Search Results (10)

Water-soluble cellulosic oligomers (WCOs) are increasingly recognized for their prebiotic benefits, but their efficient enzymatic production is hindered by the high crystallinity of cellulose, which limits enzyme accessibility. This study introduced an efficient and scalable strategy combining phosphoric acid pretreatment with enzymatic hydrolysis to produce high-purity WCOs. Microcrystalline cellulose treated with 85 wt% phosphoric acid at 10 °C exhibited significantly reduced crystallinity and crystallite size, improving its susceptibility to enzymatic degradation. Subsequent hydrolysis of the hydrated regenerated cellulose (HRC85-10) using Celluclast^® at pH 7.0 for 1 h resulted in a WCO selectivity of 93.5%, with cellobiose and cellotriose identified as major oligomeric products via electrospray ionization mass spectrometry. Maintaining cellulose in a hydrated form significantly improved both the yield and selectivity of WCOs. In vivo studies further confirmed the prebiotic potential, with a significant increase in fecal Lactobacillus spp. and Bifidobacterium spp. (p < 0.05) following WCO supplementation. These findings demonstrated a practical and effective approach for producing functional WCOs for use in dietary and gut health applications. Full article

In this study, we report the molecular and enzymatic characterisation of Spg103, a novel bifunctional β-glucanase from the marine bacterium Streptomyces sp. J103. Recombinant Spg103 (rSpg103) functioned optimally at 60 °C and pH 6. Notably, Spg103 exhibited distinct stability properties, with increased activity in the presence of Na+ and EDTA. Spg103 displays both lichenase and cellobiohydrolase activity. Despite possessing a GH5 cellulase domain, FN3 and CBM3 domains characteristic of cellulases and CBHs, biochemical assays showed that rSpg103 exhibited higher activity towards mixed β-1,3-1,4-glucan such as barley β-glucan and lichenan than towards beta-1,4-linkages. The endolytic activity of the enzyme was confirmed by TLC and UPLC-MS analyses, which identified cellotriose as the main hydrolysis product. In addition, Spg103 exhibited an exo-type activity, selectively releasing cellobiose units from cellooligosaccharides, which is characteristic of cellobiohydrolases. These results demonstrate the potential of Spg103 for a variety of biotechnological applications, particularly those requiring tailor-made enzymatic degradation of mixed-linked β-glucans. This study provides a basis for further structural and functional investigations of the bifunctional enzyme and highlights Spg103 as a promising candidate for industrial applications. Full article

The degradation of cellulose generates cellooligomers, which function as damage-associated molecular patterns and activate immune and cell wall repair responses via the CELLOOLIGOMER RECEPTOR KINASE1 (CORK1). The most active cellooligomer for the induction of downstream responses is cellotriose, while cellobiose is around 100 times less effective. These short-chain cellooligomers are also metabolized after uptake into the cells. In this study, we demonstrate that CORK1 is mainly expressed in the vascular tissue of the upper, fully developed part of the roots. Cellooligomer/CORK1-induced responses interfere with chitin-triggered immune responses and are influenced by BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE1 and the receptor kinase FERONIA. The pathway also controls sugar transporter and metabolism genes and the phosphorylation state of these proteins. Furthermore, cellotriose-induced ROS production and WRKY30/40 expression are controlled by the sugar transporters SUCROSE-PROTON SYMPORTER1, SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER11 (SWEET11), and SWEET12. Our data demonstrate that cellooligomer/CORK1 signaling is integrated into the pattern recognition receptor network and coupled to the primary sugar metabolism in Arabidopsis roots. Full article

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182–7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin’s phosphorolysis. Full article

Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-β-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-β-1,4-glucanase gene (CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6–9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley β-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G₄) and cellppentaose (G₅) to cellobiose (G₂) and cellotriose (G₃). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications. Full article

Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new β-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent T_m value was 48.5 °C. The percentages of α-helix (36.3%) and β-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50–250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC₅₀ = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg⁻¹), pNPG (340.5 ± 18.6 U mg⁻¹), cellobiose (89.3 ± 5.1 U mg⁻¹), and lactose (45.1 ± 0.5 U mg⁻¹), so it had broad specificity. The V_max values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg⁻¹ toward p-nitrophenyl-β-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast^® 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L⁻¹) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus cellulases already characterized by our research group—CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification. Full article

