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.

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.

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.

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.

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.

The characteristic structure of funoran could be hydrolyzed by a GH86 family enzyme (Aga86A_Wa): Discovery of the funoran hydrolase.

Funoran, agarose and porphyran all belong to agaran, and share the similar skeleton. Although the glycoside hydrolase for agarose and porphyran, i.e. agarase and porphyranase, have been extensively studied, the enzyme hydrolyzing funoran has not been reported hitherto. The crystal structure of a previously characterized GH86 ß-agarase Aga86A_Wa showed a large cavity at subsite -1, which implied its ability to accommodate sulfate ester group. By using glycomics and NMR analysis, the activity of Aga86A_Wa on the characteristic structure of funoran was validated, which signified the first discovery of funoran hydrolase, i.e. funoranase. Aga86A_Wa hydrolyzed the ß-1,4 glycosidic bond between ß-d-galactopyranose-6-sulfate (G6S) and 3,6-anhydro-α-l-galactopyranose (LA) unit of funoran, and released disaccharide LA-G6S as the predominant end product. Considering the hydrolysis pattern, we proposed to name the activity represented by Aga86A_Wa on funoran as "ß-funoranase" and suggested to assign it an EC number.

Sulfated fucan could serve as a species marker of sea cucumber with endo-1,3-fucanase as the essential tool.

In the past few decades, sulfated fucan from sea cucumber had attracted considerable interest owing to its abundant physiological activities. Nevertheless, its potential for species discrimination had not been investigated. Herein, particular attention was given to sea cucumber Apostichopus japonicus, Acaudina molpadioides, Holothuria hilla, Holothuria tubulosa, Isostichopus badionotus and Thelenota ananas to examine the feasibility of sulfated fucan as a species marker of sea cucumber. The enzymatic fingerprint suggested that sulfated fucan exhibited significant interspecific discrepancy and intraspecific stability, which revealed that sulfated fucan could serve as the species marker of sea cucumber, by utilizing the overexpressed endo-1,3-fucanase Fun168A and the ultra-performance liquid chromatography-high resolution mass spectrum. Moreover, oligosaccharide profile of sulfated fucan was determined. The oligosaccharide profile combined with hierarchical clustering analysis and principal components analysis further confirmed that sulfated fucan could serve as a marker with a satisfying performance. Besides, load factor analysis showed that the minor structure of sulfated fucan also contributed to the sea cucumber discrimination, besides the major structure. The overexpressed fucanase played an indispensable role in the discrimination, due to its specificity and high activity. The study would lead to a new strategy for species discrimination of sea cucumber based on sulfated fucan.

Discovery of a sulfated fucan-specific carbohydrate-binding module: The first member of a new carbohydrate-binding module family.

Sulfated fucan is an important functional polysaccharide with various physiological activities. Carbohydrate-binding module (CBM) is a representative class of carbohydrate-binding protein, which could be employed as a favorable tool for the investigations and applications of polysaccharides. Nevertheless, only one confirmed sulfated fucan-binding CBM has been hitherto reported. In the present study, an unknown domain with a predicted ß-sandwich fold was discovered from a fucanase Fun174A, and further cloned and heterologously expressed in Escherichia coli. The expressed protein Fun174A-CBM displayed a specific binding capacity to sulfated fucan. The bio-layer interferometry assays showed that the protein could bind to the sulfated fucan tetrasaccharide with an affinity constant of 2.83 × 10-4 M. Fun174A-CBM shared no significant sequence similarity to any identified CBMs, indicating that it represents a new CBM family. The discovery of Fun174-CBM and the novel CBM family would be beneficial to the investigations of sulfated fucan-binding proteins.

Characterization of an endo-1,3-fucanase from marine bacterium Wenyingzhuangia aestuarii: The first member of a novel glycoside hydrolase family GH174.

Sulfated fucans are important marine polysaccharides with various biological and biomedical activities. Fucanases are favorable tools to establish the structure-activity relationships of sulfated fucans. Herein, gene fun174A was discovered from the genome of marine bacterium Wenyingzhuangia aestuarii OF219, and none of the pre-defined glycosidic hydrolase domains were predicted in the protein sequence of Fun174A. Recombinant Fun174A demonstrated a low optimal reaction pH at 5.5. It might degrade sulfated fucans in an endo-processive manner. Glycomics and NMR analyses proved that it specifically hydrolyzed α-1,3-l-fucoside bonds between 2-O-sulfated and non-sulfated fucose residues in the sulfated fucan from sea cucumber Isostichopus badionotus. D119, E120 and E218 were critical for the activity of Fun174A, as identified by site-directed mutagenesis. Three homologs of Fun174A were confirmed to exhibit endo-1,3-fucanase activities. The novelty on sequences of Fun174A and its homologs reveals a new glycoside hydrolase family, GH174.

