Beta-glycosidic Bond Research Articles

Organocatalyzed Stereoselective Glycosylation: An Overview of the Last Decade

Abstract: Even though there has been an important evolution in the synthesis of oligosaccharides, the efficient and stereoselective study of glycosidic bonds through non-toxic, moderate, and inexpensive techniques is one of the most challenging fields in organic synthesis. Glycosyl reactions play a fundamental role in biological material and structure-activity relationships, having numerous medicinal chemistry applications. For this, interesting strategies have evolved over the years to control the stereoselectivity of glycosidic bonds, including the manipulation of different reaction elements, mainly promoters or catalysts, but also involving the nature of donors and solvents. This review looks at glycosylation methodologies in the last decade resulting in the specific formation of alpha or beta glycosidic bonds.

Immune-enhancing effect of water-soluble beta-glucan derived from enzymatic hydrolysis of yeast glucan

Immunostimulants play an important role in the treatment of immunodeficiency. Macrophages are the first line in our immune defense system and play a critical role in the immune response. Therefore, finding new and better substances to induce an immune response by activating macrophages is an attractive research topic, especially in the fields of immunopharmacology and cancer prevention. Keratinocytes actively crosstalk with immune cells during wound repair, so enhancing the function of keratinocytes is also an important part of improving immunity. Beta-glucans are naturally occurring polysaccharides, consisting of d-glucose monomers linked by beta-glycosidic bonds. Several studies have investigated the immunomodulatory effects of beta-glucan, such as its anti-inflammatory and antibacterial properties. However, the use of yeast cell wall glucan has been limited because it is not soluble in water. In this study, we produced low-molecular-weight water-soluble yeast glucan (WSY glucan) and confirmed various aspects of its immune-enhancing effect. The structure of the beta-(1→3) and (1→6) bonds of WSY glucan were confirmed by nuclear magnetic resonance spectroscopy (1H-NMR) analysis. Our results showed that treatment with WSY glucan significantly and dose-dependently induced the production of inflammatory mediators (prostaglandin E2 (PGE2) and nitric oxide (NO)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) in macrophages. In addition, WSY glucan treatment showed changes in the morphological structure of the macrophages and promoted phagocytic activity of the macrophages and wound healing in keratinocytes. Based on these results, WSY glucan is considered as a potential candidate for the treatment of diseases related to the weakening of the immune system without the limitation of insolubility.

Investigation of extraction method effect on yeast beta glucan production

Nowadays, due to the stressful, tiring and busy lives of humans, the immune system becomes weak and can get sick easily. Therefore, scientists have been doing researches about new, natural and healthy products that can strengthen their immune system and can provide to adapt life standards. One of these products is the beta glucan. It is a polysaccharide molecule that consists of D-glucose monomers bonding with beta glycosidic bonds. Beta (β) glucans have being produced from different sources (microorganisms, cereals and mushrooms) so they have different branched structures such as (1→3) (1→6), (1→3) (1→4), (1→3) (1→2). Thus, different branched beta glucans show different physicochemical properties and biological activities that designate their usage purposes. Especially yeast beta glucan has lots of biological activities. On the other hand, it is fact that the extraction method affects the molecular weight, yield, purity and other properties of beta glucans. The main purpose of this study is to compare the performances of ultrasonically assisted alkali-acidic and autolysis extraction methods to produce a high yield of beta glucan. Also, it was aimed to determine the effect of extraction method on the molecular weight of yeast beta glucan. As a result of this work, it was found that the yeast beta glucan yield for ultrasonic supported autolysis extraction (12 %) is higher than that of the ultrasonic supported alkali-acidic extraction (8 %). On the other hand, having the lower molecular weight of yeast beta glucan (87 kDa) was produced by using an ultrasonic supported autolysis extraction method.

Production, immobilization and characterization of beta-glucosidase for application in cellulose degradation from a novel Aspergillus versicolor

Beta-glucosidase (EC 3.2.1.21) catalyzes the hydrolysis of cellobiose and cellooligosaccharides containing (1 → 4)-beta-glycosidic bonds to glucose, which is crucial in cellulosic ethanol production. In this study, Aspergillus versicolor, a novel highly productive beta-glucosidase strain, was first isolated from Camptotheca acuminata seeds. The highest beta-glucosidase activity with 812.86 U/mL was obtained by using the response surface methodology, and a 14.4-fold has increased compared to the control. The beta-glucosidase was then purified to homogeneity with recovery yield and specific activity of 25.98% and 499.15 U/mg, respectively. To enhance its stability and recyclability, the purified beta-glucosidase was first immobilized onto magnetic MnO2 by electrostatic adsorption. The immobilized materials were characterized by FR-IT, TEM and FE-SEM. Compared with the free beta-glucosidase, the immobilized enzyme exhibited enhanced thermal stability (1.5-fold raise in half-life at 50 °C), and reusability (holding over 60% activity after eight cycles), besides, the optimum pH has increased to 6.0. Substrate specificity research suggested that the enzyme had high hydrolytic activity on cellobiose. It also had a hydrolysis effect on (1 → 3) and (1 → 6)-beta-glycosidic linkages. Application trials in cellulose hydrolysis revealed that the immobilized enzyme was comparatively more effective. Our results suggested this novel immobilized beta-glucosidase makes a promising alternative for the cellulosic ethanol production.

