could be effectively prepared by the oxidative hydrolysis of CS powder using hydrogen peroxide under ambient conditions.

Biochemical characterization of a new ulvan lyase and its applicability in utilization of ulvan and preparation of ulva oligosaccharides.

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva, which has a variety of biological properties. Ulvan lyase degrades ulvan through a β-elimination mechanism which cleaves the β-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97kDa, and it had the highest catalytic activity at 45°C and pH8.0 in Tris-HCl buffer. The Km and Vmax values were 2.24mg·mL-1 and 2.048μmol·min-1·mL-1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20~30°C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides.

Improving the thermostability of a novel PL-6 family alginate lyase by rational design engineering for industrial preparation of alginate oligosaccharides

Alginate is degraded into alginate oligosaccharides with various biological activities by enzymes. However, the thermostability of the enzyme limits its industrial application. In this study, a novel PL-6 alginate lyase, AlyRm6A from Rhodothermus marinus 4252 was expressed and characterized. In addition, an efficient comprehensive strategy was proposed, including automatic design of heat-resistant mutants, multiple computer-aided ΔΔGfold value calculation, and conservative analysis of mutation sites. AlyRm6A has naturally high thermostability. Compared with the WT, T43I and Q216I kept their original activities, and their half-lives were increased from 3.68 h to 4.29 h and 4.54 h, melting point temperatures increased from 61.5 °C to 62.9 °C and 63.5 °C, respectively. The results of circular dichroism showed that both the mutants and the wild type had the characteristic peaks of β-sheet at 195 nm and 216 nm, which indicated that there was no significant effect on the secondary structure of the protein. Molecular dynamics simulation (MD) analyses suggest that the enhancement of the hydrophobic interaction network, improvement of molecular rigidity, and denser structure could improve the stability of AlyRm6A. To the best of our knowledge, our findings indicate that AlyRm6A mutants exhibit the highest thermostability among the characterized PL-6 alginate lyases, making them potential candidates for industrial production of alginate oligosaccharides.

Nuclear magnetic resonance-based structural elucidation of novel marine glycans and derived oligosaccharides.

Marine glycans of defined structures are unique representatives among all kinds of structurally complex glycans endowed with important biological actions. Besides their unique biological properties, these marine sugars also enable advanced structure-activity relationship (SAR) studies given their distinct and defined structures. However, the natural high molecular weights (MWs) of these marine polysaccharides, sometimes even bigger than 100 kDa, pose a problem in many biophysical and analytical studies. Hence, the preparation of low MW oligosaccharides becomes a strategy to overcome the problem. Regardless of the polymeric or oligomeric lengths of these molecules, structural elucidation is mandatory for SAR studies. For this, nuclear magnetic resonance (NMR) spectroscopy plays a pivotal role. Here, we revisit the NMR-based structural elucidation of a series of marine sulfated poly/oligosaccharides discovered in our laboratory within the last 2 years. This set of structures includes the α-glucan extracted from the bivalve Marcia hiantina; the two sulfated galactans extracted from the red alga Botryocladia occidentalis; the fucosylated chondroitin sulfate isolated from the sea cucumber Pentacta pygmaea; the oligosaccharides produced from the fucosylated chondroitin sulfates from this sea cucumber species and from another species, Holothuria floridana; and the sulfated fucan from this later species. Specific 1H and 13C chemical shifts, generated by various 1D and 2D homonuclear and heteronuclear NMR spectra, are exploited as the primary source of information in the structural elucidation of these marine glycans.