Glycosides Research Articles

Two‐Phase Extraction, Structural Characterization of the Polysaccharide from Raspberry Pomace and Its Protection Effects against Erythrocyte Hemolysis

AbstractIn present study, a new polysaccharide (RPP) from raspberry pomace is prepared through ultrasound‐assisted aqueous two‐phase extraction (UAATPE) and purification using two‐step column chromatography, the extraction conditions are optimized by response surface method (RSM). Under the optimal parameters: liquid to material ratio of 80 mL g−1, ultrasonic power of 340 W, and ultrasonic time of 39 min, the yield of the polysaccharides is 11.72 ± 0.05%. RPP with average molecular weight (Mw) of 59 300 Da is composed of six monosaccharides. Nuclear magnetic resonance (NMR) analyses confirmed that RPP contains six main glycosidic linkages including α‐l‐Araf‐(1→, →2)‐α‐l‐Rhap‐(1→, →4)‐β‐d‐Manp‐(1→, →4)‐α‐d‐GalAp‐(1→, →3)‐β‐d‐Galp‐(1→, →4)‐β‐d‐Glcp‐(1→. RPP exhibits remarkable protective effects against H2O2‐induced erythrocyte hemolysis, the generations of reactive oxygen species, and malondialdehyde are inhibited, and the activities of glutathione peroxidase and catalase are effectively enhanced. The current findings proved that UAATPE is an effective method to extract the polysaccharide from raspberry pomace and provides a new idea for the utilization of nutrient‐rich raspberry fruit residue.

Tissue-specific alternative splicing and the functional differentiation of LmLPMO15-1 in Locusta migratoria.

Insect lytic polysaccharide monooxygenases (LPMO15s) are newly discovered copper-dependent enzymes that promote chitin degradation in insect through oxidative cleavage of glycosidic bonds. They are potential pesticide targets due to their critical role for chitin turnover in the integument, trachea, and peritrophic matrix of the midgut during insect molting. However, the knowledge about whether and how LPMO15s participate in chitin turnover in other tissues is still insufficient. Here, using the orthopteran pest Locusta migratoria as a model, a novel alternative splicing site of LmLPMO15-1 was discovered and it produces 2 variants, LmLPMO15-1a and LmLPMO15-1b. The transcripts of LmLPMO15-1a and LmLPMO15-1b were specifically expressed in the trachea and foregut, respectively. RNA interference targeting LmLPMO15-1 (a common fragment shared by both LmLPMO15-1a and LmLPMO15-1b), a specific region of LmLPMO15-1a or LmLPMO15-1b all significantly reduced survival rate of nymphs and induced lethal phenotypes with developmental stasis or molt failure. Ultrastructure analysis demonstrated that LmLPMO15-1b was specifically involved in foregut old cuticle degradation, while LmLPMO15-1a was exclusively responsible for the degradation of the tracheal old cuticle. This study revealed LmLPMO15-1 achieved tissue-specific functional differentiation through alternative splicing, and proved the significance of the spliced variants during insect growth and development. It provides new strategies for pest control targeting LPMO15-1.

The Effects of Agaro-Oligosaccharides Produced by Marine Bacteria (Rheinheimera sp. (HY)) Possessing Agarose-Degrading Enzymes on Myotube Function

Agarase and its metabolites are reported to have applications in a variety of fields, but there have been few studies of the effects of agaro-oligosaccharide hydrolysate on muscle function. In this study, we analyzed the functionality of agarase and its metabolites in bacteria isolated from seawater. A bacterium with agar-degrading activity was isolated from Shimabara, Nagasaki, Japan. Through 16S rRNA sequence alignment, it was identified as being closely related to Rheinheimera sp. WMF-1 and was provisionally named Rheinheimera sp. (HY). Crude enzymes derived from this bacterium demonstrated an ability to hydrolyze various polysaccharides, including agar, agarose, and starch, with the highest specificity observed for agarose. The optimum pH and temperature were pH 10 and 50 °C. A glycoside bond specificity analysis of enzymatic activity indicated the cleavage of the α-linkage. Next, we investigated the functional effects of agaro-oligosaccharides on C2C12 myotubes. Treatment with 10–30 kDa oligosaccharides significantly increased the hypertrophy rate, diameter, and expression of myosin heavy-chain genes in C2C12 myotubes. These results indicate that the agaro-oligosaccharides produced by the enzymes identified in this study improve muscle mass, suggesting their potential contribution to muscle function.

