Xyloglucan (Tamarind)

There is growing interest in members of the genus Segatella (family Prevotellaceae) as members of a well-balanced human gut microbiota (HGM). Segatella are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in Segatella species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain Segatella copri DSM 18205 (previously Prevotella copri CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (viz. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences vis-à-vis homologs in the better-studied Bacteroides (sister family Bacteroidaceae), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, S. copri is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of Bacteroides species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic in vitro diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between S. copri and representative HGM Bacteroides species (Bacteroides thetaiotaomicron and Bacteroides ovatus) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.

Few aerobic hyperthermophilic microorganisms degrade polysaccharides. Here, we describe the genome-enabled enrichment and optical tweezer-based isolation of an aerobic polysaccharide-degrading hyperthermophile, Fervidibacter sacchari, previously ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides for growth at 65-87.5 °C and expresses 191 carbohydrate-active enzymes (CAZymes) according to RNA-Seq and proteomics, including 31 with unusual glycoside hydrolase domains (GH109, GH177, GH179). Fluorescence in-situ hybridization and nanoscale secondary ion mass spectrometry confirmed rapid assimilation of ¹³C-starch in spring sediments. Purified GHs were optimally active at 80-100 °C on ten different polysaccharides. Finally, we propose reassigning Fervidibacteria as a class within phylum Armatimonadota, along with 18 other species, and show that a high number and diversity of CAZymes is a hallmark of the phylum, in both aerobic and anaerobic lineages. Our study establishes Fervidibacteria as hyperthermophilic polysaccharide degraders in terrestrial geothermal springs and suggests a broad role for Armatimonadota in polysaccharide catabolism.

Bacterial cellulose (BC) hydrogels exhibit nanofibril porous network with good viscoelasticity for use as food ingredients and medical materials. Xyloglucan (XG), a hemicellulose with branching residues, can hybridize with BC to improve the hydrogel's extensibility. Thus, modifying the molecular structure of XG can fine-tune the viscoelastic properties of BC hydrogels. In this study, tamarind seed XG subjected to ultrasonic and enzymatic treatment was hybridized with BC to form composite materials. The results indicated that incorporating modified XG reduced the modulus and enhanced the viscous behaviour of BC to varying degrees. XG modified via ultrasonic treatment demonstrated a higher binding efficiency (19-22%) with cellulose compared to enzymatically treated XG (11–13%). The enzymatically treated XG improved the maximum elongation ratio to 57%, but reduced the storage modulus to 30 kPa. Although ultrasonic-treated XG had a similar effect on the shear modulus, it had less impact on the extensibility of BC, with an elongation ratio of 38%. Additionally, the incorporation of modified XG also regulated the nonlinear viscoelasticity of BC. These findings advance our understanding of the application of XG as a regulator of mechanical and rheological properties, broadening its utility in BC hydrogel formulations for the food industry and medical material development.

Trametes hirsuta GMA-01 was cultivated in a culture medium supplemented with orange waste, starch, wheat bran, yeast extract, and salts. The fungus produced several holoenzymes, but the laccase levels were surprisingly high. Given the highlighted applicability of laccases in various biotechnological areas with minimal environmental impact, we provided a strategy to increase its production using response surface methodology. The immobilization of laccase into ionic supports (CM-cellulose, DEAE-agarose, DEAE-cellulose, DEAE-Sephacel, MANAE-agarose, MANAE-cellulose, and PEI-agarose) was found to be efficient and recuperative, showcasing the technical prowess of research. The crude extract laccase (CE) and CM-cellulose-immobilized crude extract (ICE) showed optimum activity in acidic conditions (pH 3.0) and at 70°C for the CE and 60°C for the ICE. The ICE significantly increased thermostability at 60°C for the crude extract, which retained 21.6% residual activity after 240 min. The CE and ICE were successfully applied to sugarcane bagasse hydrolysis, showing 13.83 ± 0.02 µmol mL⁻¹ reducing sugars after 48 h. Furthermore, the CE was tested for dye decolorization, achieving 96.6%, 71.9%, and 70.8% decolorization for bromocresol green, bromophenol blue, and orcein, respectively (0.05% (w/v) concentration). The properties and versatility of T. hirsuta GMA-01 laccase in different biotechnological purposes are interesting and notable, opening several potential applications and providing valuable insights into the future of biotechnological development.

