A comprehensive review of sulfated fucan from sea cucumber: Antecedent and prospect.

Sulfated fucan from sea cucumber is mainly consists of L-fucose and sulfate groups. Recent studies have confirmed that the structure of sulfated fucan mainly consists of repeating units, typically tetrasaccharides. However, there is growing evidence indicating the presence of irregular domains with heterogeneous units that have not been extensively explored. Moreover, as a key contributor to the nutritional benefits of sea cucumbers, sulfated fucan demonstrates a range of biological activities, such as anti-inflammatory, anticancer, hypolipidemic, anti-hyperglycemic, antioxidant, and anticoagulant properties. These biological activities are profoundly influenced by the structural features of sulfated fucan including molecular weight and distribution patterns of sulfate groups. The latest research indicates that sulfated fucan is dispersed in the extracellular matrix of the body wall of sea cucumbers. This article aimed to review the research progress on the in-situ distribution, structures, structural elucidation strategies, functions, and structure-activity relationships of sulfated fucan, especially in the last decade. It also provided insights into the major challenges and potential solutions in the research and development of sulfated fucan. Moreover, the fucanase and carbohydrate binding modules are anticipated to play pivotal roles in advancing this field.

Structural investigation of Fun168A unraveling the recognition mechanism of endo-1,3-fucanase towards sulfated fucan.

BACKGROUND: Sulfated fucan has gained interest due to its various physiological activities. Endo-1,3-fucanases are valuable tools for investigating the structure and establishing structure-activity relationships of sulfated fucan. However, the substrate recognition mechanism of endo-1,3-fucanases towards sulfated fucan remains unclear, limiting the application of endo-1,3-fucanases in sulfated fucan research. SCOPE AND APPROACH: This study presented the first crystal structure of endo-1,3-fucanase (Fun168A) and its complex with the tetrasaccharide product, utilizing X-ray diffraction techniques. The novel subsite specificity of Fun168A was identified through glycomics and nuclear magnetic resonance (NMR). KEY FINDINGS AND CONCLUSIONS: The structure of Fun168A was determined at 1.92 Å. Residues D206 and E264 acted as the nucleophile and general acid/base, respectively. Notably, Fun168A strategically positioned a series of polar residues at the subsites ranging from -2 to +3, enabling interactions with the sulfate groups of sulfated fucan through salt bridges or hydrogen bonds. Based on the structure of Fun168A and its substrate recognition mechanisms, the novel subsite specificities at the -2 and +2 subsites of Fun168A were identified. Overall, this study provided insight into the structure and substrate recognition mechanism of endo-1,3-fucanase for the first time and offered a valuable tool for further research and development of sulfated fucan.

Identification and characterization of a chondroitinase ABC with a novel carbohydrate-binding module.

Chondroitinases play important roles in structural and functional studies of chondroitin sulfates. Carbohydrate-binding module (CBM) is generally considered as an accessory module in carbohydrate-active enzymes, which promotes the association of the appended enzyme with the substrate and potentiates the catalytic activity. However, the role of natural CBM in chondroitinases has not been investigated. Herein, a novel chondroitinase ChABC29So containing an unknown domain with a predicted ß-sandwich fold was discovered from Segatella oris. Recombinant ChABC29So showed enzyme activity towards chondroitin sulfates and hyaluronic acid and acted in a random endo-acting manner. The unknown domain exhibited a chondroitin sulfate-binding capacity and was identified as a CBM. Biochemical characterization of ChABC29So and the CBM-truncated enzyme revealed that the CBM enhances the catalytic activity, thermostability, and disaccharide proportion in the final enzymatic products of ChABC29So. These findings demonstrate the role of the natural CBM in a chondroitinase and will guide future modification of chondroitinases.

Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a ß-sandwich fold comprising two antiparallel ß-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

The α-linkage in funoran and agarose could be hydrolyzed by a GH96 family enzyme: Discovery of the α-funoranase.

Agarans represent a group of galactans extracted from red algae. Funoran and agarose are the two major types and commercially applied polysaccharides of agaran. Although the glycoside hydrolases targeting ß-glycosidic bonds of agaran have been widely investigated, those capable of degrading α-glycosidic bonds of agarose were limited, and the enzyme degrading α-linkages of funoran has not been reported till now. In this study, a GH96 family enzyme BiAF96A_Aq from a marine bacterium Aquimarina sp. AD1 was heterologously expressed in Escherichia coli. BiAF96A_Aq exhibited dual activities towards the characteristic structure of funoran and agarose, underscoring the multifunctionality of GH96 family members. Glycomics and NMR analysis revealed that BiAF96A_Aq hydrolyzed the α-1,3 glycosidic bonds between 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose-6-sulfate (G6S) of funoran, as well as LA and ß-d-galactopyranose (G) of agarose, through an endo-acting manner. The end products of BiAF96A_Aq were majorly composed of disaccharides and tetrasaccharides. The identification of the activity of BiAF96A_Aq on funoran indicated the first discovery of the funoran hydrolase for α-1,3 linkage. Considering the novel catalytic reaction, we proposed to name this activity as "α-funoranase" and recommended the assignment of a dedicated EC number for its classification.