Glycosylium Ions in Superacid Mimic the Transition State of Enzyme Reactions.

The hydrolysis of glycosides is a biochemical transformation that occurs in all living organisms, catalyzed by a broad group of enzymes, including glycoside hydrolases. These enzymes cleave the glycosidic bond via a transition state with substantial oxocarbenium ion character, resulting in short-lived oxocarbenium ion-like species. While such transient species have been inferred through theoretical studies and kinetic isotope effect measurements, their direct spectroscopic characterization remains challenging. In this study, we exploit a superacid environment to generate, accumulate, and fully characterize nonprotected 2-deoxy glycosyl cations in the d-glucopyranose, d-galactopyranose, and l-arabinofuranose series using low-temperature NMR spectroscopy, supported by DFT calculations. Additionally, QM/MM MD simulations reveal that the properties of these glycosyl cations in superacid closely resemble those within the active sites of glycosidase enzymes, particularly in terms of conformation and anomeric charge distribution. These findings highlight a parallel between the stabilizing effect of counterions in superacid media and the network of multidentate noncovalent interactions within glycosidase active sites, which stabilize transition states with pronounced oxocarbenium ion character.

MARTINI Coarse-Grained Force Field for Thermoplastic Starch Nanocomposites.

Thermoplastic starch (TPS) is an excellent film-forming material, and the addition of fillers, such as tetramethylammonium-montmorillonite (TMA-MMT) clay, has significantly expanded its use in packaging applications. We first used an all-atom (AA) simulation to predict several macroscopic (Young's modulus, glass transition temperature, density) and microscopic (conformation along 1-4 and 1-6 glycosidic linkages, composite morphology) properties of TPS melt and TPS-TMA-MMT composite. The interplay of polymer-surface (weakly repulsive), plasticizer-surface (attractive), and polymer-plasticizer (weakly attractive) interactions leads to conformational and dynamics properties distinct from those in systems with either attractive or repulsive polymer-surface interactions. A subset of AA properties was used to parametrize the MARTINI-2 coarse-grained (CG) force field (FF) for the melt and composite systems. The missing bonded parameters of amylose and amylopectin and the bead types for 1-4 and 1-6 linked α-D glucose were determined using two-body excess entropy, density, and bond and angle distributions in the AA TPS melt. This new MARTINI-2 CG model was also compared with the MARTINI-3 model for the TPS melt. However, the requirement of a polarizable water model necessitates the use of MARTINI-2 FF for the composite system. This liquid-liquid partitioning-based FF shows freezing and compaction of polymer chains near the clay surface, further accentuated by lowering of dispersive interactions between pairs of high-covalent-coordination ring units of TPS polymers and the montmorillonite sheet. A rescaling of the effective dispersive component of TPS-MMT cross interactions was used to optimize the MARTINI-2 FF for the composite system with structural (chain size distribution), thermodynamic (chain conformational entropy and density), and dynamic (self-diffusion coefficient) properties obtained from long AA simulations forming the constraints for optimization. The obtained CG FF parameters provided excellent estimates for several other properties of the melt and composite systems not used in parameter estimation, thus establishing the robustness of the developed model.

CAZac: an activity descriptor for carbohydrate-active enzymes.

