Xyloglucan (Tamarind)

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that break the glycosidic linkage in recalcitrant polysaccharides such as cellulose and chitin. The LPMO LsAA9A (AA9 family lytic polysaccharide monooxygenase A) from the basidiomycete fungus Lentinus similis is biochemically and structurally well characterized, with crystallographic complexes with oligosaccharides having been obtained. Chloride ions from the crystallization solution are known to bind to the LsAA9A-substrate complex in crystals at the copper equatorial coordinating position, where activation of the co-substrate oxygen species is expected. An investigation of the effect of high concentration salts on LsAA9A activity showed that salts containing chloride and other halide anions, except for fluoride, had a clear inhibitory effect on the activity at concentrations > 100 mm, although chloride ions are known to increase the LPMO affinity for oligosaccharide binding. Surprisingly, LsAA9A crystals can be transferred for short times to considerably different chemical environments, allowing crystallographic analysis at reduced chloride concentrations. Unfortunately, these washing steps do not eliminate the chloride binding at the copper equatorial coordinating position. Furthermore, we observed that citrate buffer, also present, bound under these changed chemical conditions at the copper active site. This interaction completely blocks access to the oligosaccharide substrate and is additionally supported here by citrate inhibition of LsAA9A activities against azurine cross-linked hydroxyethylcellulose (AZCL-HEC), tamarind xyloglucan, and cellopentaose. The conclusions from our study indicate that citrate should be absolutely avoided in LPMO research, not only because of possible abstraction of copper ions from the LPMO active site but also because it might directly compete with binding of LPMOs to their target substrates.

Bacteria and fungi are well known for efficient degradation of plant polysaccharides thanks to various enzymes involved in plant cell wall decomposition. However, little is known about the role of archaea in this process or the repertoire and features of their polysaccharide-degrading enzymes. In our previous work, we discovered an archaeal multidomain glycosidase (MDG) composed of three catalytic domains (GH5 and two GH12) and two cellulose-binding modules (CBM2). The recombinant MDG and individual GH5 catalytic domain were active against cellulose and a number of other polysaccharides at a wide range of temperatures, with optimum temperatures (T_opt) of 60°C and 80°C, respectively. The present study was focused on the characterization of two GH12 domains of the MDG. Purified recombinant TMDG_GH12-1 and TMDG_GH12-2 proteins were active as individual enzymes but exhibited distinct catalytic properties. Both enzymes were thermostable and active at extremely high temperatures: TMDG_GH12-1 was active at 40-130°C (T_opt 100°C), and its half-life (t_½) at 100°C was 42 h, which makes it one of the most thermostable glycosidases known so far, whereas TMDG_GH12-2 was active at 50-100°C (T_opt 90°C) with t_½ at 100°C being 30 min. Phylogenetic and structural analysis of both TMDG_GH12 proteins together with molecular docking and site-directed mutagenesis suggested that the presence of two disulfide bridges and the W → Q mutation in the active site contribute to the exceptional thermostability of TMDG_GH12-1. Further structural and mutational studies of the TMDG_GH12-1 domain will help to gain a better understanding of the molecular mechanisms of its extraordinary thermostability and substrate specificity.

Wood-degrading white-rot fungi can efficiently degrade all plant biomass components, but the molecular mechanisms behind the degradation of plant polysaccharides remain poorly understood. For example, the gene sets and expression levels induced by the plant polysaccharide-derived monosaccharides in white-rot fungi do not reflect those induced by crude plant biomass substrates. To explore the molecular response of the white-rot fungus Dichomitus squalens to plant-derived oligo- and polysaccharides, we investigated the transcriptomes from mono- and dikaryotic strains of the fungus on 10 substrates and compared the expression of carbohydrate-active enzyme-encoding genes to that previously reported for different monosaccharides and cellobiose. Our results revealed that in D. squalens, a robust response to cellulose leads to its effective depolymerization, with an orthologue of the ascomycete Trichoderma reesei ACE3 likely acting as a central transcriptional regulator. The conserved response between cellulose and cellobiose further confirms cellobiose as the main cellulase inducer in D. squalens. Surprisingly, despite low abundance of pectin in the natural wood substrate of D. squalens, we identified polygalacturonic acid as a major inducer of a broad-targeted pectinolytic response including pectinase, pectin-related sugar transporter and catabolism genes, and four fungal specific transcription factors. This indicates that D. squalens has not only maintained its ability to degrade minor polysaccharide components in its biotope, but also a regulatory system spanning from extracellular degradation to metabolic conversion. Our study contributes to a deeper understanding of the molecular mechanisms behind white-rot fungal plant polysaccharide degradation and provides leads for functional studies of potential transcriptional regulators in basidiomycetes.