Physi-Chemical Property Research of Polysaccharides from Pomegranate Flowers

Pomegranate flowers as row materials were used for extraction of polysaccharides by water-extraction and alcohol-precipitation method. After purification, the physical and chemical properties, structure, monosaccharide composition and molecular weight were studied. The results showed that the polysaccharides from pomegranate flowers mainly contained two kinds of water soluble acidic polysaccharides, and monosaccharide composition were arabinose and galactose, both contained hydroxyl, carboxyl, amino, hydroxyl radical, sulfate, beta glycosidic bond and alpha glycosidic bond structure. The molecular weight of PP1 and PP2 were 6.16 × 104 (±6.6%) and 9.01 × 104 (±3.2%), respectively. The results of this study laid the foundation for further development and application of polysaccharides from pomegranate flowers.

Control of leached beta-glucan levels from depth filters by an improved depth filtration flush strategy.

Beta-glucans are polysaccharides of D-glucose monomers linked by (1-3) beta-glycosidic bonds, are found to have a potential immunogenicity risk in biotherapeutic products, and are labeled as process contaminants. A common source of beta-glucans is from the cellulose found in traditional depth filter media. Typically, beta-glucan impurities that leach into the product from the primary clarification depth filters can be removed by the subsequent bind-and-elute affinity chromatography capture step. Beta-glucans can also be removed by a bind-and-elute cation exchange chromatography step, which is useful for removing beta-glucans introduced by a post-Protein A depth filtration step. However, the increasing prevalence of flowthrough polishing chromatography poses a challenge for beta-glucan removal due to the lack of any bind-and-elute chromatography steps after the post-Protein A depth filter. In this work, a depth filter flush strategy was developed to control beta-glucan leaching into the product pool. Different loading conditions for the depth filtration and subsequent chromatography steps were evaluated to determine the robustness of the optimized flush strategy. Carry through runs demonstrated greater than two-fold reduction in beta-glucan levels using the optimized wash as compared to standard filter flush conditions.

Właściwości prozdrowotne i mechanizmy działania beta-glukanów zbożowych

Beta-glukany to polisacharydy zbudowane z jednostek D-glukozy połączonych wiązaniami beta-glikozydowymi. Związki te są obecne w różnych produktach spożywczych, takich jak zboża, grzyby, drożdże czy wodorosty. Pochodzące ze zbóż beta-glukany są liniowymi polimerami dobrze rozpuszczalnymi w wodzie, które wykazują szereg prozdrowotnych właściwości. W niniejszej pracy dokonano przeglądu najnowszej literatury dotyczącej korzystnego wpływu beta-glukanów obecnych w zbożach na organizm ludzi i zwierząt oraz mechanizmów ich działania. Powszechnie wiadomo, że związki te skutecznie obniżają poziom cholesterolu i zmniejszają glikemię poposiłkową, co umożliwiło dopuszczenie stosowania oświadczeń zdrowotnych na produktach spożywczych zawierających odpowiednie ilości beta-glukanów pochodzących z owsa i/lub jęczmienia. Ponadto jest wiele dowodów wskazujących na silne właściwości immunomodulujące i przeciwzapalne tych związków, które wydają się być szczególnie istotne w terapii coraz powszechniej występujących, nieswoistych zapaleń jelit. Nieliczne badania z ostatnich lat wykazały również działanie prebiotyczne i przeciwnowotworowe tych polisacharydów.