The Carbohydrate-Active enZYme database (CAZy; www.cazy.org) has been providing the reference classification of carbohydrate-active enzymes (CAZymes) for >30 years. Based on literature survey, the sequence-based families of CAZymes are enriched with functional data by using the International Union of Biochemistry and Molecular Biology Enzyme Commission (EC) number system. However, this system was not developed to search or compare functional information. To better harness functional information, we have developed CAZac (CAZyme activity descriptor), a multicriterion system that describes CAZymes' mechanisms, glycosidic bond orientations, subsites and inter-residue connectivities. This new system, implemented for glycoside hydrolases, glycoside phosphorylases, transglycosidases, polysaccharide lyases and lytic polysaccharide monooxygenases allows complex searches in the CAZy database to uncover the evolution of substrate specificity and mechanisms of CAZymes across families.

Investigating the Substrate Oxidation Mechanism in Lytic Polysaccharide Monooxygenase: H2O2- versus O2-Activation.

Lytic polysaccharide monooxygenases (LPMOs) form a copper-dependent family of enzymes classified under the auxiliary activity (AA) superfamily. The LPMOs are known for their boosting of polysaccharide degradation through oxidation of the glycosidic bonds that link the monosaccharide subunits. This oxidation has been proposed to be dependent on either O2 or H2O2 as cosubstrate. Theoretical investigations have previously supported both mechanisms, although this contrasts with recent experiments. A possible explanation is that the theoretical results critically depend on how the Cu active site is modeled. This has also led to different results even when employing only H2O2 as cosubstrate. In this paper, we investigate both the O2- and H2O2-driven pathways, employing LsAA9 as the underlying LPMO and a theoretical model based on a quantum mechanics/molecular mechanics (QM/MM) framework. We ensure to consistently include all residues known to be important by using extensive QM regions of up to over 900 atoms. We also investigate several conformers that can partly explain the differences seen in previous studies. We find that the O2-driven reaction is unfeasible, in contrast with our previous QM/MM calculations with smaller QM regions. Meanwhile, the H2O2-driven pathway is feasible, showing that for LsAA9, only H2O2 is a viable cosubstrate as proposed experimentally.

Preparation and characterization of Tobacco polysaccharides and its modulation on hyperlipidemia in high-fat-diet-induced mice.

This study aimed to investigate the structural properties of tobacco polysaccharide (TP) and its mechanism of modulating hyperlipidemia in high-fat diet-induced mice. The structural properties of TP were characterized by FT-IR, 1HNMR, SEM, AFM and thermogravimetric analysis. And the regulatory mechanism of TP on lipid metabolism was investigated in hyperlipidemia mice. These results showed that TP had a high composition of reducing monosaccharide and the glycosidic bond type was α-glycosidic bond. The intervention by TP resulted in a significant reduction of body weight and improvement in lipid accumulation. And the modulation mechanism by which TP ameliorated the abnormalities of lipid metabolism was associated with the expression levels of lipid metabolism-related genes and serum exosomes miRNA-128-3p, as well as the modulation of structure and abundance of the gut microbiota in mice. In addition, TP treatment significantly increased the content of short-chain fatty acids (SCFAs) in mice feces. The results of molecular docking and dual-luciferase assay exhibited a good interaction between propionic acid and PPAR-α, and it was hypothesized that the interaction might further ameliorate the hyperlipidemia. Therefore, TP can regulate the expression levels of lipid metabolism-related genes through miRNAs from serum exosomes and SCFAs from gut microbiota.

Auricularia Auricula Polysaccharide-Mediated Green Synthesis of Highly Stable Au NPs

Polysaccharide-functionalized gold nanoparticles (Au NPs) exhibit a promising application in biomedical fields due to their excellent stability and functional properties. The Au NPs from Auricularia auricula polysaccharide (AAP) were successfully synthesized using a straightforward method. By controlling the mass fraction of AAP, pH, reaction temperature, reaction time, and concentration of gold precursor, the highly dispersed spherical AAP-functionalized Au NPs (AAP-Au NPs) were prepared. The Fourier transform infrared spectrometer (FT-IR) and X-ray photoelectron spectroscopy (XPS) indicated that the synthesis mechanism of AAP-Au NPs was as follows: the molecular chain of AAP undergoes a glycosidic bond breakage to expose the reduction terminus in the presence of gold precursor, which reduced Au(III) to Au(0), and itself was oxidized to carboxylate compounds for maintaining the stability of AAP-Au NPs. Additionally, based on the electrostatic interactions and steric forces, as-prepared AAP-Au NPs exhibit excellent stability at various pH (5–11), temperature (25–60 °C), 5 mmol/L glutathione, and 0.1 mol/L Na+ and K+ solutions. Furthermore, AAP-Au NPs retained the ability to scavenge DDPH and ABTS radicals, which is expected to expand the application of Au NPs in biomedical fields.

