Arabinose Residues Research Articles

Structural characterization of three acidic polysaccharides from Opuntia dillenii Haw. fruits and their protective effect against hydrogen peroxide-induced oxidative stress in Huh-7 cells

Three novel acidic polysaccharide fractions (OFPP-1, OFPP-2, OFPP-3) with different molecular weights (803.7, 555.1 and 414.5 kDa) were isolated from the peeled Opuntia dillenii Haw. fruits by alkali-extraction, graded alcohol precipitation and column chromatography. Structural analysis indicated that OFPPs were pectic polysaccharides consisting of rhamnose, arabinose and galactose residues. The backbone of OFPP-1 consisted of a repeating unit →6-α-D-GalpA-(1→2)-α-L-Rhap-(1→ with T-α-D-GalpA-(1→6)-α-D-GalpA-(1→4)-α-D-Glcp-(1→, T-β-D-Xylp-(1→6)-α-D-GalpA-(1→4)-α-D-Glcp-(1→ or T-α-D-GalpA-(1→3)-α-L-Araf-(1→ as the side chains. The backbone of OFPP-2 consisted of a disaccharide repeating unit →2)-α-L-Rhap-(1→4)-β-D-GalpA-(1→ with T-β-L-Araf-(1→ as the branches substituted at the O-4 position of →2,4)-α-L-Rhap-(1→. Whereas the backbone of OFPP-3 was →2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-β-L-Araf-(1→ or →2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-β-D-GalpA-(1→, which was branched at the O-4 position of →2,4)-α-L-Rhap-(1→. Moreover, these 3 polysaccharide fractions could protect Huh-7 cells against H2O2-induced oxidative stress to different extents by decreasing the MDA content and increasing the SOD, CAT, GSH-Px activities and the GSH level in the Huh-7 cells. These results suggest that OFPPs have the potential to be used as natural antioxidants.

Antioxidant Active Polysaccharides Extracted with Oxalate from Wild Blackthorn Fruits (Prunus spinosa L.).

Although several therapeutic effects have been attributed to wild blackthorn fruits, their use is still negligible. Purification of the antioxidant-active fraction, obtained from wild blackthorn fruits by hot ammonium oxalate extraction (Ao), yielded seven fractions after successive elution with water, sodium chloride and sodium hydroxide solutions. The purified fractions differ in carbohydrates, proteins, and phenolics. About 60% of the applied Ao material was recovered from the column, with the highest yields eluted with 0.25 M NaCl solution, accounting for up to 70 wt% of all eluted material. Analyses have shown that two dominant fractions (3Fa and 3Fb) contain 72.8-81.1 wt% of galacturonic acids, indicating the prevalence of homogalacturonans (HG) with a low acetyl content and a high degree of esterification. The low content of rhamnose, arabinose and galactose residues in both fractions indicates the presence of RG-I associated with arabinogalactan. In terms of yield, the alkali-eluted fraction was also significant, as a dark brown-coloured material with a yield of ~15 wt% with the highest content of phenolic compounds of all fractions. However, it differs from other fractions in its powdery nature, which indicates a high content of salts that could not be removed by dialysis.

ISOLATION AND STUDY OF POLYSACCHARIDES OF MARGILAN RADISH RAPHANUS SATIVUS

The purpose of this research is to isolate and study the physicochemical characteristics of polysaccharides in the seeds of the Margilan radish of the Brassicaceae family cultivated in the Republic of Uzbekistan.
 For the first time, a water-soluble fraction of polysaccharides was isolated from radish seeds by sowing Margilan successive extraction after the extraction of proteins and polyphenols. Anion exchange chromatography and gel filtration were used to obtain homogeneous polysaccharides. The polysaccharide fraction was separated by ion-exchange chromatography, purified on a Sephadex G-100 column. As a result, two polysaccharides RSP1 and RSP2 were obtained. The monosaccharide composition of the isolated polysaccharides was established. Analysis of monosaccharide residues showed that the composition of the RSP1 polysaccharide is represented by monosaccharides in the following composition: rhamnose - 0.7%, ribose - 4.7%, arabinose - 55.6%, xylose - 1.5%, mannose - 3.7%, glucose - 5.1% and galactose - 28.7%. The composition of the RSP2 polysaccharide is represented by monosaccharides: ribose - 2.8%, arabinose - 9.4%, mannose - 0.9%, glucose - 64.9% and galactose - 18.4%. From the obtained results, it can be seen that the RSP1 polysaccharide consists mainly of arabinose (55.6%) and galactose (28.7%) residues and belongs to the type of arabinogalactans, and the RSP2 polysaccharide mainly consists of glucose residues (64.9%) and galactose (18.4%).