The primary plant cell wall (PCW) is a specialized structure composed predominantly of cellulose, hemicelluloses and pectin. While the role of cellulose and hemicelluloses in the formation of the PCW scaffold is undeniable, the mechanisms of how hemicelluloses determine the mechanical properties of PCW remain debatable. Thus, we produced bacterial cellulose–hemicellulose hydrogels as PCW analogues, incorporated with hemicelluloses. Next, we treated samples with hemicellulose degrading enzymes, and explored its structural and mechanical properties. As suggested, difference of hemicelluloses in structure and chemical composition resulted in a variety of the properties studied. By analyzing all the direct and indirect evidences we have found that glucomannan, xyloglucan and arabinoxylan increased the width of cellulose fibers both by hemicellulose surface deposition and fiber entrapment. Arabinoxylan increased stresses and moduli of the hydrogel by its reinforcing effect, while for xylan, increase in mechanical properties was determined by establishment of stiff cellulose–cellulose junctions. In contrast, increasing content of xyloglucan decreased stresses and moduli of hydrogel by its weak interactions with cellulose, while glucomannan altered cellulose network formation via surface deposition, decreasing its strength. The current results provide evidence for structure–dependent mechanisms of cellulose–hemicellulose interactions, suggesting the specific structural role of the latter.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize polysaccharides, leading to their cleavage. LPMOs are classified into eight CAZy families (AA9-11, AA13-17), with the functionality of AA16 being poorly characterized. This study presents biochemical and structural data for an AA16 LPMO (PnAA16) from the marine sponge symbiont Peniophora sp. Phylogenetic analysis revealed that PnAA16 clusters separately from previously characterized AA16s. However, the structural modelling of PnAA16 showed the characteristic immunoglobulin-like fold of LPMOs, with a conserved his-brace motif coordinating a copper ion. The copper-bound PnAA16 showed greater thermal stability than its apo-form, highlighting copper's role in enzyme stability. Functionally, PnAA16 demonstrated oxidase activity, producing 5 μM H₂O₂ after 30 min, but showed 20 times lower peroxidase activity (0.27 U/g) compared to a fungal AA9. Specific activity assays indicated that PnAA16 acts only on cellohexaose, generating native celloligosaccharides (C3 to C5) and oxidized products with regioselective oxidation at C1 and C4 positions. Finally, PnAA16 boosted the activity of a cellulolytic cocktail for cellulose saccharification in the presence of ascorbic acid, hydrogen peroxide, or both. In conclusion, the present work provides insights into the AA16 family, expanding the understanding of their structural and functional relationships and biotechnological potential.

The therapeutic capabilities of autologous stem cells can be fully exploited if their survival after implantation is improved. For the first time, we compared three hydrogels, with different chemical structure, morphology, and viscoelastic properties, where the same differentiation factors were immobilized and spheroids from adipose stem cells (SASCs) were incorporated. The aim is to understand if hydrogel characteristics could influence the viability of the embedded stem cells. Specifically, hydrogels of partially degalactosylated xyloglucan (dXG), sodium alginate (Alg) and k-carrageenan (kC) were produced. The structure of the networks was probed by swelling/erosion measurements, rheological and morphological analysis. Cell viability was measured after 7 and 21 days. When SASCs were incubated under stemness conditions, dXG and kC hydrogels provide the optimal environment for cell viability. When incubated in the chondrogenic or osteogenic medium, a clear correlation was found between the storage and loss moduli and cell viability. Hydrogels with the lowest shear stiffness promote stem-cell differentiation and proliferation. The systems, particularly dXG, seem more similar to natural ECM and able to recreate niches, that colonized with stem cells could represent a real support in regenerative therapies. The injectability of formulations was evaluated to determine if they could be used for minimally invasive regenerative medicine interventions.