Molecular Modeling of Glycosylated Catalytic Domain of Human Carbonic Anhydrase IX.

Glycans exhibit significant structural diversity due to the flexibility of glycosidic bonds linking their constituent monosaccharides and the formation of numerous hydrogen bonds. The present work searches a simulated ensemble of glycan chain conformations attached to the catalytic domain of N-glycosylated human carbonic anhydrase IX (HCA IX-c) to identify conformations pointed away or back-folded toward the protein surface guided by different amino acid residues. A series of classical molecular dynamics (MD) simulation studies for a total of 30 μs followed by accelerated MD simulations for a total of 2 μs have been performed using two different force fields to capture varying degrees of fluctuations of both glycan chain and HCA IX. From the underlying free energy profile and kinetics derived using hidden Markov state model, several stable glycan orientations are identified that extend away from the protein surface and convert among each other with rate constants of the order 107-1010 S-1. Most importantly, we have identified a rare glycan conformation which reaches close to a catalytically important amino acid residue, Glu-106. We further enlist the protein residues that couple such less frequent event of the glycan chain back-folding toward the surface of the protein.

Oudemansiella radicata polysaccharides alleviated LPS-induced liver damage via regulating TLR4/NF-κB and Bax/Bcl-2 signaling pathways

The study was aimed to develop natural non-toxic substances to prevent LPS-induced liver damages and its complications. In present work, a pyranose polysaccharide of Oudemansiella radicata polysaccharides (ORP) with typical characteristics of α-type glycosidic linkage was isolated from the O. radicata fruiting body by physico-chemical analysis, and the potential impact against LPS-induced liver damage were performed in mice model. The results demonstrated that ORP showed significant hepatoprotective effects through its potential anti-oxidative, anti-inflammatory and anti-apoptosis activities via regulating the TLR4/NF-κB and Bax/Bcl-2 signaling pathway, independently or synergistically. These findings had established a robust theoretical framework for promoting the comprehensive utilization of ORP as supplements in the development of functional foods or drugs targeting LPS-induced liver damage and its associated complications.

A novel mechanocatalytic process by vibratory disk mill for efficient hydrolysis of cellulose, chitin and xylan

Polysaccharides are primary sources to manufacture chemical products with low carbon footprints, owing to their carbons from atmospheric carbon dioxide fixed through photosynthesis. Breaking down such polysaccharides into water-soluble saccharides through the hydrolysis of glycosidic bonds is one of key steps to produce valuable chemicals; however, previous chemical and enzymatic processes have been suffered from their fundamental drawbacks (e.g. not environmentally benign, high production cost) and are not regarded as feasible approaches. Recently, mechanocatalytic process, which proceeds hydrolysis of polysaccharides by simply milling with solid acids, attracts attentions owing to its cost-effectiveness and environmental beingness. Herein, we propose the novel mechanocatalytic system to efficiently proceed the hydrolysis of glycosidic bonds in polysaccharides by using “vibratory disk mill”, which can produce strong shear and impact force. By milling polysaccharides (cellulose, chitin and xylan) with solid acid kaolin, the polysaccharides can be efficiently hydrolysed to water-soluble saccharides with a rate twice higher than the previous milling devices, achieving quantitative conversion of polysaccharide to water soluble products (solubility: >99 %). We elucidated the reaction mechanism of polysaccharides with kaolin by X-ray diffractograms: forces produced by the vibratory disk mill can efficiently delaminate layered aluminosilicate nanosheets in kaolin, leading to better accessibility of polysaccharides on the acidic surface of the nanosheets. This novel mechanocatalytic system can also be applicable to produce oligosaccharides directly from crude biomass samples (birchwood chips and shrimp shells) without any chemical purification steps, demonstrating that this system can be used in industrial biomass processing.

