Research of saccharides and related biocomplexes: A review with recent techniques and applications.

Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono-, di-, oligo-, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on-line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.

Interaction Effects between the Main Components of Protein-Rich Biomass during Microwave-Assisted Pyrolysis.

The interaction effects between the main components (proteins (P), carbohydrates (C), and lipids (L)) of protein-rich biomass during microwave-assisted pyrolysis were investigated in depth with an exploration of individual pyrolysis and copyrolysis (PC, PL, and CL) of model compounds. The average heating rate of P was higher than those of C and L, and the interactions in all copyrolysis groups reduced the max instant heating rate. The synergistic extent (S) of PC and PL for bio-oil yield was 16.78 and 18.24%, respectively, indicating that the interactions promoted the production of bio-oil. Besides, all of the copyrolysis groups exhibited a synergistic effect on biochar production (S = 19.43-28.24%), while inhibiting the gas generation, with S ranging from -20.17 to -6.09%. Regarding the gaseous products, apart from H2, P, C, and L primarily generated CO2, CO, and CH4, respectively. Regarding bio-oil composition, the interactions occurring within PC, PL, and CL exhibited a significantly synergistic effect (S = 47.81-412.96%) on the formation of N-heterocyclics/amides, amides/nitriles, and acids/esters, respectively. Finally, the favorable applicability of the proposed interaction effects was verified with microalgae. This study offers valuable insights for understanding the microwave-assisted pyrolysis of protein-rich biomass, laying the groundwork for further research and process optimization.

PeSTo-Carbs: Geometric Deep Learning for Prediction of Protein-Carbohydrate Binding Interfaces.

The Protein Structure Transformer (PeSTo), a geometric transformer, has exhibited exceptional performance in predicting protein-protein binding interfaces and distinguishing interfaces with nucleic acids, lipids, small molecules, and ions. In this study, we introduce PeSTo-Carbs, an extension of PeSTo specifically engineered to predict protein-carbohydrate binding interfaces. We evaluate the performance of this approach using independent test sets and compare them with those of previous methods. Furthermore, we highlight the model's capability to specialize in predicting interfaces involving cyclodextrins, a biologically and pharmaceutically significant class of carbohydrates. Our method consistently achieves remarkable accuracy despite the scarcity of available structural data for cyclodextrins.

Friedel-Crafts reactions for biomolecular chemistry.

Chemical tools and principles have become central to biological and medical research/applications by leveraging a range of classical organic chemistry reactions. Friedel-Crafts alkylation and acylation are arguably some of the most well-known and used synthetic methods for the preparation of small molecules but their use in biological and medical fields is relatively less frequent than the other reactions, possibly owing to the notion of their plausible incompatibility with biological systems. This review demonstrates advances in Friedel-Crafts alkylation and acylation reactions in a variety of biomolecular chemistry fields. With the discoveries and applications of numerous biomolecule-catalyzed or -assisted processes, these reactions have garnered considerable interest in biochemistry, enzymology, and biocatalysis. Despite the challenges of reactivity and selectivity of biomolecular reactions, the alkylation and acylation reactions demonstrated their utility for the construction and functionalization of all the four major biomolecules (i.e., nucleosides, carbohydrates/saccharides, lipids/fatty acids, and amino acids/peptides/proteins), and their diverse applications in biological, medical, and material fields are discussed. As the alkylation and acylation reactions are often fundamental educational components of organic chemistry courses, this review is intended for both experts and nonexperts by discussing their basic reaction patterns (with the depiction of each reaction mechanism in the ESI) and relevant real-world impacts in order to enrich chemical research and education. The significant growth of biomolecular Friedel-Crafts reactions described here is a testament to their broad importance and utility, and further development and investigations of the reactions will surely be the focus in the organic biomolecular chemistry fields.

Unveiling the Dissolution Regularities of the Lignin-Carbohydrate Complex in Bamboo Cell Walls during Alkali Pretreatment.