Enzymatic arabinose depletion of wheat arabinoxylan regulates in vitro fermentation profiles and potential microbial degraders

Arabinoxylan (AX) is a major dietary fibre in wheat/rye consisting of a xylan backbone with one or two single arabinose units attached to selected backbone xylose units. Whether these substitution patterns affect AX-degrading gut microbes, and to what extent, is incompletely understood. Herein an in vitro fermentation study is reported where a porcine faecal inoculum (as a model for human large intestine fermentation) was used to investigate the fermentability of wheat AX before and after hydrolysis by enzymes that selectively hydrolyse mono- or di-substituted arabinose residues, or both. Results showed that lower arabinose-to-xylose (A:X) ratios and lower di-substitution levels contributed to slower substrate disappearance, but higher microbial diversity evenness (alpha diversity). AX structure also affected microbial beta diversity, where genera regarded as potential AX degraders differed between the enzymatically-modified AX samples. Interestingly, higher Prevotella relative abundance was associated with higher A:X ratios and faster substrate fermentability, while higher relative abundances of Lachnospiraceae XPB1014 group, Lachnospiraceae MK4A136 group and Lachnospiraceae UCG-009 were linked to more slowly fermentable AX. Gas kinetics and end-product analyses revealed: i) gas production rates but not total gas production depended on both the A:X ratio and substitution patterns; ii) faster fermentation rates led to higher propionate proportions but lower acetate and butyrate proportions; iii) ammonia concentration resulting from different substrates reflected fermentation kinetics. Correlation analyses revealed the potential link between microbial AX degraders and fermentation outcomes specific to AX fine structures. Overall, this study reveals that gut microbial composition and fermentation outcomes can be altered by modifying the fine structure of arabinoxylan.

Crystal structure of the Fusarium oxysporum tannase-like feruloyl esterase FaeC in complex with p-coumaric acid provides insight into ligand binding.

Feruloyl esterases (FAEs) hydrolyze the ester bonds between hydroxycinnamic acids and arabinose residues of plant cell walls and exhibit considerable diversity in terms of substrate specificity. Here, we report the crystal structure of an FAE from Fusarium oxysporum (FoFaeC) at 1.7 Å resolution in complex with p-coumaric acid, which is the first ligand-bound structure of a tannase-like FAE. Our data reveal local conformational changes around the active site upon ligand binding, suggesting alternation between an active and a resting state of the enzyme. A swinging tyrosine residue appears to be gating the substrate binding pocket, while the lid domain of the protein exerts substrate specificity by means of a well-defined hydrophobic core that encases the phenyl moiety of the substrate.

Glucuronic acid: not just another brick in the cell wall.

This article is a Commentary on Debra‐Maceluch et al. (2023), 238: 297–312.

Polysaccharides of Salsola passerina: Extraction, Structural Characterization and Antioxidant Activity.