Plant colonization by Streptomyces scabiei, the main cause of common scab disease on root and tuber crops, is triggered by cello-oligosaccharides, cellotriose being the most efficient elicitor. The import of cello-oligosaccharides via the ATP-binding cassette (ABC) transporter CebEFG-MsiK induces the production of thaxtomin phytotoxins, the central virulence determinants of this species, as well as many other metabolites that compose the ‘virulome’ of S. scabiei. Homology searches revealed paralogues of the CebEFG proteins, encoded by the cebEFG2 cluster, while another ABC-type transporter, PitEFG, is encoded on the pathogenicity island (PAI). We investigated the gene expression of these candidate alternative elicitor importers in S. scabiei 87-22 upon cello-oligosaccharide supply by transcriptomic analysis, which revealed that cebEFG2 expression is highly activated by both cellobiose and cellotriose, while pitEFG expression was barely induced. Accordingly, deletion of pitE had no impact on virulence and thaxtomin production under the conditions tested, while the deletion of cebEFG2 reduced virulence and thaxtomin production, though not as strong as the mutants of the main cello-oligosaccharide transporter cebEFG1. Our results thus suggest that both ceb clusters participate, at different levels, in importing the virulence elicitors, while PitEFG plays no role in this process under the conditions tested. Interestingly, under more complex culture conditions, the addition of cellobiose restored thaxtomin production when both ceb clusters were disabled, suggesting the existence of an additional mechanism that is involved in sensing or importing the elicitor of the onset of the pathogenic lifestyle of S. scabiei. Full article

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical and chemical distortions, pH changes, and breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers that induce Ca²⁺-dependent CWI responses. We show that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis and are preferentially activated by cellotriose (CT). CORK1 is required for cellooligomer-induced cytoplasmic Ca²⁺ elevation, reactive oxygen species (ROS) production, mitogen-associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes, including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that CORK1 is required for CWI and immune responses activated by cellulose breakdown products. Full article

In this paper, we describe a novel method for preparation of polymer composites with homogeneous dispersion of natural fibers in the polymer matrix. In our approach, Williamson ether synthesis is used to chemically modify cellulose with polymerizable styrene moieties and transform it into a novel multifunctional cellu-mer that can be further crosslinked by copolymerization with styrene. Reactions with model compounds (cellobiose and cellotriose) successfully confirm the viability of the new strategy. The same approach is used to transform commercially available cellulose nanofibrils (CNFs) of various sizes: Sigmacell and Technocell™ 40, 90 and 150. The styrene-functionalized cellulose oligomers and CNFs are then mixed with styrene and copolymerized in bulk at 65 °C with 2,2′-azobisisobutyronitrile as initiator. The resulting composites are in a form of semi-interpenetrating networks (s-IPN), where poly(styrene) chains are either crosslinked with the uniformly dispersed cellulosic component or entangled through the network. Non-crosslinked poly(styrene) (31–41 w%) is extracted with CHCl₃ and analyzed by size-exclusion chromatography to estimate the extent of homopolymerization and reveal the mechanism of the whole process. Electron microscopy analyses of the networks show the lack of cellu-mer agglomeration throughout the polymer matrix. The homogeneous distribution of cellulose entities leads to improved thermal and mechanical properties of the poly(styrene) composites compared to the physical mixtures of the same components and linear poly(styrene) of similar molecular mass. Full article

The beta-glucosidase BglC fulfills multiple functions in both primary metabolism and induction of pathogenicity of Streptomyces scabiei, the causative agent of common scab in root and tuber crops. Indeed, this enzyme hydrolyzes cellobiose and cellotriose to feed glycolysis with glucose directly and modifies the intracellular concentration of these cello-oligosaccharides, which are the virulence elicitors. The inactivation of bglC led to unexpected phenotypes such as the constitutive overproduction of thaxtomin A, the main virulence determinant of S. scabiei. In this work, we reveal a new target substrate of BglC, the phytoalexin scopolin. Removal of the glucose moiety of scopolin generates scopoletin, a potent inhibitor of thaxtomin A production. The hydrolysis of scopolin by BglC displayed substrate inhibition kinetics, which contrasts with the typical Michaelis–Menten saturation curve previously observed for the degradation of its natural substrate cellobiose. Our work, therefore, reveals that BglC targets both cello-oligosaccharide elicitors emanating from the hosts of S. scabiei, and the scopolin phytoalexin generated by the host defense mechanisms, thereby occupying a key position to fine-tune the production of the main virulence determinant thaxtomin A. Full article