Glucose Units Research Articles

Characterization of a Thermophilic Monosaccharide Stimulated β-Glucosidase from Acidothermus cellulolyticus

The gene(ABK51908) from Acidothermus cellulolyticus encodes a mature protein of 484 residues with a calculated molecular mass of 53.0 kDa. Sequence analysis revealed that the protein had 59% identity to the β-glucosidases CAA82733, which belongs to glycoside hydrolase family 1(GH1). We cloned and expressed the gene in Escherichia coli BL21-Gold(DE3). The recombinant protein(AcBg) had an optimal pH and temperature of 7.0 and 70 °C, respectively. The specific activities of AcBg under optimal conditions were 290 and 33 U/mg for p-nitrophenyl-β-D-glucopyranoside(pNPG) and cellobiose, respectively. AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis. AcBg showed high salt tolerance and monosaccharide-stimulation properties. Its activity rose more than 2-fold when 5 mol/L NaCl/KCl were added. The activity of the β-glucosidase was remarkably enhanced in the presence of 0.2 mol/L D-glucose(increased more than 1.9-fold), 0.1 mol/L α-methyl-D-glucose(increased more than 1.4-fold) and 1.0 mol/L D-xylose(increased more than 1.9-fold). The catalysis kinetics and structural changes in various concentrations of glucose were determined. The results indicate that glucose reduces substrate affinity and causes conformational changes.

A vibrational study of inulin by means of experimental and theoretical methods

Inulin, a natural polymer formed by several units of fructose and just one unit of glucose, is found in different plants or directly in some fruits or vegetables. Due to its structure it has been used in many applications from medicine, pharmacology or food industry. In spite of this, a complete vibrational analysis of the molecule is missing in the literature. Moreover, there are contradictory results regarding the assignment of certain vibrational modes. Therefore, the aim of this study was to obtain a comprehensive vibrational investigation of inulin by means of experimental (FT-IR and Raman spectroscopy) and theoretical (density functional theory -DFT simulations) methods.

Eudesmane-Type Sesquiterpene Glycosides from Dictamnus dasycarpus Turcz.

Eudesmane-type sesquiterpenes have been reported to exhibit varieties of biological activities. During the process of investigating this kind of natural product from the root bark of Dictamnus dasycarpus Turcz., 13 eudesmane-type sesquiterpene glycosides including six new isolates, named as dictameudesmnosides A1 (1), A2 (2), B (3), C (4), D (5), and E (6), together with seven known ones (7–13), were obtained. Herein, their structures were determined by the analysis of physical data, spectroscopic analysis, and chemical methods. The existence of α-configuration glucose units in their structures (1–5, 8) is not very common in natural glycosidic components. Meanwhile, compounds 3–5, 7, and 9–13 displayed TG accumulation inhibitory effects on HepG2 cells.

Is Starch or Maltodextrin “Glucose?”

On the face of it, starch granules are polymeric structures containing two polysaccharides – amylose and amylopectin with one constituent monomer only – glucose. The number of glucose units bonded together and their bond profile affects the functionality of the polysaccharides. This brief review is written to highlight similarities and differences between the three forms (starch granules, dextrins, and molecular) of glucose. It is designed to question if glucose, maltodextrin, and starch are comparable from a chemical, physical, or nutritional perspective.

Initiation, Elongation, and Termination of Bacterial Cellulose Synthesis.