A polysaccharide from Periplaneta americana promotes macrophage M2 polarization, exhibiting anti-inflammatory and wound-healing activities

A miscellaneous polysaccharide, PAP55–3-1, with a molecular weight of 23.03 kDa, was isolated from Periplaneta americana through extraction with dilute alkali solution, ethanol precipitation, and column chromatography purification. Structural analysis shows that PAP55–3-1 is mainly composed of five monosaccharides: galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose. Its main glycosidic bonds are: Manp-(1→, Galp-(1→, →3)-Galp-(1→, →3,6)-Manp-(1→, →2,6)-Manp-(1→, →6)-Manp-(1→, →4)-Galp-(1→, →6-Glcp-(1→, →6)-Galp-(1→, →2)-Manp-(1 →, →3,4)-Glcp-(1→, →3,6)-Galp-(1→. In vitro experiments demonstrated that PAP55–3-1 can effectively inhibit reactive oxygen species (ROS) and O2− production following H2O2−induction. After H2O2−induction, HIF-1α (hypoxia-inducible factor) was translocated in mitochondria PAP55–3-1 increased localization of HIF-1α was located on mitochondria to maintain the stability of mitochondrial function stability, thereby effectively inhibiting H2O2-induced mitochondrial oxidative damage. Additionally, PAP55–3-1 inhibited the M1 polarization of macrophages stimulated by H2O2 and promoted the phenotype polarization of macrophages from M1 to M2, displaying anti-inflammatory and pro-repair properties. In vivo experimental results indicated that PAP55–3-1 promoted wound healing in mice. Immunohistochemical experiments revealed a reduction in CD68 expression and increase in CD206 expression in both positive and the high-dose polysaccharide group control group. This further demonstrated that PAP55–3-1 promotes the phenotype polarization of macrophages from M1 to M2, exerting anti-inflammatory and wound-healing activities.

Systematic Characteristics of Fucoidan: Intriguing Features for New Pharmacological Interventions.

Fucoidan, a sulfated polysaccharide found primarily in brown algae, is known for exhibiting various biological activities, many of which have been attributed to its sulfate content. However, recent advancements in techniques for analyzing polysaccharide structures have highlighted that not only the sulfate groups but also the composition, molecular weight, and structures of the polysaccharides and their monomers play a crucial role in modulating biological effects. This review comprehensively provides the monosaccharide composition, degree of sulfation, molecular weight distribution, and linkage of glycosidic bonds of fucoidan, focusing on the diversity of its biological activities based on various characteristics. The implications of these findings for future applications and potential therapeutic uses of fucoidan are also discussed.

Chemical Characterization and In Vitro Evaluation of Glucans from Fermentation-Produced Nutraceutical Bionutri-AR1®: Antioxidant and Immunomodulatory Properties

Background: The consumption of nutraceuticals or food supplements has increased crucially, aiming to address nutrient deficits and enhance immune system function. To develop safe food products with unique nutritional and functional benefits, new production methods of these nutraceuticals such as the fermentative process have been gaining prominence for industrial applications. Bionutri-AR1® is a nutraceutical produced via this bioprocess, featuring a complex composition, that has been used to improve the immune systems of debilitated people. Objectives: Considering the various biological properties attributed to glucans, one of its main components, this study aims to structurally characterize and evaluate, in vitro, the antioxidant and immunomodulatory potential of the polymers from this nutraceutical to assess whether these polymers contribute to the product’s reported biological effects. Methods/Results: Unlike previous reports, this study characterized by NMR, GC-MS, and Congo Red assay techniques two main glucans: a water-insoluble linear α-D-glucan with glycosidic bonds (1→4) and a soluble branched (1→3)- and (1→6)-linked β-glucan with a triple helix. Both glucans showed significant antioxidant activity, measured by their capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. They were also capable of inducing the secretion of cytokines such as tumoral necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and interleukin 6 (IL-6), determined through capture enzyme-linked immunosorbent assay (ELISA), especially when co-stimulated with lipopolysaccharide (LPS). Conclusions: This suggests a dual action of these glucans in both proinflammatory and regulatory pathways. Future studies will describe the mechanisms by which these glucans, especially the insoluble ones, enhance immune system function, highlighting their potential use in immunotherapy.

