Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I.

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.

Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property.

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.

Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc.

Alginate lyase degrades alginate by the ß-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

Preliminary identification and semi-quantitative characterization of a multi-faceted high-stability alginate lyase from marine microbe Seonamhaeicola algicola with anti-biofilm effect on Pseudomonas aeruginosa.

Alginate lyases with unique characteristics for degrading alginate into size-defined oligosaccharide fractions, were considered as the potential agents for disrupting Pseudomonas aeruginosa biofilms. In our study, a novel endolytic PL-7 alginate lyase, named AlyG2, was cloned and expressed through Escherichia coli. This enzyme exhibited excellent properties: it maintained more than 85% activity at low temperatures of 4 °C and high temperatures of 70 °C. After 1 h of incubation at 4 °C, it still retained over 95% activity, demonstrating the ability to withstand low temperature. The acid-base and salt tolerance properties shown it preserves more than 50% activity in the pH range of 5.0 to 11.0 and in a high salt environment at 3000 mM NacCl, indicating its high stability in several aspects. More importantly, AlyG2 in our research was revealed to be effective at removing mature biofilms and inhibiting biofilm formation produced by Pseudomonas aeruginosa, and the inhibition and disruption rates were 47.25 ± 4.52% and 26.5 ± 6.72%, respectively. Additionally, the enzyme AlyG2 promoted biofilm disruption in combination with antibiotics, particularly manifesting the synergistic effect with erythromycin (FIC=0.5). In all, these results offered that AlyG2 with unique characteristics may be an effective technique for the clearance or disruption of biofilm produced by P. aeruginosa.

A Novel Bifunctional Alginate Lyase and Antioxidant Activity of the Enzymatic Hydrolysates.

Alginate lyase Aly448, a potential new member of the polysaccharide lyase (PL) 7 family, which was cloned and identified from the macroalgae-associated bacterial metagenomic library, showed bifunctionality. The molecular docking results revealed that Aly448 has two completely different binding sites for alginate (polyMG), poly-α-l-guluronic acid (polyG), and poly-ß-d-mannuronic acid (polyM) substrates, respectively, which might be the molecular basis for the enzyme's bifunctionality. Truncational results confirmed that predicted key residues affected the bifunctionality of Aly448, but did not wholly explain. Besides, Aly448 presented excellent biochemical characteristics, such as higher thermal stability and pH tolerance. Degradation of polyMG, polyM, and polyG substrates by Aly448 produced tetrasaccharide (DP4), disaccharide (DP2), and galactose (DP1), which exhibited excellent antioxidant activity. These findings provide novel insights into the substrate recognition mechanism of bifunctional alginate lyases and pave a new path for the exploitation of natural antioxidant agents.

Advances in alginate lyases and the potential application of enzymatic prepared alginate oligosaccharides: A mini review.

Alginate is mainly a linear polysaccharide composed of randomly arranged ß-D-mannuronic acid and α-L-guluronic acid linked by α, ß-(1,4)-glycosidic bonds. Alginate lyases degrade alginate mainly adopting a ß-elimination mechanism, breaking the glycosidic bonds between the monomers and forming a double bond between the C4 and C5 sugar rings to produce alginate oligosaccharides consisting of 2-25 monomers, which have various physiological functions. Thus, it can be used for the continuous industrial production of alginate oligosaccharides with a specific degree of polymerization, in accordance with the requirements of green exploitation of marine resources. With the development of structural analysis, the quantity of characterized alginate lyase structures is progressively growing, leading to a concomitant improvement in understanding the catalytic mechanism. Additionally, the use of molecular modification methods including rational design, truncated expression of non-catalytic domains, and recombination of conserved domains can improve the catalytic properties of the original enzyme, enabling researchers to screen out the enzyme with the expected excellent performance with high success rate and less workload. This review presents the latest findings on the catalytic mechanism of alginate lyases and outlines the methods for molecular modifications. Moreover, it explores the connection between the degree of polymerization and the physiological functions of alginate oligosaccharides, providing a reference for enzymatic preparation development and utilization.

Characterization and Mechanism Study of a Novel PL7 Family Exolytic Alginate Lyase from Marine Bacteria Vibrio sp. W13.

Alginate lyase can degrade alginate into oligosaccharides through ß-elimination for various biological, biorefinery, and agricultural purposes. Here, we report a novel PL7 family exolytic alginate lyase VwAlg7A from marine bacteria Vibrio sp. W13 and achieve the heterologous expression in E. coli BL21 (DE3). VwAlg7A is 348aa with a calculated molecular weight of 36 kDa, containing an alginate lyase 2 domain. VwAlg7A exhibits specificity towards poly-guluronate. The optimal temperature and pH of VwAlg7A are 30 °C and 7.0, respectively. The activity of VwAlg7A can be significantly inhibited by the Ni2+, Zn2+, and NaCl. The Km and Vmax of VwAlg7A are 36.9 mg/ml and 395.6 µM/min, respectively. The ESI and HPAEC-PAD results indicate that VwAlg7A cleaves the sugar bond in an exolytic mode. Based on the molecular docking and mutagenesis results, we further confirmed that R98, H169, and Y303 are important catalytic residues.

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.

Rational design for thermostability improvement of a novel PL-31 family alginate lyase from Paenibacillus sp. YN15.

