Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Binding of Akkermansia muciniphila to mucin is O-glycan specific.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galß1-4GlcNAcß1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production.

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

Metabolism of Inulin via Difructose Anhydride I Pathway in Microbacterium flavum.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by ß-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A ß-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan ß-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Enzymatic construction of a library of even- and odd-numbered heparosan oligosaccharides and their N-sulfonated derivatives.

Low-molecular-weight heparins (LMWHs), especially the specific-sized heparin oligosaccharides, are attractive for the therapeutic applications, while their synthesis remains challenging. In the present study, unsaturated even-numbered heparosan oligosaccharides were firstly prepared by cleaving high-molecular-weight heparosan using recombinant heparinase III (HepIII). The conversion rates of the unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were 33.9 %, 47.9 %, 78.7 %, 71.8 %, and 53.4 %, respectively. After processing the aforementioned heparosan oligosaccharides with the Δ4,5 unsaturated glycuronidase, saturated odd-numbered heparosan trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides were produced. It was observed that among them, the pentasaccharides were the smallest units of saturated odd-numbered oligosaccharides recognized by HepIII. These oligosaccharides were further catalyzed with bifunctional heparan sulfate N-deacetylase/N-sulfotransferase (NDST) under optimized reaction conditions. It was found that the tetrasaccharide was defined as the smallest recognition unit for NDST, obtaining the N-sulfonated heparosan tetrasaccharides, pentasaccharides, and hexasaccharides with a single sulfonate group, as well as N-sulfonated heparosan heptasaccharides, octasaccharides, and nonasaccharides with multiple sulfonate groups. These results provide an easy pathway for constructing a library of specific-sized N-sulfonated heparosan oligosaccharides that can be used as the substrates for the enzymatic synthesis of LMWHs and heparin oligosaccharides, shedding new light on the substrate preference of NDST.

Catalytic properties characterization and degradation mode elucidation of a polyG-specific alginate lyase OUC-FaAly7.

Polymerized guluronates (polyG)-specific alginate lyase with lower polymerized mannuronates (polyM)-degrading activity, superior stability, and clear action mode is a powerful biotechnology tool for the preparation of AOSs rich in M blocks. In this study, we expressed and characterized a polyG-specific alginate lyase OUC-FaAly7 from Formosa agariphila KMM3901. OUC-FaAly7 belonging to polysaccharide lyase (PL) family 7 had highest activity (2743.7 ± 20.3 U/µmol) at 45 °C and pH 6.0. Surprisingly, its specific activity against polyG reached 8560.2 ± 76.7 U/µmol, whereas its polyM-degrading activity was nearly 0 within 10 min reaction. Suggesting that OUC-FaAly7 was a strict polyG-specific alginate lyase. Importantly, OUC-FaAly7 showed a wide range of temperature adaptations and remarkable temperature and pH stability. Its relative activity between 20 °C and 45 °C reached >90 % of the maximum activity. The minimum identifiable substrate of OUC-FaAly7 was guluronate tetrasaccharide (G4). Action process and mode showed that it was a novel alginate lyase digesting guluronate hexaose (G6), guluronate heptaose (G7), and polymerized guluronates, with the preferential generation of unsaturated guluronate pentasaccharide (UG5), although which could be further degraded into unsaturated guluronate disaccharide (UG3) and trisaccharide (UG2). This study contributes to illustrating the catalytic properties, substrate recognition, and action mode of novel polyG-specific alginate lyases.

Innovative application of a novel di-D-fructofuranose 1,2':2,3'-dianhydride hydrolase (DFA-IIIase) from Duffyella gerundensis A4 to burdock root to improve nutrition.

Burdock is native to Europe and Asia and rich in many functional ingredients, including biomacromolecule polysaccharide inulin. The prebiotic fructan inulin can provide energy to organisms via several pathways. One pathway is that inulin fructotransferase (IFTase) first converts inulin to III-type difructose anhydride (DFA-III), which has many beneficial physiological functions. Then, DFA-III is hydrolyzed to inulobiose, which is a Fn-type prebiotic fructo-oligosaccharide, via difructose anhydride hydrolase (DFA-IIIase). However, there has been no study on the application of IFTase or DFA-IIIase to process burdock to increase DFA-III or inulobiose. Moreover, only five DFA-IIIases have been reported to date and all of them are from the Arthrobacter genus. Whether other microbes except for the Arthrobacter genus can utilize DFA-III through DFA-IIIase is unknown. In this work, a DFA-IIIase from Duffyella gerundensis A4 (D. gerundensis A4), abbreviated as DgDFA-IIIase, was identified and characterized in detail. DgDFA-IIIase is a bifunctional enzyme, that is, besides its hydrolytic ability to DFA-III, it has the same catalytic ability as IFTase to inulin. The enzyme was applied to the burdock root aiming at inulin and DFA-III, and inulobiose was produced with an increase in Gn-type fructo-oligosaccharide. The work verifies that microorganisms of the non-Arthrobacter genus also have the potential ability to use DFA-III by DFA-IIIase, and DFA-IIIase is feasible to increase functional substances of burdock root instead of IFTase and endo-inulinase, which paves the way for the production of functional food utilizing the polysaccharide inulin to improve nutrition and health.