Elimination of Ethanol for the Production of Fucoidans from Brown Seaweeds: Characterization and Bioactivities

Fucoidan, a sulphated polysaccharide from brown seaweed composed of several monosaccharides, has been stated to have several bioactive properties such as antioxidant, antiviral, anticancer, antithrombic, anti-inflammatory, and immunomodulatory effects. This paper provides research findings on green extraction methods, structural analysis of fucoidan, and its associated bioactivities. Fucoidans from brown seaweeds, Fucus vesiculosus and Ascophyllum nodosum, were extracted using green solvents such as citric acid (CA) followed by MWCO (molecular weight cut-off) filtration to obtain high-purity polysaccharides. The presence of functional groups typical to fucoidans, namely, fucose, sulfate, and glycosidic bonds, in the extracts were confirmed through the data obtained from FTIR (Fourier-transform infrared spectroscopy), TGA (thermogravimetric analysis), DSC (differential scanning calorimetry), and solid-state CP–MAS (cross-polarization magic angle spinning) analysis. The MWCO analysis identified that the >300 kDa fraction can have better content of fucoidan (FV-CA 79.16%, FV-HCl 63.59%, AN-CA 79.21%, AN-HCl 80.70%) than the conventional extraction process. Furthermore, the >300 kDa fraction showed significantly higher antioxidant activities compared to crude fucoidan extracts. Crude fucoidan extracts showed significant inhibition of cell viability in human lung (A459 lung carcinoma cells) and colorectal adenocarcinoma (Caco-2) cells at higher concentrations. The fucoidan extracted with green solvents and avoiding alcohol-based precipitation has substantial antioxidant/antitumor action, so, due to this activity, it can be employed as functional foods in food applications.

Structural basis for human OGG1 processing 8-oxodGuo within nucleosome core particles

Base excision repair (BER) is initialized by DNA glycosylases, which recognize and flip damaged bases out of the DNA duplex into the enzymes active site, followed by cleavage of the glycosidic bond. Recent studies have revealed that all types of DNA glycosylases repair base lesions less efficiently within nucleosomes, and their repair activity is highly depended on the lesion’s location within the nucleosome. To reveal the underlying molecular mechanism of this phenomenon, we determine the 3.1 Å cryo-EM structure of human 8-oxoguanine-DNA glycosylase 1 (hOGG1) bound to a nucleosome core particle (NCP) containing a common oxidative base lesion, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Our structural analysis shows that hOGG1 can recognize and flip 8-oxodGuo even within NCPs; however, the interaction between 8-oxodGuo and hOGG1 in a NCP context is weaker than in free DNA due to competition for nucleosomal DNA by the histones. Binding of OGG1 and the flipping of 8-oxodGuo by hOGG1 leads to a partial detachment of DNA from the histone core and a ratchet-like inward movement of nucleosomal DNA. Our findings provide insights into how the dynamic structure of nucleosomes modulate the activity of repair enzymes within chromatin.