Currently, alginate oligosaccharides (AOS) become attractive due to their excellent physiological effects. AOS has been widely used in food, pharmaceutical, and cosmetic industries. Generally, AOS can be produced from alginate using alginate lyase (ALyase) as the biocatalyst. However, most ALyase display poor thermostability. In this study, a thermostable ALyase from Paenibacillus sp. YN15 (Payn ALyase) was characterized. It belonged to the polysaccharide lyase (PL) 31 family and displayed poly ß-D-mannuronate (Poly M) preference. Under the optimum condition (pH 8.0, 55 °C, 50 mM NaCl), it exhibited maximum activity of 90.3 U/mg and efficiently degraded alginate into monosaccharides and AOS with polymerization (DP) of 2-4. Payn ALyase was relatively stable at 55 °C, but the thermostability dropped rapidly at higher temperatures. To further improve its thermostability, rational design mutagenesis was carried out based on a combination of FireProt, Consensus Finder, and PROSS analysis. Finally, a triple-point mutant K71P/Y129G/S213G was constructed. The optimum temperature was increased from 55 to 70 °C, and the Tm was increased from 62.7 to 64.1 °C. The residual activity after 30 min incubation at 65 °C was enhanced from 36.0 % to 83.3 %. This study provided a promising ALyase mutant for AOS industrial production.

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 ß-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.

Elucidating structure of pectin in ramie fiber to customize enzyme cocktail for high-efficiency enzymatic degumming.

Pectin is one of the main components of bast fiber including ramie fiber, and must be removed before use. Enzymatic degumming is the preferred process as it is an environment-friendly, simple and controllable process for ramie degumming. However, an important problem limiting wide application of this process is the high cost due to the low efficiency of enzymatic degumming. In this study, pectin samples were extracted from raw ramie fiber and degummed ramie fiber, respectively, and their structures were characterized and compared to allow tailoring of an enzyme cocktail for degrading the pectin. It was elucidated that pectin from ramie fiber is composed of low esterified homogalacturonan (HG) and low branched rhamnogalacturonan I (RG-I), and the ratio of HG/RG-I is 1.72:1. Based on the pectin structure, potential enzymes to be used for enzymatic degumming of ramie fiber were proposed and an enzyme cocktail was customized. Degumming experiments confirmed that the customized enzyme cocktail can effectively remove pectin from ramie fiber. To our knowledge, this is the first time the structural characteristics of pectin in ramie fiber have been clarified, and it also provides an example of tailoring a specific enzyme system to achieve high-efficiency degumming for biomass containing pectin.

Improving thermostability of a PL 5 family alginate lyase with combination of rational design strategies.

Alginate lyases with strict substrate specificity possess potential in directed production of alginate oligosaccharides with specific composition. However, their poor thermostability hampered their applications in industry. In this study, an efficient comprehensive strategy including sequence-based analysis, structure-based analysis, and computer-aid ΔΔGfold value calculation was proposed. It was successfully performed on alginate lyase (PMD) with strict poly-ß-D-mannuronic acid substrate specificity. Four single-point variants A74V, G75V, A240V, and D250G with increased Tm of 3.94 °C, 5.21 °C, 2.56 °C, and 4.80 °C, respectively, were selected out. After ordered combined mutations, a four-point mutant (M4) was finally generated which displayed remarkable increase on thermostability. The Tm of M4 increased from 42.25 °C to 51.59 °C and its half-life at 50 °C was about 58.9-fold of PMD. Meanwhile, there was no obvious loss of enzyme activity (more than 90% retained). Molecular dynamics simulation analysis insisted that the improvement of thermostability might be attribute to the rigidified region A which might be caused by the newly formed hydrogen bonds and salt bridges introduced by mutations, the lower distance of original hydrogen bonds, and the more compact overall structures.

High-level expression and characterization of a highly active hyaluronate lyase HylC with significant potential in hyaluronan oligosaccharide preparation.

Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca2+, K+, Zn2+, Ni2+ and Li+. Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 104 U/mL to 2.64 × 104 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×105 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.

The specificity of pectate lyase VdPelB from Verticilium dahliae is highlighted by structural, dynamical and biochemical characterizations.

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by ß-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel ß-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

Antimicrobial high molecular weight pectin polysaccharides production from diverse citrus peels using a novel PL10 family pectate lyase.

The discovery of environmentally friendly enzymes that can convert inexpensive and abundant citrus peel pectin into high value-added product is a potential avenue for the citrus peel application. In this study, a novel PL10-family pectate lyase (pelA) was characterized from marine bacterium Echinicola pacifica. PelA was a Ca2+ dependent pectate lyase whose activity was highest at pH 8 and 40 °C. It was capable of degrading polygalacturonic acid (PGA) and citrus peel pectin (CPP), but not apple peel pectin. Notably, PelA hydrolyzed PGA to high molecular weight polysaccharide (average molecular weight 111.4 kDa). Moreover, PelA was also able to degrade CPP from nine distinct citrus species into polysaccharides (average molecular weight ranging from 84.7 to 539.2 kDa) that showed antimicrobial activity against Staphylococcus epidermidis (88.8 %), Bacillus subtilis (99.8 %), Staphylococcus aureus (92.1 %), Escherichia coli (100.0 %) and Klebsiella pneumoniae (86.4 %). Considering the high market value of pectin in the food industry, PelA's capacity to convert citrus pectin into high molecular weight polysaccharides lays a foundation for its applications.

A heterodimeric hyaluronate lyase secreted by the activated sludge bacterium Haliscomenobacter hydrossis.

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-ß-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.

NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.