Sustainable cellobionic acid biosynthesis from starch via artificial in vitro synthetic enzymatic biosystem.

Cellobionic acid (CBA), a kind of aldobionic acid, offers potential applications in the fields of pharmaceutical, cosmetic, food, and chemical industry. To tackle the high cost of the substrate cellobiose in CBA production using quinoprotein glucose dehydrogenase, this study developed a coenzyme-free and phosphate-balanced in vitro synthetic enzymatic biosystem (ivSEBS) to enable the sustainable CBA synthesis from cost-effective starch in one-pot via the CBA-synthesis module and gluconic acid-supply module. The metabolic fluxes of this artificial biosystem were strengthened using design-build-test-analysis strategy, which involved exquisite pathway design, meticulous enzyme selection, module validation and integration, and optimization of the key enzyme dosage. Under the optimized conditions, a remarkable concentration of 6.2 g/L CBA was achieved from initial 10 g/L maltodextrin with a starch-to-CBA molar conversion rate of 60 %. Taking into account that the biosystem simultaneously accumulated 3.6 g/L of gluconic acid, the maltodextrin utilization rate was calculated to be 93.3 %. Furthermore, a straightforward scaling-up process was performed to evaluate the industrial potential of this enzymatic biosystem, resulting in a yield of 21.2 g/L CBA from 50 g/L maltodextrin. This study presents an artificial ivSEBS for sustainable production of CBA from inexpensive starch, demonstrating an alternative paradigm for biomanufacturing of other aldobionic acids.

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes. METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay. RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05). CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

Physicochemical properties and antimicrobial activities of MytiLec-1, a member from the mytilectin family of mussels.

MytiLec-1, the recombinant form of a mussel lectin from Mytillus galloprovincialis, was purified by affinity chromatography and showed the maximum hemagglutination activity at a temperature range of 10 °C to 40 °C and at pH 7.0 to 9.0. Denaturants like urea and acidic-guanidine inhibited its hemagglutination activity significantly. MytiLec-1 was found to be metal-independent though Ca2+ slightly increased the activity of chelated MytiLec-1. The lectin suppressed 65 % growth of Pseudomonas aeruginosa (ATCC 47085) at 200 µg/ml and reduced the formation of biofilm (15 % at 200 µg/ml). Comparing to Shigella sonnei (ATCC 29930), Shigella boydii (ATCC 231903) and Shigella dysenteriae (ATCC 238135), Bacillus cereus (ATCC 14579) was slightly more sensitive to MytiLec-1. At a concentration of 200 µg/disc and 100 µg/ml, MytiLec-1 prevented the growth of Aspergillus niger and agglutinated the spores of Aspergillus niger and Trichoderma reesei, respectively. Amino acid sequences, physicochemical properties and antimicrobial activities of MytiLec-1 were compared with three other lectins (CGL, MTL and MCL from Crenomytilus grayanus, Mytilus trossulas and Mytilus californianus, respectively) from the mytilectin family of bivalve mollusks. It reconfirms the function of these lectins to recognize pathogens and perform important roles in innate immune response of mussels.

Systematic Engineering of Saccharomyces cerevisiae Chassis for Efficient Flavonoid-7-O-Disaccharide Biosynthesis.

Flavonoids are an essential class of secondary metabolites found in plants and possess various nutritional, medicinal, and agricultural properties. However, the poor water solubility of flavonoid aglycones limits their potential applications. To overcome this issue, glycosylation is a promising approach for improving water solubility and bioavailability. In this study, we constructed a flavonoid-7-O-disaccharide biosynthetic pathway with flavonoid aglycones as substrates in Saccharomyces cerevisiae. Subsequently, through metabolic engineering and promoter strategies, we constructed a UDP-rhamnose regeneration system and optimized the UDP-glucose (UDPG) synthetic pathway. The optimized strain produced up to 131.3 mg/L eriocitrin. After this, the chassis cells were applied to other flavonoids, with substrates such as (2S)-naringenin, (2S)-hesperetin, diosmetin, and (2S)-eriodictyol, which resulted in the synthesis of 179.9 mg/L naringin, 276.6 mg/L hesperidin, 249.0 mg/L neohesperidin, 30.4 mg/L diosmin, and 100.7 mg/L neoeriocitrin. To the best of our knowledge, this is the first report on the biosynthesis of flavonoid-7-O-disaccharide.