Phenolic Content, Antioxidant and Antimicrobial Properties of Hawthorn (Crataegus orientalis) Fruit Extracts Obtained via Carbohydrase-Assisted Extraction

The enzyme-assisted approaches for plant phenolics extraction are more eco-friendly methods compared to acid or alkaline hydrolysis. Carbohydrase enzymes can release free phenolics from plant materials by cleaving the glycosidic bonds between phenolic compounds and cell wall polymers. In this study, the efficiency of carbohydrase-assisted treatment approaches was evaluated to extract bioactive phenolics from hawthorn (Crataegus orientalis) fruit residues. Enzymatic treatment of the fruits was operated by using a crude cellulolytic enzyme cocktail from Rhizomucor miehei NRRL 5282 and a pectinase preparate from Aspergillus niger. Both cellulase and combined cellulase–pectinase treatments improved the total phenolic content (TPC) and antioxidant activity of extracts. The TPC increased to 1899 ± 27 mg gallic acid equivalents/100 g dry matter during the combined enzyme treatment, showing a strong correlation with the average antioxidant capacity determined by ferric-reducing antioxidant power (1.7-fold increment) and 2,2-diphenyl-1-picrylhydrazyl (1.15-fold increment) reagents. The major phenolics in enzyme-treated extracts were vanillic and ferulic acids, the concentrations of which increased 115.6-fold and 93.9-fold, respectively, during carbohydrase treatment. The planktonic growth of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Chromobacterium violaceum was slightly inhibited by the extracts with minimum inhibitory concentration values between 15.0 and 77.9 mg/mL, while the yeasts tested were quite resistant to the samples. B. subtilis and yeast biofilms were sensitive to the enzyme-treated extracts, which also showed quorum-sensing inhibitory effects against C. violaceum. The obtained bioactive hawthorn extracts hold potential as a natural source of antioxidants and antimicrobials.

Structural Characterization and Antioxidant Activity of Exopolysaccharide Produced from Beet Waste Residue by Leuconostoc pseudomesenteroides

Lactic acid bacteria exopolysaccharide (EPS) is a large molecular polymer produced during the growth and metabolism of lactic acid bacteria. EPS has multiple biological functions and is widely used in fields such as food and medicine. However, the low yield and high production cost of EPS derived from lactic acid bacteria limit its widespread application. In this study, we used beet waste residue as a substrate to produce EPS by fermentation with Leuconostoc pseudomesenteroides to improve the utilization rate of agricultural waste and reduce the production cost of lactic acid bacterial EPS. After purification, the molecular weight (Mw) of EPS was determined to be 417 kDa using high-performance size exclusion chromatography (HPSEC). High-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy revealed that the EPS was composed of glucose subunits with α-1,6 glycosidic linkages. The thermal analysis and heavy metal adsorption capacity revealed a relatively high degradation temperature of 315.54 °C and that the material could effectively adsorb Cu2+. Additionally, the findings indicated that the EPS exhibited a significant ability to neutralize free radicals, a property that was found to be concentration dependent. Furthermore, the results of the intracellular study showed the protective effect of freshly isolated EPS on tBHP-induced cellular oxidative stress at a concentration of 50 µg/mL. These results suggest that the EPS from L. pseudomesenteroides may be developed as antioxidant agents for functional food products and pharmaceutical applications due to its capacity to scavenge free radicals.

Crystal structures of Aspergillus oryzae exo-β-(1,3)-glucanase reveal insights into oligosaccharide binding, recognition, and hydrolysis.

Exo-β-(1,3)-glucanases are promising enzymes for use in the biofuel industry as they hydrolyse sugars such as laminarin, a major constituent of the algal cell wall. This study reports structural and biochemical characterizations of Aspergillus oryzae exo-β-(1,3)-glucanase (AoBgl) belonging to the GH5 family. Purified AoBgl hydrolyses β-(1,3)-glycosidic linkages of the oligosaccharide laminaritriose and the polysaccharide laminarin effectively. We have determined three high-resolution structures of AoBgl: (a) the apo form at 1.75 Å, (b) the complexed form with bound cellobiose at 1.73 Å and (c) the glucose-bound form at 1.20 Å. The crystal structures, molecular dynamics simulation studies and site-directed mutagenesis reveal the mode of substrate binding and interactions at the active site. The results also indicate that AoBgl effectively hydrolyses trisaccharides and higher oligosaccharides. The findings from our structural and biochemical studies would aid in rational engineering efforts to generate superior AoBgl variants and similar GH5 enzymes for their industrial use.