Extraction of yellow pear residue polysaccharides and effects on immune function and antioxidant activity of immunosuppressed mice

Yellow pear residue polysaccharides were extracted via water bath alcohol precipitation, and the extraction conditions were optimized through the response surface method. The yield of polysaccharides maximized to 56.88 mg/g after extraction at 67.45 °C and material-liquid ratio of 1:34.91 for 3.66 h. The crude polysaccharide was further purified to obtain high-purity polysaccharide LPB-C, it was a kind of pyranoid polysaccharide containing a beta glycosidic bond. It consisted of mannose, glucose, rhamnose and xylose and the molar ratio was 3.3:2.3:10.6:23.2. Then the test mice were grouped, modeled and intragastrically administrated and the immune function and antioxidant capacity in immunosuppressed mice were evaluated. The results showed that the administration of yellow pear residue polysaccharides could markedly improve the immune organ index, carbon clearance ability and earlap swelling rate in immunosuppressed mice, significantly increased the secretion of TNF-α, INF-γ, IL-4 and the activities of CAT, SOD, GSH-Px, and decreased MDA levels in liver, kidney and heart. The present study indicated that yellow pear residue polysaccharides might be used as a potential natural immunomodulator and have great development values in the food and medicine fields.

Development of downstream processing to minimize beta‐glucan impurities in GMP‐manufactured therapeutic antibodies

The presence of impurities or contaminants in biological products such as monoclonal antibodies (mAb) could affect efficacy or cause adverse reactions in patients. ICH guidelines (Q6A and Q6B) are in place to regulate the level of impurities within clinical drug products. An impurity less often reported and, therefore, lacking regulatory guideline is beta-glucan. Beta-glucans are polysaccharides of d-glucose monomers linked by (1-3) beta-glycosidic bonds, and are produced by prokaryotic and eukaryotic organisms, including plants. They may enter manufacturing processes via raw materials such as cellulose-based membrane filters or sucrose. Here we report the detection of beta-glucan contamination of a monoclonal IgE antibody (MOv18), manufactured in our facility for a first-in-human, first-in-class clinical trial in patients with cancer. Since beta-glucans have potential immunostimulatory properties and can cause symptomatic infusion reactions, it was of paramount importance to identify the source of beta-glucans in our product and to reduce the levels to clinically insignificant concentrations. We identified beta-glucans in sucrose within the formulation buffer and within the housing storage buffer of the virus removal filter. We also detected low level beta-glucan contamination in two of four commercially available antibodies used in oncology. Both formulation buffers contained sucrose. We managed to reduce levels of beta-glucan in our product 10-fold, by screening all sucrose raw material, filtering the sucrose by Posidyne® membrane filtration, and by incorporating extra wash steps when preparing the virus removal filter. The beta-glucan levels now lie within a range that is unlikely to cause clinically significant immunological effects. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1494-1502, 2016.

Stability of aminooxy glycosides to glycosidase catalysed hydrolysis

The stability of the amino(methoxy) beta-glycosidic bond to glycosidase catalysed hydrolysis is reported. Beta-O-benzyl glucose and beta-O-benzyl galactose are substrates hydrolysed by beta-glucosidase and beta-galactosidase from almonds and Escherichia coli, respectively. However their beta-N-benzyl-(O-methoxy)-glucoside and beta-N-benzyl-(O-methoxy)-galactoside derivatives are competitive inhibitors.

Extracellular β-D-glucosidase of the Penicillium canescens marine fungus

Extracellular beta-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70 degrees C. Glucosidase catalyzed the hydrolysis of beta-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbon-containing compounds.

Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the beta (1-4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc-hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc-hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc-hly may have important potential application as a future safely administered human drug and food additive.

Enhancedin vitroandin vivoantioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi

To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different beta-glucosidases-producing filamentous fungi to enhance their antioxidant activity. Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger, Aspergillus niveus and Aspergillus awamori. The fungi studied produced approximately the same beta-glucosidase activity units amount when p-nitrophenyl-beta-d-glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-beta-d-glucopyranoside as substrate, showed that beta-glucosidase produced by A. niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus. Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H(2)O(2) generated in vivo by the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate. A. niveus synthesized a beta-glucosidase with higher specificity to hydrolyse genistin beta-glycosidic bond than those produced by A. awamori and A. niger. The utilization of these beta-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.

Conserved Cys residue influences catalytic properties of potato endo-(1-->3)-beta-glucanase GLUB20-2.

The synthesis and degradation of (1-->3)-beta-glycosidic bonds between glucose moieties are essential metabolic processes in plant cell architecture and function. We have found that a unique, conserved cysteine residue, positioned outside the catalytic centre of potato endo-(1-->3)-beta-glucanase - product of the gluB20-2 gene, participates in determining the substrate specificity of the enzyme. The same residue is largely responsible for endo-(1-->3)-beta-glucanase inhibition by mercury ions. Our results confirm that the spatial adjustment between an enzyme and its substrate is one of the essential factors contributing to the specificity and accuracy of enzymatic reactions.

