Agarose, a hydrophilic polysaccharide derived from red algae, is widely used in biochemical and agricultural applications, but its intrinsic gelation below ∼40 °C limits enzymatic conversion into high-value neoagarooligosaccharides (NAOS). Here, we report the systematic characterization of a novel thermophilic β-agarase, SaAgaD, identified from Salegentibacter agarivorans. SaAgaD is a 398-residue GH16 enzyme that expresses solubly in Escherichia coli and exhibits a high catalytic efficiency (kcat/Km = 1.76 × 104 mM-1 s-1) toward agarose. Distinct from many characterized agarases, SaAgaD displays optimal activity at pH 8.0 and 55 °C, retains ∼90% activity at 65 °C, and remains stable under neutral to mildly alkaline conditions, enabling effective hydrolysis above the agarose gelation point. The enzyme preferentially degrades agar and crude Gracilaria agar, with minimal activity on sulfated porphyran. Product profiling by HPAEC-PAD and ESI-MS demonstrates an endolytic mechanism yielding predominantly neoagarotetraose (NA4) and neoagarohexaose (NA6), with a scale-up NAOS yield of ∼75%. Molecular dynamics simulations of SaAgaD and its NA8 complex reveal a stable substrate binding mode and highlight flexible loop regions that coordinate substrate recognition. The combination of high thermal tolerance, catalytic efficiency, and product specificity positions SaAgaD as a promising biocatalyst for industrial NAOS production from marine biomass.

β-1,3-Glucans are ubiquitous polysaccharides whose enzymatic hydrolysis yields β-1,3-gluco-oligosaccharides (GOS) with immunomodulatory, antioxidant, and prebiotic properties. These bioactivities highlight their potential as functional food ingredients. However, conventional enzymatic methods often yield structurally heterogeneous GOS, complicating their application. Glycoside hydrolase family 64 (GH64) enzymes are unique in producing laminaripentaose as a single dominant product, enabling the preparation of structurally uniform GOS. Here, a novel GH64 enzyme, CsGH64, from Candidatus saccharibacteria was biochemically and structurally characterized. CsGH64 exhibited strict β-1,3-glucanase activity, producing laminaripentaose at > 80% yield, with optimal activity at pH 6.0 and 20 °C. Kinetic analysis revealed a Km of 27.9 mg mL⁻¹ and a Vmax of 16.3 U mg⁻¹. Comparative structural analysis with previously characterized GH64 enzymes uncovered distinct features underlying its cold-adapted catalytic properties. These results identify CsGH64 as a promising biocatalyst for the food and nutraceutical industries, enabling the efficient and mild production of structurally defined, bioactive oligosaccharides.

BACKGROUND:Conventional pesticide formulations are often limited by poor targeting efficiency and low utilization rates, resulting in excessive application and severe environmental pollution. To overcome these drawbacks, smart stimuli-responsive delivery systems based on nanotechnology have become a forefront research area in agrochemicals. Of particular interest are systems capable of responding to specific biochemical stimuli at the site of infection. RESULTS:In this study, we developed a dual-responsive system, methoxy polyethylene glycol (mPEG)-dithiobisethanol (SS)-thioketal (TK)-chitosan oligosaccharide (COS), designed for the targeted release of pesticides and the integrated delivery of plant immunity inducers, COS. The system utilizes reactive oxygen species and glutathione to cleave TK and SS bonds, thereby triggering the release of both pesticide and COS. Structural characterization confirmed the formation of spherical nanoparticles with an average diameter of 300 nm. Biological evaluations demonstrated that the system effectively enhanced disease control through targeted payload delivery and specific activation of the salicylic acid signaling pathway. This chemico-immunological synergy achieved a control efficiency of 74.28%, representing a 147.60% improvement compared to validamycin alone. The synergistic effect was supported by a reduction in lesion area on detached leaves. DISCUSSION:This work presents an effective strategy for crop disease management by combining prompt and targeted pesticide release with induced plant immunity. The dual-responsive system enhances pesticide efficiency through precise and timely release, while simultaneously mitigating environmental risks. This approach represents a promising and sustainable solution for modern pest management in agriculture. © 2026 Society of Chemical Industry.