Bamboo is a promising biomass resource. However, the complex multilayered structure and chemical composition of bamboo cell walls create a unique anti-depolymerization barrier, which increases the difficulty of separation and utilization of bamboo. In this study, the relationship between the connections of lignin-carbohydrate complexes (LCCs) within bamboo cell walls and their multilayered structural compositions was investigated. The chemical composition, structural properties, dissolution processes, and migration mechanisms of LCCs were analyzed. Alkali-stabilized LCC bonds were found to be predominantly characterized by phenyl glycoside (PhGlc) bonds along with numerous p-coumaric acid (PCA) linkage structures. As demonstrated by the NMR and CLSM results, the dissolution of the LCC during the alkaline pretreatment process was observed to migrate from the inner secondary wall (S-layer) of the bamboo fiber cell walls to the cell corner middle lamella (CCML) and compound middle lamella (CML), ultimately leading to its release from the bamboo. Furthermore, the presence of H-type lignin-FA-arabinoxylan linkage structures within the bamboo LCC was identified with their primary dissolution observed in the S-layer of the bamboo fiber cell walls. The study results provided a clear target for breaking down the anti-depolymerization barrier in bamboo, signifying a major advancement in achieving the comprehensive separation of bamboo components.

Characterization of polyphenols and carbohydrates exuded by Phaeodactylum tricornutum diatom grown under Cu stress.

This study is focused on analysing polyphenols and carbohydrates released by Phaeodactylum tricornutum (P. tricornutum) diatoms cultured in natural seawater enriched with sublethal and lethal Cu doses. Cu concentrations of 0.31, 0.79 and 1.57 µM reduced cell densities by 37, 82 and 91%, respectively, compared to the control. The total sum of all identified polyphenols and total carbohydrates released by cells grown under lethal Cu levels increased up to 18.8 and 107.4 times, respectively, compared to data from a control experiment. Four different in vitro assays were used to estimate the antioxidant activities of the extracellular compounds: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition, cupric ion reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power and Cu complexing ability (CCA). The highest antioxidant activities were observed in the Cu lethal treatments, where the CCA assay exhibited a greater increase (up to 32.2 times higher than that found in the control experiment) to reduce the concentration of free Cu in the medium and its toxicity. The presence of Cu stimulated the release of polyphenols and carbohydrates to the medium as a detoxification mechanism to survive under lethal levels of Cu regulating its speciation.

Characterization of flours from the aroeira leaf (Schinus terebinthifolius Raddi), obtained by different drying methods.

The present work aimed at the development and characterization of aroeira leaf flour (Schinus terebinthifolius Raddi), obtained by lyophilization and drying in an air circulation oven. The technological, physical, physico-chemical, morphological, functional, and microbiological aspects were analyzed. Physico-chemical analysis identified the following properties with values provided respectively for fresh leaves (FOin) and flours (FES and FLIO): low water activity (0.984, 0.370, 0.387 g/100 g), moisture (64.52, 5.37, 7.97 g /100 g), ash (2.69, 6.51, and 6.89 g/100 g), pH (0.89, 4.45, 4.48 g/100 g), lipids (0.84, 1.67, 5.23 g/100 g), protein (3.29, 8.23, 14.12 g/100 g), carbohydrates (17.02, 53.12, 33.02 g/100 g), ascorbic acid (19.70, 34.20, 36.90 mg/100 g). Sources of fiber from plant leaves and flours (11.64, 25.1, 32.89 g/100 g) showed increased levels of luminosity. For NMR, the presence of aliphatic and aromatic compounds with olefinic hydrogens and a derivative of gallic acid were detected. The most abundant minerals detected were potassium and calcium. Micrographs identified the presence of irregular, non-uniform, and sponge-like particles. The main sugars detected were: fructose, glucose, and maltose. Malic, succinic, citric, lactic, and formic acids were found. Fifteen phenolic compounds were identified in the samples, highlighting: kaempferol, catechin, and caffeic acid. The values ​​found for phenolics were (447, 716.66, 493.31 mg EAG/100 g), flavonoids (267.60, 267.60, 286.26 EC/100 g). Antioxidant activity was higher using the ABTS method rather than FRAP for analysis of FOin, FES, and FLIO. Since the flours of the aroeira leaf have an abundant matrix of nutrients with bioactive properties and antioxidant activity, they have a potential for technological and functional use when added to food.