Systematic Analysis of Galactinol Synthase and Raffinose Synthase Gene Families in Potato and Their Expression Patterns in Development and Abiotic Stress Responses.

Raffinose family oligosaccharides (RFOs) are very important for plant growth, development, and abiotic stress tolerance. Galactinol synthase (GolS) and raffinose synthase (RFS) are critical enzymes involved in RFO biosynthesis. However, the whole-genome identification and stress responses of their coding genes in potato remain unexplored. In this study, four StGolS and nine StRFS genes were identified and classified into three and five subgroups, respectively. Remarkably, a total of two StGolS and four StRFS genes in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. Subsequent analysis revealed that StGolS4 exhibited significantly high expression levels in transport-related tissues, PEG-6000, and ABA treatments, with remarkable upregulation under salt stress. Additionally, StRFS5 showed similar responses to StGolS4, but StRFS4 and StRFS8 gene expression increased significantly under salt treatment and decreased in PEG-6000 and ABA treatments. Overall, these results lay a foundation for further research on the functional characteristics and molecular mechanisms of these two gene families in response to ABA, salt, and drought stresses, and provide a theoretical foundation and new gene resources for the abiotic-stress-tolerant breeding of potato.

Regional and disease-specific glycosaminoglycan composition and function in decellularized human lung extracellular matrix.

Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor ß to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.

Effect of Marine-Derived Saccharides on Human Skin Fibroblasts and Dermal Papilla Cells.

The skin is the largest organ of the human body, composed of a diverse range of cell types, non-cellular components, and an extracellular matrix. With aging, molecules that are part of the extracellular matrix undergo qualitative and quantitative changes and the effects, such as a loss of skin firmness or wrinkles, can be visible. The changes caused by the aging process do not only affect the surface of the skin, but also extend to skin appendages such as hair follicles. In the present study, the ability of marine-derived saccharides, L-fucose and chondroitin sulphate disaccharide, to support skin and hair health and minimize the effects of intrinsic and extrinsic aging was investigated. The potential of the tested samples to prevent adverse changes in the skin and hair through stimulation of natural processes, cellular proliferation, and production of extracellular matrix components collagen, elastin, or glycosaminoglycans was investigated. The tested compounds, L-fucose and chondroitin sulphate disaccharide, supported skin and hair health, especially in terms of anti-aging effects. The obtained results indicate that both ingredients support and promote the proliferation of dermal fibroblasts and dermal papilla cells, provide cells with a supply of sulphated disaccharide GAG building blocks, increase ECM molecule production (collagen and elastin) by HDFa, and support the growth phase of the hair cycle (anagen).

Trehalose counteracts the dissociation of tetrameric rabbit lactate dehydrogenase induced by acidic pH conditions.

The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K0.5 and Vmax when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of Vmax when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features.

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.

Glycosylated quantum dots as fluorometric nanoprobes for trehalase.

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.

Possible Association Between Anti-Trisulfated-Heparin-Disaccharide (TS-HDS) Immunoglobulin M Autoantibody and Fibromyalgia Syndrome.

BACKGROUND: Fibromyalgia syndrome (FMS) is estimated to affect 2-4% of the general population. While FMS has some known environmental and genetic risk factors, the disorder has no clear etiology. A common coexisting disorder with FMS is small fiber neuropathy (SFN). High levels of serum immunoglobulin M (IgM) binding to trisulfated-heparin-disaccharide (TS-HDS) were recently found to be associated with SFN. OBJECTIVES: To evaluate potential differences in anti-TS-HDS antibody titers in women with FMS compared to healthy controls. METHODS: In this cross-sectional study, we evaluated 51 female participants: 30 with a diagnosis of FMS and 21 healthy controls who had been recruited at the Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Israel. All of the participants were older than 18 years of age. Anti-TS-HDS IgM levels were measured in their sera using the enzyme immunoassay technique. RESULTS: The mean anti-TS-HDS IgM levels were significantly lower in the FMS group, compared with the control group (7.7 ± 5 vs. 13.2 ± 8.6 U/ml, respectively; P = 0.013). CONCLUSIONS: There is a possible association between FMS and anti-TS-HDS IgM. This association might be the missing link for the coexistence of SFN and FMS, but further study should be performed to assess this association and this auto-antibody characteristic.

Functional and structural characterization of Norovirus GII.6 in recognizing histo-blood group antigens.

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 â€‹P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors' saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 â€‹Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.