Structural characterization on a ß-agarase Aga86A_Wa from Wenyingzhuangia aestuarii reveals the prevalent methyl-galactose accommodation capacity of GH86 enzymes at subsite -1.

Agarans are sulfated galactans extracted from red algae with high structural complexity, of which natural methylation often occurs on the O-6 position of its ß-d-galactopyranose units. Although many agaran degrading enzymes, including agarases and porphyranases, have been characterized, little attention has been paid to the tolerance of methyl groups at cleavage subsites. In this study, the structure of GH86 ß-agarase Aga86A_Wa from Wenyingzhuangia aestuarii was determined by X-ray crystallography and investigated from a structural biology perspective. The structure indicated that an accommodation pocket formed by F367, Y280, and Q326 at subsite -1 contributes to the methyl-galactose tolerance of Aga86A_Wa. Furthermore, we found that similar accommodation pockets were present in the structures of two other GH86 enzymes BuGH86 from Bacteroides uniformis and BpGH86A from Phocaeicola plebeius, and their previously undisclosed methyl-galactose tolerance was verified, validating the function of the pockets. Phylogenetic analysis, structural modeling, and hydrolysis product characterization suggested that the methyl-galactose accommodation capacity at subsite -1 was prevalent in GH86 members. These findings achieve a better understanding of the function and mechanism of GH86 agaran degrading enzymes, and will facilitate the precise preparation of agaran oligosaccharides by employing defined tools.

Establishment of a carbohydrate binding module-based lateral flow immunoassay method for identifying hyaluronic acid.

Hyaluronic acid is a commercially important polysaccharide with wide applications. Along with the rapid development of hyaluronic acid-based products, their authenticity has aroused considerable attention from consumers. In the present study, a carbohydrate-binding module (CBM) SrCBM70 was cloned and expressed. The protein could specifically bind to hyaluronic acid with a strong affinity. A novel method for the identification of hyaluronic acid was subsequently established by integrating SrCBM70 into the lateral flow immunoassay (LFIA). Its detection limit for hyaluronic acid was approximately 0.1 µg/mL, and the assay could be completed in 5 min. The feasibility of this method in the authenticity identification of commercialized products containing hyaluronic acid was confirmed. The establishment of the SrCBM70-based LFIA method provided a solution for the on-site authenticity identification and would facilitate the market supervision of hyaluronic acid-based products.

Characterization of a Novel Carrageenan-Specific Carbohydrate-Binding Module: a Promising Tool for the In Situ Investigation of Carrageenan.

Carrageenan is a commercially important polysaccharide widely applied in the food industry. Specific probes are critical tools for the in situ investigation of polysaccharides, whereas the carrageenan-specific probes are limited at present. Carbohydrate-binding modules (CBMs) could serve as specific probes for the in situ investigation of polysaccharides. In the present study, an unknown function module from the κ-carrageenase Cgk16A was cloned and expressed in Escherichia coli. The expressed protein Cgk16A-CBM92 could specifically bind to carrageenan. Its novelty sheds light on a new CBM family (CBM92) as the founding member. Furthermore, a fluorescent probe was successfully constructed by fusing Cgk16A-CBM92 with a green fluorescent protein. The application potential of Cgk16A-CBM92 as a probe served in the in situ visualization of carrageenan was evaluated. The discovery of Cgk16A-CBM92 provided a promising tool for the in situ investigation of carrageenan.

Characterization of a sulfated fucan-specific carbohydrate-binding module: A promising tool for investigating sulfated fucans.

Sulfated fucans are important polysaccharides with diverse biological and biomedical activities. Carbohydrate-binding modules (CBMs) could serve as beneficial tools for the investigation of polysaccharides. Nevertheless, no sulfated fucan-binding CBM has been hitherto discovered. In the present study, a novel CBM47 domain was cloned from the marine bacterium Wenyingzhuangia fucanilytica, and heterologously expressed in Escherichia coli. The expressed protein WfCBM47 exhibited a specific binding capacity to sulfated fucans with the backbone composed of 1,3-α-l-fucopyranose residues. Furthermore, a fluorescent probe was successfully constructed by fusing WfCBM47 with a green fluorescent protein, based on which the in situ visualization of sulfated fucan in the sea cucumber (Apostichopus japonicus) body wall was implemented for the first time. The discovery of WfCBM47 provided a promising tool for future investigations on sulfated fucans.