Plant pathogens of the Xanthomonas genus are capable of infecting a wide array of economically important plant species. Among their virulence factors, the GH10 family xylanase, XynB, is typically responsible for the predominant extracellular xylanase activity. However, the detailed biochemical characteristics of this enzyme in most Xanthomonas pathogens remain insufficiently understood. In this study, we conducted a comprehensive characterization of the enzymatic and immunogenic properties of XynB from Xanthomonas campestris pv. campestris (Xcc). We found that XccXynB functions as an endoxylanase with a K-m of 16.93 +/- 4.93 (mg/mL) and K-cat of 1.42 +/- 0.17 (min(-1)), primarily producing xylooligosaccharides (XOSs) ranging from xylobiose (X2) to xylohexaose (X6). Site-directed mutagenesis confirmed that residues E138 and E254 are essential for its catalytic activity. Furthermore, we found that XccXynB can act as a pathogen-associated molecular pattern (PAMP) in the model plants N. benthamiana and A. thaliana, triggering immune responses such as reactive oxygen species (ROS) burst comparable to the elicitor Flg22, without inducing cell death. Heterologous expression of XccXynB in A. thaliana led to constitutive immune activation and significantly enhanced disease resistance, with the in planta bacterial population of Pst DC3000 being reduced to similar to 80%. Taken together, these findings could provide a rationale for developing novel strategies against Xanthomonas diseases by targeting the conserved xylanase function or utilizing the protein as an immunogen.

Oligogalacturonides (OGs), recognized as damage-associated molecular patterns (DAMPs), play critical roles in plant disease resistance and growth regulation. This study aimed to elucidate the regulatory mechanism by which short OGs induce resistance against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis thaliana. Arabidopsis plants were pretreated with either individual OGs with degrees of polymerization 2 (DP2) and 7 (DP7), or a mixture of OGs (DP2-7) at 25 mg/L, prior to infection with Pst DC3000. Water-pretreated plants served as controls. All tested oligosaccharides and mixture enhanced resistance against Pst DC3000 infection, as evidenced by reduced phenotypic symptoms, suppressed bacterial growth, and decreased disease indices. Analysis of salicylic acid (SA) and jasmonic acid (JA) levels, along with expression of associated genes, revealed that DP2 and DP7 conferred resistance by elevating hormone levels and upregulating defense-related genes in the Pst DC3000-Arabidopsis system. Notably, DP7 and OGs (DP2-7) mixture triggered a reactive oxygen species (ROS) burst, while DP2 did not. Transcriptomic profiling demonstrated that DP2, DP7, and OGs (DP2-7) activated defense pathways, including JA, SA, and H₂O₂ signaling, while modulating different key genes to bolster resistance. Gene Ontology analysis revealed that DP2 upregulated more genes in nucleus, while DP7 upregulated more genes in space outside the nucleus. Furthermore, these short-chain OGs (DP2-7) regulated growth-related genes, suggesting a dual role in stress adaptation and developmental balance in Arabidopsis-Pst DC3000. Our findings highlight that both DP2 and DP7 effectively prime early defense responses against pathogens in Arabidopsis.

The rapid degradation of the cell wall leads to firmness loss in fruit, resulting in a shorter shelf life and quality deterioration. Here, we evaluated the impact of preharvest foliar application of alginate oligosaccharides (AOS) on a perishable fruit kiwiberry (Actinidia arguta) over a 42 d storage period using physiological, molecular, and biochemical approaches. The selected plants were sprayed with the AOS solution 15 d before harvest, covering both the leaves and fruit surfaces. The preharvest AOS application significantly reduced decay incidence by about 60 %, maintained nearly twice the firmness, and 22 % less weight loss compared with control fruit after 42 d of storage. Transcriptome analysis reveals that AOS treatment downregulates the genes related to cell wall degradation. Moreover, biochemical analysis indicates that preharvest application suppressed the activation of cell wall degrading enzymes, reduced the solubilization of cell wall polysaccharides, and preserved their monosaccharide composition. These results reveal that preharvest application of AOS ensured the storability of kiwiberry by controlling cell wall degradation enzymes and gene expression. These findings highlight the significant potentiality of AOS preharvest treatments for delaying postharvest softening of kiwiberry.