The above-ground part of the Salsola passerine was found to contain ~13% (w/w) of polysaccharides extractable with water and aqueous solutions of ammonium oxalate and sodium carbonate. The fractions extracted with aqueous sodium carbonate solutions had the highest yield. The polysaccharides of majority fractions are characterized by similar monosaccharide composition; namely, galacturonic acid and arabinose residues are the principal components of their carbohydrate chains. The present study focused on the determination of antioxidant activity of the extracted polysaccharide fractions and elucidation of the structure of polysaccharides using nuclear magnetic resonance (NMR) spectroscopy. Homogalacturonan (HG), consisting of 1,4-linked residues of α-D-galactopyranosyluronic acid (GalpA), rhamnogalacturonan-I (RG-I), which contains a diglycosyl repeating unit with a strictly alternating sequence of 1,4-linked D-GalpA and 1,2-linked L-rhamnopyranose (Rhap) residues in the backbone, and arabinan, were identified as the structural units of the obtained polysaccharides. HMBC spectra showed that arabinan consisted of alternating regions formed by 3,5-substituted and 1,5-linked arabinofuranose residues, but there was no alternation of these residues in the arabinan structure. Polysaccharide fractions scavenged the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 0.2-1.8 mg/mL. The correlation analysis showed that the DPPH scavenging activity of polysaccharide fractions was associated with the content of phenolic compounds (PCs).

Hydration of Wheat Flour Water-Unextractable Cell Wall Material Enables Structural Analysis of Its Arabinoxylan by High-Resolution Solid-State 13C MAS NMR Spectroscopy.

To enable its structural characterization by nuclear magnetic resonance (NMR) spectroscopy, the native structure of cereal water-unextractable arabinoxylan (WU-AX) is typically disrupted by alkali or enzymatic treatments. Here, WU-AX in the wheat flour unextractable cell wall material (UCWM) containing 40.9% ± 1.5 arabinoxylan with an arabinose-to-xylose ratio of 0.62 ± 0.04 was characterized by high-resolution solid-state NMR without disrupting its native structure. Hydration of the UCWM (1.7 mg H2O/mg UCWM) in combination with specific optimizations in the NMR methodology enabled analysis by solid-state 13C NMR with magic angle spinning and 1H high-power decoupling (13C HPDEC MAS NMR) which provided sufficiently high resolution to allow for carbon atom assignments. Spectral resonances of C-1 from arabinose and xylose residues of WU-AX were here assigned to the solid state. The proportions of un-, mono-, and di-substituted xyloses were 59.2, 19.5, and 21.2%, respectively. 13C HPDEC MAS NMR showed the presence of solid-state fractions with different mobilities in the UCWM. This study presents the first solid-state NMR spectrum of wheat WU-AX with sufficient resolution to enable assignment without prior WU-AX solubilization.

Characterisation, rheological properties and immunomodulatory efficiency of corn silk polysaccharides

SummaryCorn silk polysaccharide (CSP) was isolated by heated 80 °C ethanol 70%. The extracted light (83.70 L*) with a yellowish (13.09 b*) reddish (1.98 a*) CSP had an ash content of 2.53 ± 0.03%. It is mainly composed of seven monosaccharides (xylose 23.64, glucose 16.65, ribose 17.35, galactose 13.64, rhamnose 11.93, arabinose 11.87 and mannose 4.85 mol%) and showed four peaks in 13C NMR correlated to the anomeric carbon of arabinose and/or xylose residues. The thermal characteristics exhibited the breakdown of CSP glycosidic bonds at 321.16 °C. The rheological properties showed that the raising of share rate (γ) caused a reduction in apparent viscosity for all tested concentrations, whilst the elastic G′ and viscous G″ storage moduli of CSP were enhanced by increasing the oscillation frequency. The immunomodulatory effects of CSP on RAW264.7 cells showed a significant (P < 0.05) increase in NO secretion up to a concentration of 80 μg mL−1 from CSP. Additionally, the phagocytosis index and acid phosphatase activity were enhanced (P < 0.05) in a dose‐dependent manner by increasing CSP concentrations.