Cellulose is the major component of the plant cell wall and composed of β-linked glucose units. Use of cellulose is greatly impacted by its physical properties, which are dominated by the number of individual cellulose strand within each fiber and the average length of each strand. Our work described herein provides a complete mechanism for cellulose synthase accounting for its processivity and mechanism of initiation. Using ionic liquids and gel permeation chromatography, we obtain kinetic constants for initiation, elongation, and termination (release of the cellulose strand from the enzyme) for two bacterial cellulose synthases (Gluconacetobacter hansenii and Rhodobacter sphaeroides). Our results show that initiation of synthesis is primer-independent. After initiation, the enzyme undergoes multiple cycles of elongation until the strand is released. The rate of elongation is much faster than that of steady-state turnover. Elongation requires cyclic addition of glucose (from uridine diphosphate-glucose) and then strand translocation by one glucose unit. Translocations greater than one glucose unit result in termination requiring reinitiation. The rate of the strand release, relative to the rate of elongation, determines the processivity of the enzyme. This mechanism and the measured rate constants were supported by kinetic simulation. With the experimentally determined rate constants, we are able to simulate steady-state kinetics and mimic the size distribution of the product. Thus, our results provide for the first time a mechanism for cellulose synthase that accounts for initiation, elongation, and termination.

Structural characterization of the exopolysaccharides from water kefir

Water kefir is a beverage which is produced by initiating fermentation of a fruit extract/sucrose solution with insoluble kefir grains. Exopolysaccharides that are formed from sucrose play a major role in the kefir grain formation, but the exopolysaccharides in the kefir beverage and the detailed structural composition of the whole kefir grains have not been studied yet. Therefore, kefir grains and the corresponding kefir beverage were analyzed for exopolysaccharides by multiple chromatographic approaches and two-dimensional NMR spectroscopy. Furthermore, different fractionation techniques were applied to obtain further information about the exopolysaccharides. The exopolysaccharide-fraction of the investigated kefir beverage was predominantly composed of O3- and O2-branched dextrans as well as lower amounts of levans. The insoluble dextrans from the kefir grains were mostly O3-branched and contained an elevated portion of 1,3-linked glucose units compared to the soluble dextrans. The structurally different exopolysaccharides in water kefir suggest the involvement of multiple bacteria.

Water soluble glucose derivative of thiocarbohydrazone acts as ionophore with cytotoxic effects on tumor cells

A novel water-soluble ionophore based on the thiocarbohydrazone moiety conjugated with glucose (GluTch) was synthesized through a simple two-step procedure. Structural elucidation was carried out in water solution by means of various spectroscopic techniques (NMR, UV–Vis, and CD), electrospray ionization mass spectrometry and density functional theory calculations. The flexible nature of the thiocarbohydrazone moiety of the new glycoderivative compound induced both different coordination motifs and stoichiometry towards copper and zinc. Cytotoxicity assays of the ligands on the human normal keratinocyte NCTC-2544, MDA-MB-231 breast cancer and PC-3 human prostate adenocarcinoma cell lines demonstrated that i) higher activity on cancer cells growth inhibition compared to a normal cell line; ii) the introduction of the glucose unit does not alter the cytotoxic activity of the underivatized ionophore ligand and iii) the presence of copper ion improves the activity of the thiocarbohydrazones.

Stereocontrolled synthesis of oleanolic saponin ladyginoside A isolated from Ladyginia bucharica

Efficient stereocontrolled synthesis of ladyginoside A isolated from Ladyginia bucharica is described. The presented methodology bases on the β-selective glycosylation to construct oleanate-3-O-β-glycoside from selectively protected d-cellobiose comprising desired β-linkage in carbohydrate unit. By using this procedure, dimethyl ester of ladyginoside A (1) (methyl oleanate 3-O-(β-d-glucopyranosyl)-(1 → 4)-β-d-glucuronide methyl ester) was obtained in 16% overall yield. Elaborated synthesis is also demonstrated as useful methodology en route to saponin 2 with additional glucose unit, namely 3-O-[β-d-glucopyranosyl-(1 → 4)-β-d-glucuronide] oleanolic acid 28-O-β-d-glucopyranosyl ester.