Fucoidan from Apostichopus japonicus ameliorates alcoholic liver disease by regulating gut-liver axis homeostasis.

Long-term and excessive alcohol consumption can lead to the development of alcoholic liver disease (ALD), characterized by oxidative damage, intestinal barrier injury, and disruption of intestinal microbiota. In this study, we extracted fucoidan (Aj-FUC) from Apostichopus japonicus using enzymatic methods and characterized its structure. The ALD model was established in male Balb/c mice using 56° Baijiu, with silymarin as a positive control. Mice were orally administered 100 mg/kg·bw and 300 mg/kg·bw of Aj-FUC for 28 days to evaluate its effects on liver injury in ALD mice and explore its potential role in modulating the gut-liver axis. The results showed significant improvements in histopathological changes and liver disease in the Aj-FUC group. Aj-FUC treatment significantly increased the levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) while weakly reduced the elevation of malondialdehyde (MDA) induced by ALD. It also regulated the Nrf2/HO-1 signaling pathway, collectively alleviating hepatic oxidative stress. Aj-FUC intervention upregulated the expression of ZO-1 and Occludin, thus contributing to repair the intestinal barrier. Additionally, Aj-FUC increased the content of short-chain fatty acids (SCFAs) and regulated the imbalance in gut microbiota. These results suggested that Aj-FUC alleviates ALD by modulating the gut-liver axis homeostasis. It may prove to be a useful dietary supplement in the treatment of alcoholic liver damage.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Biochemical characterization and cleavage specificities analyses of three endo-1,3-fucanases within glycoside hydrolase family 174.

Sulfated fucans have garnered extensive research interest in recent decades due to their varied bioactivity. Fucanases are important tools for investigating sulfated fucans. This study reported the bioinformatic analysis and biochemical properties of three GH174 family endo-1,3-fucanases. Wherein, Fun174Rm and Fun174Sb showed the highest optimal reaction temperature among the reported fucanases, and Fun174Sb possessed favorable thermostability and catalysis efficiency. Fun174Rm displayed a random endo-acting manner, while Fun174Ri and Fun174Sb hydrolyzed sulfated fucan in processive manners. UPLC-MS and NMR analyses confirmed that the three enzymes catalyze cleavage of the α(1 â†’ 3)-bonds between Fucp2S and Fucp2S in the sulfated fucan from Isostichopus badionotus. These enzymes demonstrated novel cleavage specificities, which could accept α-Fucp2S residues at subsites -1 and + 1. The acquiring of these biotechnological tools would be beneficial to the in-depth research of sulfated fucans.

Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 µg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti-A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Integrated Omics Approach: Revealing the Mechanism of Auxenochlorella pyrenoidosa Protein Extract Replacing Fetal Bovine Serum for Fish Muscle Cell Culture.

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.

A comparative investigation of anionic polysaccharides on the structure and gastrointestinal digestion of collagen fibrils.

Collagen, the most abundant and widely distributed functional protein in mammals, typically assembles into collagen fibrils through side-by-side packing. The purpose of this study was to comparatively investigate the fate of sea cucumber collagen fibrils in the gastrointestinal tract when interacting with different anionic polysaccharides (fucoidan (FUC), Kappa-carrageenan (K-car), sodium alginate (SA)). Results revealed that the gel properties and viscosity values of collagen fibrils were notably enhanced, and the rate of structural alteration in collagen fibrils was reduced when K-car and SA were introduced. Conversely, in the presence of FUC, collagen fibril viscosity decreased, and the secondary structure of collagen fibrils underwent changes. FUC was found to diminish the structural stability of collagen fibrils and accelerate the gastric digestion rate, which was further exacerbated by thermal treatment. All these anionic polysaccharides were observed to facilitate the formation of collagen peptide aggregates by binding to polysaccharides during intestinal digestion. This study bridged the knowledge gap regarding the impact of anionic polysaccharides on the gastrointestinal digestion of collagen fibrils, potentially paving the way for broader applications of collagen in the food industry.

Discovery and characterization of a novel carbohydrate-binding module: a favorable tool for investigating agarose.

BACKGROUND: Agarose, mainly composed of 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose (G) units, is an important polysaccharide with wide applications in food, biomedical and bioengineering industries. Carbohydrate-binding modules (CBMs) are favorable tools for the investigations of polysaccharides. Few agarose-binding CBMs have been hitherto reported, and their binding specificity is unclear. RESULTS: An unknown domain with a predicted ß-sandwich fold was discovered from a ß-agarase of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T . The expressed protein WfCBM101 could bind to agarose and exhibited relatively weak affinity for porphyran, with no affinity for the other seven examined polysaccharides. The protein binds to the tetrasaccharide (LA-G)2 , but not to the major tetrasaccharide contained in porphyran. The sequence novelty and well-defined binding function of WfCBM101 shed light on a novel CBM family (CBM101). Furthermore, the feasibility of WfCBM101 for visualizing agarose in situ was confirmed. CONCLUSION: A novel CBM, WfCBM101, with a desired specificity for agarose was discovered and characterized, which represents a new CBM family. The CBM could be utilized as a promising tool for studies of agarose. © 2023 Society of Chemical Industry.