Arabinoxylans: A new class of food ingredients arising from synergies with biorefining, and illustrating the nature of biorefinery engineering

Arabinoxylans (AX) are hemicellulose polysaccharides comprising a linear backbone of xylose sugars with arabinose residues attached along the chain. They offer interesting functional properties that could have application as a new class of food ingredients or for non-food applications. The emergence in recent decades of biorefineries gives a context in which commercial production of a portfolio of AX products could be feasible, through integration with bioethanol production, using ethanol to precipitate the AX. Extending the concept, AX fractions of different functionality can be precipitated at different ethanol concentrations, giving further scope for efficiencies through bioethanol pinch analysis, while producing a portfolio of products with different potential markets and end uses. The current work demonstrates the potential of AX fractions extracted from sugarcane bagasse as bread ingredients. Bagasse AX fractions increased the water absorption in dough formulations, by more than double their own weight for fractions larger than 10 kDa, and increased dough development time in a Chopin Mixolab. As well as promising in their own right, AX also illustrate the more general opportunity for synergies between biorefining and the food industry, with the rise of biorefineries giving opportunities to provide the food industry with new ingredients not currently available.

Chemical characteristics and significant antitussive effect of the Erigeron canadensis polyphenolic polysaccharide-protein complex

Ethnopharmacological relevanceErigeron canadensis has been used in traditional medicine to treat a variety of respiratory diseases, including acute upper and lower respiratory tract infections and cough-related asthma. There is as yet no relevant experimental or clinical study in the scientific literature evaluating the efficacy of plants in these disorders. Aim of the studyTo investigate the active ingredients in Erigeron canadensis, a complex isolated from flowering parts of a plant was tested for airway defense reflexes, in particular for cough reflexes and airway reactivity. Both were experimentally induced by a chemical irritant that simulated the inflammatory conditions of their formation. Material and methodsThe polyphenolic polysaccharide-protein (PPP) complex was isolated from the flowering parts of Erigeron canadensis by hot alkaline extraction and a multi-stage purification process. The antitussive activity was confirmed as a decrease in the number of citric acid-induced coughs and the bronchodilator effect was verified as a decrease in specific airway resistance (sRaw) in conscious guinea pigs. ResultsThe dark brown Erigeron complex with a molecular weight of 38,000 g/mol contained phenolics (13.2% wt%), proteins (16.3% wt%), and uronic acids (6.3% wt%). The neutral carbohydrate part of Erigeron consisted mainly of xylose (12.1 wt%), glucose (13.3 wt%), arabinose (24.1 wt%), and galactose (41.0 wt%) residues. Arabinogalactan and 4-OMe-glucuronoxylan have been found to be the major polysaccharides in the Erigeron complex. Using a method of chemically-induced cough reflex and guinea pigs test system the Erigeron complex exhibited statistically significant, the dose-dependent antitussive activity, which was similar to that of the centrally-acting opioid agonist codeine. ConclusionPharmacological tests have revealed a new pharmacodynamic effect of the Erigeron complex, namely an antitussive effect. Its activity was most pronounced in comparison with all previously tested compounds from other medicinal plants and approached the effect of codeine, the most potent antitussive used in clinical practice. The results provide the scientific basis for the application of this herb in traditional medicine.

ISOLATION AND STUDY OF THE POLYSACCHARIDE COMPLEX ISOLATED FROM AESCULUS HIPPOCASTA-NUM L.

This paper presents the results of a study of the polysaccharide complex, first obtained using microwave radiation from the shells of horse chestnut seeds (Aesculus Hippocastanum L.), collected on the territory of the Tashkent region of the Republic of Uzbekistan. It is shown that the use of microwave waves makes it possible to intensify the method of obtaining polysaccharides with an increase in their yield. The yield of the obtained product reaches 13%, compared with the method of obtaining without using microwave radiation (9.85%), which indicates the prospects of using microwave radiation in the isolation of polysaccharides from this type of raw material. The molecular weight characteristics were determined, and using GC/MS analysis of trimethylsilyl derivatives, the monosaccharide composition of the complex isolated from the shells of the Semyon chestnut by alkaline extraction was established. It was found that the isolated polysaccharides consist mainly of arabinose and galactose residues (65–100%), and also, as minor monosaccharides, up to 35% of glucose, xylose, mannose, rhamnose, ribose residues. Signals and absorption bands corresponding to total polysaccharides were observed in 13C NMR and IR spectra. The NMR analysis data are consistent with the results of the analysis of the monosaccharide composition of the polysaccharide complex obtained using GC / MS. The antiradical activity of the polysaccharide complex has been established.