Novel Ent-Kaurene Glycosides with Eight Glycosyl Units from Stevia rebaudiana

Several steviol glycosides from Stevia rebaudiana are associated with extremely potent sweetness and are sought-after as low-calorie alternatives to sucrose. With the exception of a few nine sugar glycosides, steviol glycosides described in the scientific literatures contain less than a total of seven glycosyl units. We present here two new steviol glycosides, rebaudioside VIIIa (1) and VIIIb (2) containing eight sugar units isolated from S. rebaudiana Bertoni. The complete structure elucidation of these ent -kaurene diterpene glycosides with eight sugar units was completed using 1D- and 2D-NMR spectroscopy experiments (1H, 13C, 1H-1H COSY, 1H-13C HSQC-DEPT, 1H-13C HMBC, 1D TOCSY, and 1H-1H ROESY) and mass spectral data. Both compounds have four glycosyl units each attached to the C-13 and C-19 positions of aglycone core but vary in the glycosylation pattern and number of glucose and rhamnose units. This report therefore expands the catalog of highly glycosylated components from S. rebaudiana.

Starch-based polyurethane/CuO nanocomposite foam: Antibacterial effects for infection control

In the present study, a new method for the synthesis of the open cell flexible polyurethane foams (PUFs) was developed by using starch powder and the modification of closed cell foam formulation. Starch is the second largest polymeric carbohydrate as a macromolecule on this planet with a large number of glucose units. Copper oxide nanoparticles (CuO NPs) were synthesized by thermal degradation method at different temperatures of 400, 600 and 800 °C as antimicrobial agents. The antimicrobial activity of CuO NPs and commercial CuO powder against the main causes of hospital infections were tested. CuO600 was the most effective antimicrobial agent and enhanced polymer matrix tensile strength with starch powder as new polyurethane foams (PUFs) cell opener with high tensile strength. The effects of parameters on tensile strength were optimized using response surface methodology (RSM). CuO NPs and PUF had optimal conditions and were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). Foam synthesized at the optimal conditions had an open cell structure with high tensile strength and efficient antimicrobial activity that made them suitable to be used as an antimicrobial hospital mattress to control hospital infections.

Bacteria make modified cellulose

Cellulose is an abundant biopolymer made of strands of β-1,4-linked glucose units. It provides mechanical strength to plants and biofilms made by bacteria. A team led by Lynette Cegelski of Stanford University and Regine Hengge of Humboldt University of Berlin now reports that the cellulose made by bacteria is a chemically modified version (Science 2018, DOI: 10.1126/science.aao4096). Using solid-state nuclear magnetic resonance spectroscopy to analyze the structure of the cellulosic material, the researchers determined that it contains a phosphoethanolamine group (shown in blue) attached to the C6 position of a glucose ring in the repeating unit. The modification had not been observed before because standard HCl hydrolysis degrades the modification, leaving only glucose, glucose-6-phosphate, and ethanolamine. The researchers also identified the genes behind the modification. They propose that a protein known as BcsG acts as an enzyme that installs phosphoethanolamine and that two other proteins in the sa...

Biotinylated Cyclooligosaccharides for Paclitaxel Solubilization.

The poor water solubility of paclitaxel causes significant problems in producing cancer therapeutic formulations. Here, we aimed to solubilize paclitaxel using biocompatible cyclic carbohydrates. Generally recognized as safe, labeled β-cyclodextrin (β-CD), a cyclic α-1,4-glucan consisting of seven glucoses, was prepared, and bio-sourced cyclosophoraoses (CyS), which are unbranched cyclic β-1,2-glucans with 17–23 glucose units, were purified using various chromatographic methods from Rhizobium leguminosarum cultural broth. For effective targeting, CyS and β-CD were modified with a biotinyl moiety in a reaction of mono-6-amino CyS and mono-6-amino-β-CD with N-hydroxysuccinimide ester of biotinamidohexanoic acid. Interestingly, the aqueous solubility of paclitaxel was enhanced 10.3- and 3.7-fold in the presence of biotinyl CyS and biotinyl β-CD, respectively. These findings suggest that biotin-appended cyclooligosaccharides can be applied to improve the delivery of paclitaxel.

Syntheses of 1,2,3-triazole-BODIPYs bearing up to three carbohydrate units

BODIPYs bearing up to three glucose units were synthesized and their cytotoxicity and uptake investigated in human HEp2 cells.