Chemoenzymatic labeling strategies, relying on azide-modified sugar donors and subsequent Cu-based click chemistry to introduce enrichable handles, have greatly advanced the study of O-GlcNAcylation. However, multistep labeling and enrichment procedures often lead to substantial sample loss. While a recent one-step labeling approach using biotinylated UDP-GalNAc donors simplifies the procedures, the strong biotin-avidin interactions, as well as bulky tags, limited this strategy to protein-level identification rather than site-specific glycopeptide mapping. Herein, we developed an oxime-based reversible O-GlcNAc enrichment (ORO-GlcNAc) approach employing a ketone-functionalized UDP-GalNAc analogue (UDP-GalNLeV) for one-step chemoenzymatic labeling with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, oxime-based capture using hydroxylamine-functionalized beads, followed by reversible methoxyamine-mediated release, overcomes the limitations of conventional ketone-based enrichment strategies and eliminates the need for azide-based chemistry. Using the ORO-GlcNAc strategy, we identified 4129 potential O-GlcNAcylation sites in HeLa cells, of which 64.3% were annotated in the O-GlcNAc Atlas, demonstrating the high coverage and enrichment performance of ORO-GlcNAc. Functional studies suggested a potential role of O-GlcNAcylation in the nucleocytoplasmic redistribution of the chromatin remodeler EP400 during stress-granule assembly. This platform thus provides a powerful and versatile tool for advancing the O-GlcNAcylation research.

Members of the Rhodophyta such as Kappaphycus alvarezii and Eucheuma denticulatum are rich sources of carrageenans, and enzymatic degradation of κ-carrageenan into oligosaccharides has attracted increasing attention as a mild and environmentally friendly alternative to chemical methods. In this study, a novel κ-carrageenase, VsCgkA, was identified from Vibrio sp. C7 and heterologously expressed in E. coli. The enzyme has 262 amino acid residues with a theoretical molecular weight of 30.54 kDa. The recombinant enzyme exhibited optimal activity at 40 °C and pH 7.0. VsCgkA retained over 75% of its activity after incubation at 40 °C for 30 min or incubated under a broad pH range of 6.0–8.0 at 40 °C for 4 h. Enzyme activity was significantly enhanced (~47%) in the presence of Ca2+, whereas ions such as Mg2+, Cu2+, K+, and NH4+ exerted significant inhibitory effect. Kinetic analysis yielded a Km of 1.69 mg·mL−1, kcat of 60.36 ± 5.48 s−1, and a kcat/Km of 35.72 s−1·mL·mg−1 toward κ-carrageenan. The specificity activity of VsCgkA toward κ-carrageenan was 95.94 ± 4.80 U·mg−1 under optimal reaction conditions. HPLC and MALDI-TOF-MS analyses demonstrated that VsCgkA acts as an endo-type κ-carrageenase, predominantly producing κ-neocarrabiose and κ-neocarratetraose. Notably, the enzymatic hydrolysates significantly promoted plant growth, enhancing rice seedling development in a concentration-dependent manner and improving agronomic traits and biomass accumulation in mature rapeseed. These findings highlight the potential of VsCgkA in degrading κ-carrageenan and producing oligosaccharides with agricultural applications.

The phytopathogen endoglucanases play vital roles in pathogenesis, likely due to their capacity in deconstructing plant cell wall. While acting as virulence factors, some endoglucanases can also be recognized by the plant immune system and activate plant defense responses against possible pathogens. However, many of these enzymes and their roles in inducing plant defense responses are not characterized, especially those from Erwinia amylovora. In this study we cloned a putative glycoside hydrolase family 8 (GH8) endoglucanase (EaCel8) from E. amylovora. The enzymatic properties of EaCel8 and its induction of plant immune responses were comprehensively investigated. The biochemical characterization of the recombinant EaCel8 showed high activity towards barley β-glucan and lower activity on carboxymethyl cellulose. EaCel8 was active within a broad range of pH and temperature, and it hydrolyzed barley β-glucan mainly to mixed-linked glucan (MLG) oligosaccharides with a degree of polymerization ≥3. Interestingly, EaCel8 triggered immune responses in Arabidopsis thaliana, rice, soybean, and pepper, and pretreating tobacco and rice with the purified recombinant EaCel8 significantly enhanced the disease resistance of these plants against Rhizoctonia solani. These findings deepen our mechanistic understanding of plant pathogen derived cell wall degrading enzymes, and the novel GH8 endoglucanase EaCel8 has the potential to be developed as a plant immune inducer.

Strawberry ripening and softening during storage are important postharvest metabolic mechanisms, mainly associated with to the activities of several enzymes. In our previous study, we observed that alginate oligosaccharide (AOS) postharvest treatment effectively preserved fruit texture and quality and extended the shelf life of strawberry. The present study aimed to evaluate the consequences of AOS postharvest treatment on cell wall-degrading enzymes and their encoding gene expression during storage of strawberry. The postharvest treatment comprised AOS at a concentration of 100 mg.L−1. Results demonstrated that AOS postharvest treatment effectively reduced protein loss as well as pectin methyl esterase (PME) and polygalacturonase (PG) enzyme activity during storage of strawberry. Moreover, AOS treatment also had a positive impact by inhibiting the relative expression of genes encoding cell wall-degrading enzymes (FaPE1, FaPG1, and FaPG 2) and ethylene biosynthesis genes (FaACO and FaACS) during storage. In addition, AOS treatment increased endogenous salicylic acid (SA) content through the upregulation of genes related to the SA signaling pathway (PR1, PR2 and NPR 1). Our results suggest that AOS postharvest treatment effectively reduce cell wall-degrading enzyme activities, inhibited ethylene biosynthesis, and delayed strawberry fruit softening by triggering the SA-dependent pathway.

