The major hemicellulose of Ulva is a β-(1→4)-linked linear polysaccharide of Xyl and Glc, which the authors term phycoxyloglucan, which has runs of up to three contiguous β-Xyl and three contiguous β-Glc residues, thus differing fundamentally from land-plant xyloglucan.
Abstract
Background
AND
Aims
The chlorophycean seaweed genus Ulva (sea lettuce and relatives) possesses cellulose and cell-wall-matrix polysaccharides operationally classified as 'pectin-like' (hot-water-extractable anionic ulvans and glucuronans) and hemicelluloses. As Ulva is only distantly related to streptophytes (land-plants plus charophytic algae), their wall polysaccharides are likely to diverge significantly. We therefore explored the little-known structure and properties of Ulva hemicellulose.
Methods
Hemicellulose-b was alkali-extracted from hot-oxalate-pretreated U. linza alcohol-insoluble residue, then digested with endo-β-xylanase, Driselase or endo-cellulase, yielding several oligosaccharides. These were separated by size on gel-permeation and preparative paper chromatography. Oligosaccharides were dissected by acid hydrolysis, glycosidase digestion and alkaline peeling; products were analysed by thin-layer chromatography. Purified oligosaccharides were also characterised by NMR spectroscopy.
KEY
Results
Thirteen di- to pentasaccharides were purified. On acid hydrolysis, they yielded only xylose (Xyl) and/or glucose (Glc). Each was digestible by either β-xylosidase or β-glucosidase, identifying the non-reducing terminus as β-Xyl or β-Glc respectively. The rate of alkaline peeling, which attacks progressively from the reducing terminus, indicated (1→4)-linkages; in each case the smallest peeling product (i.e., originally the non-reducing terminus) was either Xyl or Glc, concurring with the glycosidase digestion result. Structures were deduced [β- (1→4)-linked Xyl-Xyl, Xyl-Xyl-Xyl, Xyl-Glc, Xyl-Xyl-Glc, Xyl-Xyl-Xyl-Glc, Glc-Glc, Glc-Xyl-Xyl, Glc-Glc-Xyl-Xyl, and Xyl-Glc-Glc-Xyl-Xyl], and validated by NMR spectroscopy. Unexpectedly, NMR showed one oligosaccharide to be Glc-3-Glc-4-Glc, the (1→3)-bond probably arising artefactually by trans-β-glucanase activity of the cellulase. Ulva hemicellulose hydrogen-bonded to cellulose more rapidly than any tested land-plant polysaccharide.
Conclusions
The major hemicellulose of Ulva is a β-(1→4)-linked linear polysaccharide of Xyl and Glc, which we term phycoxyloglucan. The backbone has runs of up to three contiguous β-Xyl and three contiguous β-Glc residues, thus differing fundamentally from land-plant xyloglucan. Its structure confers high affinity for cellulose, suggesting potential biotechnological applications for this seaweed and contributing to the cell wall's mechanical properties.
The findings demonstrate the considerable potential of S. griseoincarnatus SSPJ4 as a source of accessory lignocellulolytic enzymes for improving biomass saccharification and could be exploited to develop cost-effective and efficient enzymatic formulations for second-generation bioethanol production.
Prakriti Jhilta, Vikram Poria, Arjun Singh et al.· Iranian Journal of Microbiol...· 0 citations
Glucan, a potent immunomodulator with well-established pharmacological activities, can be efficiently produced through the microbial transformation of low-value substrates. Here, the psychrotolerant Antarctic fungus Penicillium chrysogenum MS-02 was cultivated on potato starch. A homogeneous glucan with molecular weight 1.293 × 104 Da (termed PPS2-A) was then isolated from the fermentation broth using gel-filtration and ion-exchange chromatography. Structural characterization revealed that PPS2-A featured a linear backbone composed of (1 → 4)-linked α-d-glucopyranosyl (Glcp) residues, with α-(1 → 4)-linked Glcp side chains at C-6 positions, and lacked a triple-helical conformation. Studies on primary mouse immune cells demonstrated that PPS2-A significantly enhanced splenic lymphocyte proliferation, potentiated peritoneal macrophage phagocytic capacity, and upregulated NO secretion and acid phosphatase activity. Moreover, PPS2-A enhanced proliferation and phagocytosis, suppressed apoptosis, and promoted M1 polarization (elevated NO, cytokines TNF-α/IL-1β/IL-6, and CD86+). Collectively, these findings demonstrate the immunostimulatory potential of PPS2-A as a pro-inflammatory macrophage activator, and support its development as a candidate natural immunomodulator derived from the Antarctic fungus.
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β-Mannanases are endo-acting glycoside hydrolases (GHs) that cleave β-1,4 glycosidic linkages in mannan-rich plant cell wall polysaccharides. They find application in the food and paper industries. Activity-based probes (ABPs) are powerful tools for GH profiling in complex biological samples, yet to date, bespoke ABPs reporting on mannanases have not been reported. Here, we describe the synthesis of cyclophellitol-inspired ABPs based on mannobiose, mannotriose, and glucomannose, and their use in reporting mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides. In addition to mannanases, our ABPs also labelled cellulases in secretomes from both Aspergillus niger and Cellvibrio japonicus, which may indicate broader (“negative-subsite”) substrate specificity in these enzymes. Mechanistic proof of active-site nucleophile labelling by our ABPs was obtained for both AnManA and CjMan26C by X-ray crystallography and for both AnManA and AnMan26A by mass spectrometry. Together, our results establish mannanase-targeted ABPs that may find use alongside existing reagents that report on retaining GHs that process other bulk polysaccharides.
Massimo Tedeschi, Vincent A. J. Lit, Nicholas G. S. McGregor et al.· Chemical Science· 1 citation
Arabinoxylans (AX) are major hemicellulosic polysaccharides of cell walls of cereal grains. Potential health benefits and techno-functional properties of AX are determined, among others, by the extent and distribution of arabinofuranose (Araf) units as substituents along the β-(1 → 4)-linked d-xylopyranose (Xylp) backbone. Conventional carbohydrate analytical methods, such as methylation analysis, are time-consuming and often insufficient to capture structural diversity. Therefore, a semi-quantitative profiling method based on enzymatic AX degradation and on high-performance anion-exchange chromatography with pulsed amperometric and mass spectrometric detection (HPAEC-PAD/MS) was developed. The method relies on enzymatic depolymerization of AX in cereal dietary fiber using endo-β-1,4-xylanases from glycoside hydrolase families 10 and 11, releasing xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS), which were analyzed by HPAEC-PAD/MS. These enzyme-dependent (A)XOS profiles, which do not cover enzymatically resistant AX regions, provide indirect insights into AX structural characteristics. Newly isolated higher substituted AXOS standards were combined with commercially available compounds, resulting in 17 structurally distinct (A)XOS standards for a more detailed characterization of AX substitution patterns with Araf. Furthermore, incubation conditions and chromatographic parameters were optimized, and relative response factors (RRF) of the (A)XOS standards relative to an internal standard were determined, providing a basis for routine semi-quantitative analysis. The applicability of the method was demonstrated using well-characterized cereal soluble and insoluble dietary fiber samples (five different wheat (Triticum) species, rye, and barley), confirming its ability to provide additional structural information on AX. Among others, it was demonstrated that the analyzed wheat species exhibited comparable (A)XOS profiles, being consistent with their close phylogenetic relationship.
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Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
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