Identification and structural characterization of a novel chondroitin sulfate-specific carbohydrate-binding module: The first member of a new family, CBM100.

Chondroitin sulfate is a biologically and commercially important polysaccharide with a variety of applications. Carbohydrate-binding module (CBM) is an important class of carbohydrate-binding protein, which could be utilized as a promising tool for the applications of polysaccharides. In the present study, an unknown function domain was explored from a putative chondroitin sulfate lyase in PL29 family. Recombinant PhCBM100 demonstrated binding capacity to chondroitin sulfates with Ka values of 2.1 ± 0.2 × 106 M-1 and 6.0 ± 0.1 × 106 M-1 to chondroitin sulfate A and chondroitin sulfate C, respectively. The 1.55 Å resolution X-ray crystal structure of PhCBM100 exhibited a ß-sandwich fold formed by two antiparallel ß-sheets. A binding groove in PhCBM100 interacting with chondroitin sulfate was subsequently identified, and the potential of PhCBM100 for visualization of chondroitin sulfate was evaluated. PhCBM100 is the first characterized chondroitin sulfate-specific CBM. The novelty of PhCBM100 proposed a new CBM family of CBM100.

An enzyme-pHBH method for specific quantification of porphyran.

Porphyran, the major polysaccharide extracted from Porphyra, exhibits tremendous potential for development as functional food or pharmaceutical due to its multiple biological activities. The quantitative analysis of porphyran is important for the quality control in product development. However, the specific quantitative method of porphyran has not been established, and the lack of reference substance makes the quantification more challenging. Here, a common component of porphyran, with high purity, similar molecular weight distribution, sourced from different Porphyra producing areas in China was first prepared by a series of isolation and purification steps, and utilized as the reference substance for porphyran quantification. Subsequently, the porphyran was fully degraded into oligosaccharides by using a ß-porphyranase, followed by employing para-hydroxybenzoic acid hydrazide (pHBH) method to detect the content of the generated reducing sugar. The enzyme-pHBH method for porphyran specific quantification was established. Results showed that this method was validated with good linearity, high accuracy and precision, and reliability. Addtionally, NaCl with a concentration below 0.5 %, alcohol under 8 % and other polysaccharide including chitosan, agarose, chondrotin sulfate, alginate, hyaluronic acid and κ-carrageenan did not interfere with this method. This approach is promising for quality control of the porphyran products and offers a feasible strategy for the specific quantification of other polysaccharides.

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 repertoire of alginate lyases in the alginate polysaccharide utilization loci of marine bacterium Wenyingzhuangia fucanilytica: biochemical properties and action pattern.

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T , and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35-50 °C and pH 8.0-9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M-specific enzyme (Aly17A). Ultra-performance liquid chromatography-mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M-blocks of oligosaccharide products, as well as the M-blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry.

Characterization of a novel carbohydrate-binding module specifically binding to the major structural units of porphyran.

Porphyran is a promising bioactive polysaccharide majorly composed of 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) disaccharide repeating units. Carbohydrate-binding modules (CBMs) have been verified to be essential tools for investigating polysaccharides. However, no confirmed CBM binding to porphyran has been hitherto reported. In this study, an unknown domain with a predicted ß-sandwich fold from a potential GH86 porphyranase was discovered, and further recombinantly expressed. The CBM protein (named FvCBM99) presented a desired specificity for porphyran tetrasaccharide with an affinity constant of 1.9 × 10-4 M, while it could not bind to agarose tetrasaccharide. The sequence novelty and well-defined function of FvCBM99 and its homologs reveal a new CBM family, CBM99. Besides, the application potential of FvCBM99 in in situ visualization of porphyran was demonstrated. The discovery of FvCBM99 provides a favorable tool for future studies of porphyran.

Characterization of a novel endo-1,3-fucanase from marine bacterium Wenyingzhuangia fucanilytica reveals the presence of diversity within glycoside hydrolase family 168.

Sulfated fucans attract increasing research interests in recent decades for their various physiological activities. Fucanases are indispensable tools for the investigation of sulfated fucans. Herein, a novel GH168 family endo-1,3-fucanase was cloned from the genome of marine bacterium Wenyingzhuangia fucanilytica. The expressed protein Fun168D was a processive endo-acting enzyme. Ultra performance liquid chromatography-high resolution mass spectrum and NMR analyses revealed that the enzyme cleaved the α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2OSO3-) in sulfated fucan from Isostichopus badionotus, and α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2,4OSO3-) in sulfated fucan from Holothuria tubulosa. Fun168D would prefer to accept α-l-Fucp(2,4OSO3-) than α-l-Fucp(2OSO3-) at subsite +1, and could tolerate the absence of fucose residue at subsite +2. The novel cleavage specificity and hydrolysis pattern revealed the presence of diversity within the GH168 family, which would facilitate the development of diverse biotechnological tools for the molecule tailoring of sulfated fucan.