Dietary oligo- and polysaccharides modulate gut microbiota and thus exert prebiotic activity, which is determined by their heterogeneous structure. To explore the correlations between monosaccharide profile and microbial community, simulated gut fermentation of different glycans, including arabinan (ArB), galactooligosaccharide (GOS), arabinogalactan (ArG), rhamnogalacturonan (RhG), and xyloglucan (XyG) that are characterized by typical sugar residues were performed. Results showed that RhG displayed high contents of galacturonic acid (344.79 mg/g), rhamnose (171.70 mg/g), and galactose (151.77 mg/g), and the degradation ratio of them after fermentation was 73.87 %, 84.96 %, and 87.11 %, respectively. Meanwhile, the relative abundance of glycan-degrading bacteria Bacteroides in the RhG was boosted from 4 h (4.97 %) to 48 h (36.45%). Butyrate-generating bacteria Megasphaera (56.69 %) and Bifidobacterium (28.02 %) are dominant genera in the ArB, which generated the highest concentration of carbohydrate-metabolite (94.58 mmol/L) in terms of acetate, propionate, butyrate and valerate, followed by the ArG (87.36 mmol/L). However, ammonia generation of the ArG increased rapidly, representing the highest content of protein-metabolite (66.36 mmol/L) including ammonia, isobutyrate, and isovalerate. As compared, metabolites generated from protein and carbohydrates grow steadily at a low level during the XyG fermentation. Correlation analysis further indicated that Bacteroides was positively correlated with propionate (p < 0.001), galacturonic acid (p < 0.001), and rhamnose (p < 0.05), while Bifidobacterium has positive correlation with butyrate and arabinose (p < 0.01). Overall, monosaccharides composition in the different oligo- and polysaccharides induces distinct responses of the dominant microbiota and thus modulates the subsequent fermentation metabolites of carbohydrate and protein, promoting a deep understanding of the structure-fermentation relationship of dietary glycans.

Xyloglucan oligosaccharides (XyGOs) are highly branched, complex carbohydrates with a variety of chemical and biotechnological applications. Due to the regular repeating pattern of sidechain substitution of the xyloglucan backbone, well-defined XyGOs are readily accessed for analytical and preparative purposes by specific hydrolysis of the polysaccharide with endo-glucanases. To broaden the application potential of XyGOs, we present here an optimized, scalable method to access large quantities of galactosylated XyGOs by treatment of the bulk agricultural by-product, tamarind kernel powder (TKP), with a highly specific endo-xyloglucanase at high-solids content. Subsequent β-galactosidase treatment reduced XyGO complexity to produce exclusively the branched heptasaccharide XXXG (Xyl₃Glc₄: [α-D-Xylp-(1 → 6)]-β-D-Glcp-(1 → 4)-[α-D-Xylp-(1 → 6)]-β-D-Glcp-(1 → 4)-[α-D-Xylp-(1 → 6)]-β-D-Glcp-(1 → 4)-D-Glcp). The challenge of removing the co-product galactose was overcome by fermentation with baker’s yeast, thereby avoiding chromatography and other fractionation steps to yield highly pure XXXG. This simplified approach employs many of the core concepts of green chemistry and engineering, enables facile production of 100 g quantities of XyGOs and XXXG for laboratory use, and serves as a guide to further production scale-up for applications, including as prebiotics, plant growth effectors and elicitors, and building blocks for glycoconjugate synthesis.

Cellulose is a major renewable resource for a wide variety of sustainable industrial products. However, for its utilization, finding new efficient enzymes for plant cell wall depolymerization is crucial. In addition to microbial sources, cellulases also exist in plants, however, are less studied. Fleshy fruit ripening includes enzymatic cell wall hydrolysis, leading to tissue softening. Therefore, bilberry (Vaccinium myrtillus L.), which produces small fruits that undergo extensive and rapid softening, was selected to explore cellulases of plant origin. We identified 20 glycoside hydrolase family 9 (GH9) cellulases from a recently sequenced bilberry genome, including four of which showed fruit ripening-specific expression and could be associated with fruit softening based on phylogenetic, transcriptomic and gene expression analyses. These four cellulases were secreted enzymes: two B-types and two C-types with a carbohydrate binding module 49. For functional characterization, these four cellulases were expressed in Pichia pastoris. All recombinant enzymes demonstrated glucanase activity toward cellulose and hemicellulose substrates. Particularly, VmGH9C1 demonstrated high activity and ability to degrade cellulose, xyloglucan, and glucomannan. In addition, all the enzymes retained activity under wide pH (6-10) and temperature ranges (optimum 70°C), revealing the potential applications of plant GH9 cellulases in the industrial bioprocessing of lignocellulose.