The recombinant 40 kDa BoExXyl43A glycoside hydrolase family 43 (GH43) from bacterium Bacteroides ovatus exhibited highest specific activity (U/mg) against corn cob xylan (136.8), followed by Beechwood xylan (81.1), Carbosynth xylan (69.3), 4-O-D-methylglucuronoxylan (61.4) and Birchwood xylan (59.9). BoExXyl43A demonstrated optimal performance at 37 °C and pH 7.6 with V_max and K_m of 141.8 U/mg and 4.0 mg/mL as well as 64.1 U/mg and 6.0 mg/mL against corn cob and Birchwood xylan, respectively. The activity of BoExXyl43A increased by 48 % by addition of 10 mM Ca²⁺ ions, while 1 mM EDTA or 1 mM EGTA decreased its activity by 100 % or 42.5 %, respectively, highlighting its calcium-ion dependence. Thin-layer chromatography (TLC) analysis of BoExXyl43A hydrolysates of Birchwood and Beechwood xylan as well as that of various xylooligosaccharides (DP2-DP9) from corn cob xylan showed the release of D-xylose, identifying it as an exo-β-1,4-xylosidase/exo-β-1,4-xylanase (EC 3.2.1.-/3.2.1.37). Moreover, the time-dependent TLC analysis of xylobiose hydrolysis showed release of D-xylose units, confirming its β-xylosidase activity. BoExXyl43A also exhibited exo-1,4-β-xylosidase activity on Larchwood and Carbosynth xylans. Notably, it released D-xylose from α-L-Araf²-xylotriose demonstrating its activity against decorated xylooligosaccharides. BoExXyl43A's exo-1,4-β-xylosidase and residual β-xylosidase activity on xylan and xylobiose, respectively, could potentially enhance xylan saccharification efficiency in bioethanol-based refineries. The molecular modeling showed that BoExXyl43A has 5-bladed β-propeller structure with a very shallow active-site having −1, +1 and + 2 subsites, which could accommodate three D-xylose units of longer xylan like xylododecaose thus supporting its exoxylosidase activity.

Bacteroidota species are enriched in the plant microbiome and provide several beneficial functions for their host, including disease suppression. Determining the mechanisms that enable bacteroidota to colonise plant roots may therefore provide opportunities for enhancing crop production through microbiome engineering. By focusing on nutrient acquisition mechanisms, we discovered Bacteroidota species lack high affinity ATP-binding cassette transporters common in other plant-associated bacteria for capturing simple carbon exudates. Instead, bacteroidota possess TonB-dependent transporters predicted to import glycans produced by plant polysaccharide breakdown. Metatranscriptomics (oat rhizosphere) identified several TonB-dependent transporters genes that were highly expressed in Flavobacterium (phylum Bacteroidota). Using Flavobacterium johnsoniae as the model, we experimentally validated the function of one highly expressed TonB-dependent transporter, identifying a conserved Xyloglucan utilisation loci conferring the ability to import and degrade xyloglucan, the major hemicellulose secreted from plant roots. Xyloglucan utilisation loci harbour an endoxyloglucanase related to family 5 subfamily 4 subclade 2D glycoside hydrolases carrying a mutation that we demonstrate is required for full activity towards xyloglucan. Based on analysing 700 soil metagenomes, subclade 2D glycoside hydrolases have radiated in soil and are prevalent among plant-associated bacteroidota and certain taxa affiliated with Gammaproteobacteria. In bacteroidota, particularly Flavobacterium species, xyloglucan utilisation loci organisation was highly conserved, which may increase their competitive ability to utilise xyloglucan. Given bacteroidota lack high-affinity nutrient transporters for simple carbon, instead possessing xyloglucan utilisation loci and similar gene clusters, our data suggests hemicellulose exudates provide them with an important carbon source in the rhizosphere.

Heat moisture treatment (HMT) of starch granules is a successful technique for enhancing rice noodle quality; however, conventional HMT is time-consuming. In this study, efficient saturated-steam HMT (SS-HMT) was employed for gel modification to enhance rice noodle quality. This treatment was performed under saturated steam (produced under atmospheric pressure in a water bath at 100°C) for brief durations (5, 10, 15, and 20 min), and the underlying mechanism was investigated by examining the variation in starch multiscale structures. SS-HMT disrupted the short double helices and single helices and promoted the formation of longer double helices through rearrangement, increasing the network tie-point size and starch thermal stability. High thermal stability reduced starch leaching and minimized damage to the gas cell walls during cooking, resulting in thicker gas cell walls that enhanced the samples' mechanical strength. SS-HMT markedly improved rice noodle quality. Compared with the control group, rice noodles treated with SS-HMT for 10 min exhibited a 56.05% reduction in cooking loss, a 100% decrease in breakage rate, a 48.46 % increase in hardness, and a 24.68% decrease in adhesiveness. This study provides a straightforward and efficient strategy for improving rice noodle quality.

The role of the hydrogels in cell differentiation and proliferation is critical to the development of tissue engineering and regenerative medicine applications. Understanding the specific mechanisms by which hydrogels interact with cells and how they might influence their behaviour could lead to the design of more effective systems. We analysed the proliferation and mRNAs expression of the spheroids from adipose stem cells (SASCs) mixed to partially degalactosylated xyloglucan (dXG) in a generic culture medium without specific stimulus to assess whether the hydrogel is an “active” or “passive” component in a regenerative context. Overall, our results showed that dXG-based niches with the lowest polymer concentration (1 %w), without recourse to biochemical induction can support cell proliferation and higher commitment toward mesenchymal differentiation.

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.