Computational analysis of glycoside hydrolase family 1 specificities

Glycoside hydrolase family 1 consists of beta-glucosidases, beta-galactosidases, 6-phospho-beta-galactosidases, myrosinases, and other enzymes having similar primary and tertiary structures but diverse specificities. Among these enzymes, beta-glucosidases hydrolyze cellobiose to glucose, and therefore they are key players in any cellulose to glucose process. All family members attack beta-glycosidic bonds between a pyranosyl glycon and an aglycon, but most have little specificity for the aglycon or for the bond configuration. Furthermore, glycon specificity is not absolute. Sixteen family members (six beta-glucosidases, two cyanogenic beta-glucosidases, one 6-phospho-beta-galactosidase, two myrosinases, and five beta-glycosidases) have known tertiary structures. We have used automated docking to computationally bind disaccharides with allopyranosyl, galactopyranosyl, glucopyranosyl, mannopyranosyl, 6-phosphogalactopyranosyl, and 6-phosphoglucopyranosyl glycons, all linked by beta-(1,2), beta-(1,3), beta-(1,4), and beta-(1,6)-glycosidic bonds to beta-glucopyranoside aglycons, along with beta-(1,1-thio)-allopyranosyl, -galactopyranosyl, -glucopyranosyl, and -mannopyranosyl) beta-glucopyranosides, into all of these structures to investigate the structural determinants of their enzyme specificities. The following are the eight active-site residues: Glu191, Thr194, Phe205, Asn285, Arg336, Asn376, Trp378, and Trp465 (Zea mays beta-glucosidase numbering), that control a significant amount of glycon specificity.

Computational and Experimental Analyses of Furcatin Hydrolase for Substrate Specificity Studies of Disaccharide-specific Glycosidases

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.2.1.161) derived from Viburnum furcatum. Amino acid sequence comparisons and model structure building revealed two residues, Ala419 and Ser504 of FH, as candidates determining the substrate specificity. These residues were specifically conserved in the diglycosidases. The model structure suggested that Ala419 is involved in the aglycone recognition, whereas Ser504 recognizes the external saccharide of the glycone. Mutations at these sites drastically decreased the diglycosidase activity. The mechanism by which the diglycosidases acquired their substrate specificity is discussed, based on these observations.

CDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N‐acetylglucosamine‐binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Anti-tumor and immunomodulating effects of Pleurotus ostreatus mycelia-derived proteoglycans

Pleurotus ostreatus is one of the widely cultivated edible mushrooms. Water-soluble proteoglycan fractions from P. ostreatus mycelia were purified by alcohol-precipitation, ion exchange and followed by gel permeation (Sephadex G-100) chromatography. Three neutral fractions were found, which had polysaccharide to protein ratios 14.2, 26.4 and 18.3, respectively. These fractions were tested for in vitro and in vivo immunomodulatory and anticancer effects on Sarcoma-180-bearing mouse model. In vivo injection of proteoglycans to Sarcoma-180-bearing mice decreased the number of tumor cells and cell cycle analysis showed that most of the cells were found to be arrested in pre-G 0/G 1 phase of cell cycle. All of the three proteoglycans elevated mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide. The Fourier transform infra red (FTIR) spectra suggested the presence of beta-glycosidic bond in all the fractions. Fraction I strongly interacted with glucose/mannose-specific lectin Concanavalin A (ConA), indicating the presence of large number of terminal sugar with glucose/mannose. Thus, the three neutral proteoglycans derived from the mushroom ( P. ostreatus) mycelia could be used as immunomodulators and anti cancer agents.

Furcatin Hydrolase from Viburnum furcatum Blume Is a Novel Disaccharide-specific Acuminosidase in Glycosyl Hydrolase Family 1

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.

The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides.

Bitter taste generally causes aversion, which protects humans from ingesting toxic substances. But bitter flavors also contribute to the palatability of food and beverages, thereby influencing nutritional habits in humans. Although many studies have examined bitter taste, the underlying receptor mechanisms remain poorly understood. Anatomical, functional and genetic data from rodents suggest the existence of a family of receptors that are responsive to bitter compounds. Here we report that a human member of this family, TAS2R16, is present in taste receptor cells on the tongue and is activated by bitter beta-glucopyranosides. Responses to these phytonutrients show a similar concentration dependence and desensitization in transfected cells and in experiments assessing taste perception in humans. Bitter compounds consisting of a hydrophobic residue attached to glucose by a beta-glycosidic bond activate TAS2R16. Thus, TAS2R16 links the recognition of a specific chemical structure to the perception of bitter taste. If the ability of TAS2R16 to detect substances with common molecular properties is typical of the bitter receptor family, it may explain how a few receptors permit the perception of numerous bitter substances.