Isolation and characterization of flavonoid C-glycosides from Prosopis glandulosa Torr. leaves

Background: Prosopis glandulosa Torr. (Fabaceae) is native to Africa, America, and Asia. In India, it is abundantly found in southern parts. The plant is reported to contain polyphenols, flavonoids, mesquitol-catechin dimers, indolizidine alkaloids, and triterpenes, imparting antitumor, antimicrobial, anti-infective, and anti-parasitic activities. Aim: The present study deals with the isolation and characterization of flavonoid C-glycosides from P. glandulosa Torr. leaves. Materials and Methods: P. glandulosa leaves were subjected to hot aqueous extraction at 90°C—100°C to yield crude aqueous extract (PGA). PGA was fractionated and then subjected to chromatography over Diaion® HP-20 column and semi-preparative high-performance liquid chromatography to provide flavonoid C-glycosides. Results: We have identified and characterized vicenin-2/isomer and schaftoside (s) from P. glandulosa leaves through liquid chromatography-electron spray ionization-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance. LC-MS/MS chromatogram displayed peaks at retention time of 9.69 and 10.37 with m/z 594 and major fragments at m/z 473 [M-H-120]− (loss of glucose unit) for “vicenin-2/isomer.” Interestingly, five major peaks were observed with retention times of 10.75, 11.17, 11.92 (major), 12.92, and 13.32 corresponding to the same mass of m/z 564 with major fragments at m/z 473 [M-H-90]− (loss of arabinose unit) and 443 [M-H-120]− (loss of glucosyl unit), together termed as “schaftosides.” Conclusion: To the best of our knowledge, this is the first report on flavonoid C-glycosides (vicenin-2/isomer and Schaftosides) from P. glandulosa leaves which may act as chemomarker(s) for the standardization and quality control of this plant. Abbreviations used: PGA: Aqueous extract of Prosopis glandulosa leaves; PGM: Methanol fraction of PGA; PGAM: Aqueous-methanolic fraction of PGA; LC-ESI-MS/MS: Liquid chromatography-electron spray ionization-mass spectrometry/mass spectrometry

Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method

The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.

Purification and Characterization of the Total Cellulase Activities (TCA) of Cellulolytic Microorganisms.

Cellulose is the earth's most abundant plant polysaccharide containing a large array of glucose units linked through β (1→4) linkages by existing in both crystalline and amorphous forms. Cellulose is widely distributed in plants, constituting up to 40-50% overall dry weight of the plant biomass. Majorly, microorganisms secrete three types of enzymes such as endoglucanases, exoglucanases, and beta-glucosidase for the hydrolysis of cellulose, contributing to the total cellulase activity. Industrially, the cellulolytic microorganisms are assessed based on their total cellulolytic activities. Similarly, total cellulase activity can also be used for the isolation and characterization of the cellulolytic microorganisms. In this chapter, we have specifically discussed about the methods used for the purification and characterization of the total cellulase activities of the microorganisms such as filter paper assay and cellulase zymogram assay. Our present chapter can be used as primer for characterizing cellulolytic abilities of cellulose-degrading microorganisms.

Towards speciation of organically bound tritium and deuterium: Quantification of non-exchangeable forms in carbohydrate molecules

An original methodology to quantitatively explore exchangeability of hydrogen isotopes in carbohydrate molecules is proposed. To access the speciation of organically bound hydrogen isotopes, isotopic exchanges were carried out under a soft path regime in the vapor phase at 20 °C with set (D,T/H) vapor pressure ratios. When steady states were reached, the fraction of exchangeable hydrogen of microcrystalline cellulose, alpha-cellulose and wheat grains were obtained and ranged from 13 to 31% (versus a theoretical value of 30%). In cellulose, and more specifically in microcrystalline cellulose, the molecular hydrogen bonds as well as the different conformations of the network seemed to decrease the hydroxyl groups of glucose units available for isotopic exchange. On the contrary, the assumed enzymatic hydrolysis of the constitutive molecules of wheat starch into low-molecular weight carbohydrate molecules enhanced the exchangeable pool. An average value of the activity between non-exchangeable organically bound tritium (NE-OBT) and non-exchangeable organically bound hydrogen was calculated for wheat grains, (TH)NE = 0.55 ± 0.03 Bq.g−1 of hydrogen atoms.