Antitubercular Potential of Novel Isoxazole Encompassed 1, 2, 4- Triazoles: Design, Synthesis, Molecular Docking Study and Evaluation of Antitubercular Activity

Background: Decaprenylphosphoryl-β-D-ribose epimerase (DprE1), a flavoprotein enzyme engaged in the biosynthesis of decaprenylphosphoryl-β-D-arabinofuranose (DPA), is the only contributor of arabinose residues which is fundamental for the mycobacterium cell wall constituents. DprE1 is an interesting target for antitubercular agent and has been exploring to develop potential chemical entities as antitubercular agents. Objective: The objective of the study is the development of novel antitubercular agents targeting Mtb Decaprenylphosphoryl-β-D-ribose epimerase (DprE1). Methods: A series of isoxazole encompassed 1, 2, 4-triazoles were designed based on the antitubercular potential of triazoles and structural features of DprE1 inhibitors. Designed 1, 2, 4- triazoles were synthesized and characterized by spectral studies. The in vitro anti-TB activity of the compounds was screened against Mycobacterium tuberculosis H37Rv strain by Microplate Almar Blue Assay and in vitro cytotoxicity against normal cell lines by MTT assay. Molecular docking study was carried out on DprE1 enzyme to understand designed compounds interactions with amino acid residues at the active site. Results: Antitubercular activity data revealed that eight compounds (6d, 6e,7d, 7e, 10d, 10e, 11d and 11e) have shown promising antitubercular activity with minimum inhibitory concentration at 1.6μg/mL. Cytotoxicity data of anti-TB active compounds demonstrate good safety profile on normal cell lines. Conclusion: Eight compounds have shown promising antitubercular activity with good safety profile on normal cell lines. Molecular docking study revealed that the synthesized compounds have shown non-covalent interactions with amino acid residues of DprE1 enzyme.

Extraction and characterization of lipids and phenolic compounds from the brans of different wheat varieties

The lipids and phenolic compounds of wheat bran (WB) and hemicellulose B (water extractable Hemi. B and alkali extractable Hemi. B) fractions from four wheat varieties were investigated. The X-ray diffraction pattern and NMR spectra of those Hemi. B were also studied. The predominant lipid components identified in WB include triacylglycerols (41.58–53.24 wt %) and free fatty acids (7.62–13.85 wt %) with the highest content in HD-2967 and C-306 varieties (9.13 and 13.85 wt %, respectively). The WE-Hemi. B had more lipids than the AE-Hemi. B fractions. The HD-3086 variety had the highest lipid content in both its WE-Hemi. B and AE-Hemi. B fractions (6.60 and 3.19 wt %, respectively). The bound phenolic content in WB was higher (1160–2848.88 μg GAE/g) than free phenolic content (564.17–724.72 μg GAE/g). The total antioxidant activity (TAA) of WB varied from 11.76 to 13.01 μmol TE/g. The bound protocatechuic and ferulic acids were the major phenolic acids with the highest content in WB of HS-490 and HD-2967 variety, respectively. The major flavonoid identified in WB was bound catechin (70.98–198.58 μg/g) with the highest content in HD-2967. The ferulic acid was quantified as the major phenolic acid in Hemi. B fractions with a significantly higher level in WE-Hem-B (439.0–627.0 μg/g) compared to AE-Hemi. B (5.7–13.0 μg/g) fractions. The level of syringaldehyde varied from 0.8 to 1.4 μg/g in AE-Hemi. B while p-coumaric acid content in AE-Hemi. B and WE-Hemi. B varied from 0.2 to 0.3 and 5.8–9.7 μg/g, respectively. Sinapic acid and syringaldehyde were detected only in WE-Hemi. B fractions of HD-2967 and C-306 varieties. The X-ray diffractograms of WE-Hemi. B and AE-Hemi. B showed an amorphous profile with a broad peak in the region of 2θ 15–23°. The structural elucidation of Hemi. B by 1H NMR analysis indicated the xylose backbone with substitution mainly at C(O)-3 and/or C(O)-2 positions by arabinose residues and presence of esterified ferulic acid residues attached to arabinose side chains.

AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), AtFUT4, and AtFUT6, are members of the plant-specific GT family 37 (GT37). AtFUT4 and AtFUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. AtFUT4 and AtFUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of AtFUT4 and AtFUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

Phenolic acid bound arabinoxylans extracted from Little and Kodo millets modulate immune system mediators and pathways in RAW 264.7 cells.

The immunomodulating effect of Phenolic acid bound arabinoxylans (PCA-AXs) extracted from Little (PCA-AX-L) and Kodo (PCA-AX-K) millet seeds in RAW 264.7 cells were investigated. The PCA-AXs were extracted from millets and their chemical characterization were carried out by GC-MS, HPLC, and FT-IR. The immunomodulatory effect of PCA-AXs in RAW 264.7 cells were investigated by estimating ROS, NO, and cytokines TNF-α, IL-1β, IL-6, and evaluation of molecular mechanism by q-PCR & western blotting techniques. The xylose: arabinose ratio of PCA-AX-L and PCA-AX-K were 1.48:1.0 and 2.26:1.0, respectively. The phenolic acids content was higher in PCA-AX-K than PCA-AX-L determined by HPLC. FT-IR analysis confirms the presence of α-glucosidic linkage with the degree of substitution of xylan backbone by arabinose residues. The evaluation of immunomodulating effect of PCA-AXs revealed that the PCA-AX-L-treated cells showed higher release of NO, ROS and cytokines than PCA-AX-K-treated cells. The mRNA expressions of TNF-α, iNOS and COX-2 were upregulated by PCA-AX-L and downregulated by PCA-AX-K in dose-dependent manner. Furthermore, in western blotting, the ERK and NF-κB were found to be activated by PCA-AX-L and inhibited by PCA-AX-K. Our findings suggest that the high branched arabinoxylans of PCA-AX-L could modulate the immune response in RAW 264.7 cells through activation of ERK and NF-κB signaling pathways and acts as an immunostimulant. The higher phenolic content in PCA-AX-K could modulate the immune response by downregulation of ERK and NF-κB signaling pathways and thus, it could act as an immunomodulator. PRACTICAL APPLICATIONS: Millets are the richest source of arabinoxylans in which they are known to be bound with phenolic acids (PCA-AX). Arabinoxylans derived from rice and wheat is known immunomodulators. This study was focused to evaluate the immunomodulatory property of PCA-AX derived from two different millets little and kodo. The study results clearly indicated the immune stimulatory action of PCA-AX-L and immunomodulatory action of PCA-AX-K. The explored mechanism indicated that the PCA-AXs modulate NF-κB & ERK pathways for their immunomodulatory action.

Structural characterization, α-amylase and α-glucosidase inhibitory activities of polysaccharides from wheat bran

In this study, two polysaccharide fractions were isolated from wheat bran by sequential extraction with water and alkaline solution, DEAE Cellulose-52 chromatography and Sephacryl S-400 gel permeation chromatography, they were named as WXA-1 and AXA-1, respectively. Structural analyses indicated that both polysaccharide fractions were heteropolysaccharides, their average molecular weights were 193 kDa and 107 kDa, respectively. The backbone of WXA-1 was → 4)-β-d-Xylp-(1→, which was substituted at O-3 positions by arabinose, glucose and galactose residues, while the backbone of AXA-1 was → 4)-β-d-Xylp-(1→, which was mainly substituted at O-3 positions by arabinose. AXA-1 exerted a stronger inhibitory effect on the activities of α-amylase and α-glucosidase compared with WXA-1. Moreover, AXA-1 exhibited a competitive inhibition of α-amylase and a mixed-type noncompetitive inhibition of α-glucosidase. These results suggest that AXA-1 can be used as α-amylase and α-glucosidase inhibitors.