Bioconversion of methane into functional polysaccharides presents a promising strategy for mitigating methane-induced greenhouse gas emissions and addressing the limitations of plant-derived polysaccharide and sugar-based microbial polysaccharides production. In this study, the novel methane-derived polysaccharide (MePS) was obtained from a newly isolated methanotrophic bacterium Alkalicoccus glycogenes WONF2802. Structural characterization found that MePS is a branched-chain glucan with a weight-average molecular weight of 283.2 kDa. Additionally, MePS exhibited considerable antioxidant capacities in both in vitro biochemical assays and the H2O2-induced oxidative stress cell model. This work establishes a potential pathway for polysaccharide production, reducing reliance on plant and sugar-based feedstocks, while offering a new strategy for methane emission reduction.

The review article presents current research on obtaining chitosan nanoparticles (NPs). It highlights their key properties and potential applications in agriculture. Various methods of chitosan nanoparticle formation were considered: emulsion method in the presence of special agents, drying method, crosslinking of molecules through covalent bonds, self-organisation processes and obtaining interpolyelectrolyte complexes, and ionotropic gelation. Some examples of the chitosan NPs application have also been reviewed.

Photothermal therapy (PTT) converts light to heat, offering a minimally invasive tumor treatment option. However, its efficacy for triple-negative breast cancer (TNBC) is limited by nanoparticle non-specificity and immunosuppression from tumor-associated macrophages (TAMs). Therefore, it is imperative to develop nanoparticles with high selectivity and precise targeting to enhance the efficacy of tumor treatment. In this study, we developed polyethylene glycol (PEG)- and aptamer (aptPD-L1)-functionalized silver nanotriangles (apt-PNTs) targeting PD-L1-overexpressing tumor cells and TAMs. Subsequently, we evaluated tumor-targeting ability, anti-tumor activity, and TAMs reprogramming effect through in vitro and in vivo studies. The study revealed that the synthesized nanoparticles exhibit exceptional photothermal properties and high stability. The apt-PNTs demonstrate selective uptake by TNBC cells and M2. In murine models, apt-PNTs accumulated maximally in tumors at 6 h. Under near-infrared laser irradiation (40-45 °C), apt-PNTs reduced M2 marker CD206 and increased M1 markers CD16/32 and iNOS, reprogramming TAMs and achieving 68.73 % tumor growth inhibition. In conclusion, in the absence of significant adverse reactions, apt-PNTs exhibit excellent photothermal properties and tumor-targeting ability. This work demonstrates the application of apt-PNTs-mediated PTT in promoting TAMs reprogramming and killing tumors, providing a potential approach for targeted therapy of TNBC.

Steviol glycosides (SGs) from Stevia rebaudiana are prized as noncaloric sweeteners, with rebaudioside M (Reb M)-a next-generation SG known for its sucrose-like sweetness and lack of off-tastes-standing out for its superior sensory profile. However, Reb M's limited natural abundance impedes its commercial production. Here, we report the identification of a glucosyltransferase, UGT76G4 that efficiently catalyzes the conversion of Reb D to Reb M with a strong preference for C19 glycosylation. Structural and functional analyses, including X-ray crystallography, molecular dynamics simulations, and mutagenesis, revealed key residues in UGT76G4 that dictate its regioselectivity, with residue 200 playing a pivotal role. Engineered UGT76G4 variants, including Q199I/ G200Y and H155S/Q199I/G200Y, enhanced Reb E and Reb D conversion efficiency by 1.46-fold and 23-fold, respectively, compared to UGT76G1. The engineered variants offer a promising pathway for increasing Reb M production, advancing biotechnological strategies for steviol glycoside biosynthesis and optimizing plant metabolic engineering approaches. Our findings deepen the understanding of SG biosynthesis and provide a basis for sustainable production of high-value sweeteners.