Cellulose synthase complex organization and cellulose microfibril structure.

Cellulose consists of linear chains of β-1,4-linked glucose units, which are synthesized by the cellulose synthase complex (CSC). In plants, these chains associate in an ordered manner to form the cellulose microfibrils. Both the CSC and the local environment in which the individual chains coalesce to form the cellulose microfibril determine the structure and the unique physical properties of the microfibril. There are several recent reviews that cover many aspects of cellulose biosynthesis, which include trafficking of the complex to the plasma membrane and the relationship between the movement of the CSC and the underlying cortical microtubules (Bringmann et al. 2012 Trends Plant Sci.17, 666-674 (doi:10.1016/j.tplants.2012.06.003); Kumar & Turner 2015 Phytochemistry112, 91-99 (doi:10.1016/j.phytochem.2014.07.009); Schneider et al. 2016 Curr. Opin. Plant Biol.34, 9-16 (doi:10.1016/j.pbi.2016.07.007)). In this review, we will focus on recent advances in cellulose biosynthesis in plants, with an emphasis on our current understanding of the structure of individual catalytic subunits together with the local membrane environment where cellulose synthesis occurs. We will attempt to relate this information to our current knowledge of the structure of the cellulose microfibril and propose a model in which variations in the structure of the CSC have important implications for the structure of the cellulose microfibril produced.This article is part of a discussion meeting issue 'New horizons for cellulose nanotechnology'.

Biochemical studies of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare and their roles in lignocellulose degradation.

Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris. The oxidization preferences and substrate specificities of these two enzymes were determined. The two LPMOs were shown to cleave different carbohydrate components of plant cell walls. HiLPMO9H was active on cellulose and oxidized the substrate at the C1 carbon of the pyranose ring at β-1,4-glycosidic linkages, whereas HiLPMO9I cleaved cellulose with strict oxidization at the C4 carbon of glucose unit at internal bonds, and also showed activity against glucomannan. We propose that the two LPMOs play different roles in the plant-cell-wall degrading system of H. irregulare for degradation of softwood and that the lignocellulose degradation mediated by this white-rot fungus may require collective efforts from multi-types of LPMOs.

Pyrolysis mechanism of glucose and mannose: The formation of 5-hydroxymethyl furfural and furfural

Fast pyrolysis of biomass will produce various furan derivatives, among which 5-hydroxymethyl furfural (5-HMF) and furfural (FF) are usually the two most important compounds derived from holocellulose. In this study, density functional theory (DFT) calculations are utilized to reveal the formation mechanisms and pathways of 5-HMF and FF from two hexose units of holocellulose, i.e., glucose and mannose. In addition, fast pyrolysis experiments of glucose and mannose are conducted to substantiate the computational results, and the orientation of 5-HMF and FF is determined by 13C-labeled glucoses. Experimental results indicate that C1 provides the aldehyde group in both 5-HMF and FF, and FF is mainly derived from C1 to C5 segment. According to the computational results, glucose and mannose have similar reaction pathways to form 5-HMF and FF with d-fructose (DF) and 3-deoxy-glucosone (3-DG) as the key intermediates. 5-HMF and FF are formed via competing pathways. The formation of 5-HMF is more competitive than that of FF, leading to higher yield of 5-HMF than FF from both hexoses. In addition, compared with glucose, mannose can form 5-HMF and FF via extra pathways because of the epimerization at C2 position. Therefore, mannose pyrolysis results in higher yields of 5-HMF and FF than glucose pyrolysis.