Expression and characterization of a novel alginate-binding protein: A promising tool for investigating alginate.

Alginate is a commercially important polysaccharide widely applied in various industries. Carbohydrate-binding proteins could be utilized as desirable tools in the investigation and further applications of polysaccharides. Few alginate-binding proteins have hitherto been characterized and reported. In the present study, a novel alginate-binding protein ABP_Wf, consisting of two "orphan" carbohydrate-binding modules, was cloned from a predicted alginate utilization locus of marine bacterium Wenyingzhuangia funcanilytica, and expressed in Escherichia coli. ABP_Wf exhibited a specific binding capacity to alginate, and the association constant (Ka) and affinity (KD) were 1.94 × 103 M-1s-1 and 1.16 × 10-6 M. It was confirmed that the binding capacity of ABP_Wf to alginate is attributed to its constituent CBM16 domain rather than the CBM44 domain. The potentials of ABP_Wf in the semi-quantitative detection and the in situ visualization of alginate were evaluated, which implied that ABP_Wf could be served as a promising tool for investigating alginate.

Discovery and Characterization of an Endo-1,3-Fucanase From Marine Bacterium Wenyingzhuangia fucanilytica: A Novel Glycoside Hydrolase Family.

Sulfated fucans are important marine polysaccharides widely distributed in brown algae and echinoderms, which gained increasing research interest for their various biological and biomedical activities. Fucanases could serve as tools in the bioconversion and structural investigation of sulfated fucans. A few gene-defined endo-1,4-fucanases have been characterized, while the sequence of endo-1,3-fucanase remain unstudied. Here, an endo-1,3-fucanase gene funA was screened from the genome of marine bacterium Wenyingzhuangia fucanilytica CZ1127T using transcriptomics. None of the previously reported glycoside hydrolase domains were predicted in the enzyme FunA, which hydrolyzed sulfated fucans in a random endo-acting manner. Ultrahigh performance size exclusion chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that FunA specifically cleaves α-1,3 glycosidic linkage between 2-O-sulfated and non-sulfated fucose residues. FunA exhibited transglycosylating activity with glycerin, methanol, and L-fucose as acceptors. D206 and E264 were critical for the functioning of FunA as identified by the site-directed mutagenesis. Another five homologs of FunA were confirmed to possess endo-1,3-fucanase activities. This is the first report on the sequence of endo-1,3-fucanase. The novelty of FunA and its homologs in sequences and activity shed light on a novel glycoside hydrolase family, GH168.

Characterization of a Novel Porphyranase Accommodating Methyl-galactoses at Its Subsites.

Porphyran is the major polysaccharide of laver and mainly composed of 3-linked ß-d-galactopyranose (G) and 4-linked α-l-galactopyranose-6-sulfate (L6S) units. Structural heterogeneity of porphyran highly originates from the natural methylation on the O-6 position of G units (GMe). Here, a GH16 porphyranase Por16C_Wf was cloned from a porphyran-related polysaccharide utilization locus of Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It hydrolyzed porphyran in a random endo-acting manner. Using a glycomics strategy combining liquid chromatography-mass spectrometry and glycoinformatics, the subsite specificity was clarified. Por16C_Wf accommodated both G and GMe at subsites -1 and +2. This is the first report on the sequence of porphyranases hydrolyzing consecutive methyl-porphyranobiose moieties, which shed light on the diversity in subsite specificity of porphyranases. Por16C_Wf was the first characterized enzyme in subfamily 14 of the GH16 family. The defined and novel activity of Por16C_Wf implied that it could serve as a favorable tool in the full degradation and structural investigation of porphyran.

Fast coarse-fine locating method for φ-OTDR.

We proposed and demonstrated a coarse-fine method to achieve fast locating of external vibration for the phase-sensitive optical time-domain reflectometer (φ-OTDR) sensing system. Firstly, the acquired backscattered traces from heterodyne coherent φ-OTDR systems are spatially divided into a few segments along a sensing fiber for coarse locating, and most of the acquired data can be excluded by comparing the phase difference between the endpoints in adjacent segments. Secondly, the amplitude-based locating is implemented within the target segments for fine locating. By using the proposed coarse-fine locating method, we have numerically and experimentally investigated a distributed vibration sensor based on the heterodyne coherent φ-OTDR system with a 50-km-long sensing fiber. We find that the computation cost of signal processing for locating is significantly reduced in the long-haul sensing fiber, showing a potential application in real-time locating of external vibration.