Photoelectrochemical water oxidation to generate H2O2 is a clean and promising method. Its performance is strongly dependent on electrolyte species, in which the Faradaic efficiency is considerably promoted by HCO3- anion. The kinetic mechanism is under debate, which is highly desired but challenging. Herein, we reveal the charge dynamics and reaction kinetics in the H2O2 evolution from photoelectrochemical water oxidation by time-resolved spectroscopic techniques. The H2O2 evolution reaction exhibits the same first-hole transfer dynamics as that in O2 evolution reaction. The rate law analysis indicates that H2O2 evolution reaction exhibits the first-order reaction kinetics, demonstrating that the rate-determining step in 2e- water oxidation reaction for H2O2 evolution is the consumption of the first-hole intermediates. Importantly, the HCO3- anion accelerates the consumption of the first-hole intermediates in 2e- water oxidation reaction by about 30 fold in rate constants or 60 fold in turnover frequency relative that in 4e- water oxidation reaction. This work sheds light on the control strategy for reaction selectivity by modulation of reaction kinetics in catalysis.

β-1,3-Glucans are widespread in plants, algae, fungi, and certain bacteria, and their enzymatic hydrolysis yields β-1,3-gluco-oligosaccharides (GOS) with diverse biological activities. However, the structural heterogeneity of GOS mixtures complicates investigations of their structure-activity relationships. Members of glycoside hydrolase family 64 (GH64) are unique in that, despite being classified as endo-β-1,3-glucanases, they predominantly produce a single product─laminaripentaose. Here, we biochemically characterized a GH64 enzyme, Mvgh64, from Massilia violaceinigra. Mvgh64 exhibited high specificity toward β-1,3-glucans and produced laminaripentaose as the major product (>85%). Structural analysis via X-ray crystallography, coupled with GPC and MS analysis of the products, revealed features indicative of an exolike endo cleavage mechanism, including a tunnel-like active site formed by region 1 and loop 1. These findings provide mechanistic insight into GH64 substrate specificity and establish a framework for engineering glycosidases to produce uniform, high-degree-of-polymerization oligosaccharides.

Chitin and its deacetylated derivative chitosan are the major components of fungal cell walls and are recognized by plant pattern-recognition receptors (PRRs) as pathogen-associated molecular patterns that induce innate immunity. Recognition of chitin oligosaccharide (CTOS) in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) requires the membrane-localized lysin-motif (LysM)-domain-containing receptors AtLYK5 and OsCEBiP, respectively. However, the mechanism underlying chitosan oligosaccharide (CSOS)-induced plant immunity remains unclear. In this study, we determined that CTOS and CSOS trigger immune responses and boost disease resistance in soybean (Glycine max) through the LysM-domain-containing protein GmNRF5a and its co-receptor GmCERK1. Surprisingly, both GmNFR5a and GmCERK1 bind directly to CTOS and CSOS, with distinct binding sites. The receptor-like kinase GmCAK1 acts downstream of GmCERK1 and is essential for CTOS/CSOS-mediated immune activation. Overall, these findings uncovered how soybean plants respond to CSOS and initiate immune signaling, demonstrating that soybean exploits shared immune sectors to transduce immune signals triggered by CTOS/CSOS, paving the way for the development of disease-resistant crops with broad-spectrum resistance.

Stevia rebaudiana, a perennial herb, is recognized not only for its sweet steviol glycosides but also for its rich flavonoid content, which confer pharmacological properties including anti-inflammatory, antimicrobial, and anticancer activities. However, the enzymatic basis underlying flavonoid modification in S. rebaudiana remains poorly understood. In this study, we identified, cloned, and heterologously expressed a novel flavonoid glycosyltransferase gene, SrUGT72B1 in E. coli. The recombinant SrUGT72B1 catalyzed the glycosylation of multiple flavonoids using UDP-glucose as the primary sugar donor, and exhibited broad substrate promiscuity toward apigenin, luteolin, phloretin and kaempferol. In addition to UDP-glucose, SrUGT72B1 also accepted UDP-xylose and UDP-rhamnose, with UDP-glucose exhibiting the highest catalytic efficiency. Biochemical characterization revealed that the enzyme functions optimally at pH 9.0 and 50 °C. Notably, SrUGT72B1 demonstrates regioselective 5-O-glycosylation toward apigenin, a rare activity among plant glycosyltransferases. Molecular docking and molecular dynamics simulations provided structural insights into this unique regioselectivity and substrate recognition. Together, these findings establish SrUGT72B1 as a previously uncharacterized flavonoid 5-O-glycosyltransferase, expanding the functional landscape of plant UGTs and offering potential applications in the biosynthesis of value-added flavonoid glycosides.