A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria.

In this work, we report the isolation and detailed functional characterization for the new non-ribosomally synthesized antibiotic 5812-A/C, which was derived from metabolites of Streptomyces roseoflavus INA-Ac-5812. According to its chemical structure, the studied 5812-A/C preliminary is composed of a cyclic peptide part covalently bounded with an arabinose residue. N-terminal amino acid sequencing of the native peptide has identified its partial structure of Leu-Asp-Gly-Ser-Gly and consisting of a Tyr residue that is supposed to have a two-component peptide nature for the molecule studied. However, the structural analysis of the antibiotic complex derived from S. roseoflavus INA-Ac-5812 is still ongoing. The mechanism of action of 5812-A/C was assessed in comparison with its most related analog, the lipopeptide antibiotic daptomycin, given the presence in both antimicrobials of an L-kynurenine amino acid residue. The inhibitory activity of 5812-A/C against Gram-positive bacteria including methicillin-resistant strain of Staphylococcus aureus was similar to daptomycin. The mechanism of action of 5812-A/C was associated with the disruption of membrane integrity, which differs in comparison with daptomycin and is most similar to the antimicrobial membrane-disturbing peptides. However, 5812-A/C demonstrated a calcium-dependent mode of action. In addition, unlike daptomycin, 5812-A/C was able to penetrate mature biofilms and inhibit the metabolic activity of embedded S. aureus cells. At the same time, 5812-A/C has no hemolytic activity toward erythrocyte, but possessed weak cytotoxic activity represented by heterochromatin condensation in human buccal epithelium cells. The biological properties of the peptide 5812-A/C suggest its classification as a calcium-dependent antibiotic effective against a wide spectrum of Gram-positive pathogenic bacteria.

Functional characterization of a novel thermophilic exo-arabinanase from Thermothielavioides terrestris

Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization.

Evaluating the Utility of Permethylated Polysaccharide Solution NMR Data for Characterization of Insoluble Plant Cell Wall Polysaccharides.

Plant cell wall polysaccharide analysis encompasses the utilization of a variety of analytical tools, including gas and liquid chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These methods provide complementary data, which enable confident structural proposals of the many complex polysaccharide structures that exist in the complex matrices of plant cell walls. However, cell walls contain fractions of varying solubilities, and a few techniques are available that can analyze all fractions simultaneously. We have discovered that permethylation affords the complete dissolution of both soluble and insoluble polysaccharide fractions of plant cell walls in organic solvents such as chloroform or acetonitrile, which can then be analyzed by a number of analytical techniques including MS and NMR. In this work, NMR structure analysis of 10 permethylated polysaccharide standards was undertaken to generate chemical shift data providing insights into spectral changes that result from permethylation of polysaccharide residues. This information is of especial relevance to the structure analysis of insoluble polysaccharide materials that otherwise are not easily investigated by solution-state NMR methodologies. The preassigned NMR chemical shift data is shown to be vital for NMR structure analysis of minor polysaccharide components of plant cell walls that are particularly difficult to assign by NMR correlation data alone. With the assigned chemical shift data, we analyzed the permethylated samples of destarched, alcohol-insoluble residues of switchgrass and poplar by two-dimensional NMR spectral profiling. Thus, we identified, in addition to the major polysaccharide components, two minor polysaccharides, namely, <5% 3-linked arabinoxylan (switchgrass) and <2% glucomannan (poplar). In particular, the position of the arabinose residue in the arabinoxylan of the switchgrass sample was confidently assigned based on chemical shift values, which are highly sensitive to local chemical environments. Furthermore, the high resolution afforded by the 1H NMR spectra of the permethylated switchgrass and poplar samples allowed facile relative quantitative analysis of their polysaccharide composition, utilizing only a few milligrams of the cell wall material. The concepts herein developed will thus facilitate NMR structure analysis of insoluble plant cell wall polysaccharides, more so of minor cell wall components that are especially challenging to analyze with current methods.