Selective Quantification of Bacteria in Mixtures by Using Glycosylated Polypyrrole/Hydrogel Nanolayers.

Here, we present a covalent nanolayer system that consists of a conductive and biorepulsive base layer topped by a layer carrying biorecognition sites. The layers are built up by electropolymerization of pyrrole derivatives that either carry polyglycerol brushes (for biorepulsivity) or glycoside moieties (as biorecognition sites). The polypyrrole backbone makes the resulting nanolayer systems conductive, opening the opportunity for constructing an electrochemistry-based sensor system. The basic concept of the sensor exploits the highly selective binding of carbohydrates by certain harmful bacteria, as bacterial adhesion and infection are a major threat to human health, and thus, a sensitive and selective detection of the respective bacteria by portable devices is highly desirable. To demonstrate the selectivity, two strains of Escherichia coli were selected. The first strain carries type 1 fimbriae, terminated by a lectin called FimH, which recognizes α-d-mannopyranosides, which is a carbohydrate that is commonly found on endothelial cells. The otherE. coli strain was of a strain that lacked this particular lectin. It could be demonstrated that hybrid nanolayer systems containing a very thin carbohydrate top layer (2 nm) show the highest discrimination (factor 80) between the different strains. Using electrochemical impedance spectroscopy, it was possible to quantify in vivo the type 1-fimbriated E. coli down to an optical density of OD600 = 0.0004 with a theoretical limit of 0.00005. Surprisingly, the selectivity and sensitivity of the sensing remained the same even in the presence of a large excess of nonbinding bacteria, making the system useful for the rapid and selective detection of pathogens in complex matrices. As the presented covalent nanolayer system is modularly built, it opens the opportunity to develop a broad band of mobile sensing devices suitable for various field applications such as bedside diagnostics or monitoring for bacterial contamination, e.g., in bioreactors.

Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4.

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.

Separation of carbohydrates using dynamically adsorbed borate stationary phase for hydrophilic interaction liquid chromatography.

In this work, a chromatographic method for the separation of carbohydrates was proposed. Tris-(hydroxymethyl)-amine (TRIS) functionalized silica-based hydrophilic interaction liquid chromatography (HILIC) stationary was synthesized. The dynamically absorbed borate layer is generated by using borate buffer as a polar modifier due to the complexation of borate with TRIS ligand in the stationary phase. The chromatographic systems were analyzed by the linear solvation energy relationship model. The calculated system constants revealed the enhancement of anionic exchange by the addition of borate in the mobile phase system. In addition, ligand exchange is critical for the retention and elution order of sugars and sugar alcohols. Carbohydrates displayed prolonged retention with different selectivity profiles relating to their complexation coefficients with borate. Experiment results showed that the effect of borate in this chromatographic system was stable within the range of pH 3-7 and borate concentration of 5-15 mM. This work provides a complementary solution for the separation of carbohydrates. It can also be extended to the separation of glycosides.

Selective enhancement of (6-4) photoproduct formation in dithymine dinucleotides driven by specific sugar puckering.

Four dinucleotide analogs of thymidylyl(3'-5')thymidine (TpT) have been designed and synthesized with a view to increase the selectivity, with respect to CPD, of efficient UV-induced (6-4) photoproduct formation. The deoxyribose residues of these analogs have been modified to increase north and south conformer populations at 5'- and 3'-ends, respectively. Dinucleotides whose 5'-end north population exceeds ca. 60% and whose 3'-end population is almost completely south display a three-fold selective enhancement in (6-4) adduct production when exposed to UV radiation, compared to TpT. These experimental results undoubtedly provide robust foundations for studying the singular ground-state proreactive species involved in the (6-4) photoproduct formation mechanism.

Metal-Assisted Carbohydrate Assembly.

The sequence-controlled assembly of nucleic acids and amino acids into well-defined superstructures constitutes one of the most revolutionary technologies in modern science. The elaboration of such superstructures from carbohydrates, however, remains elusive and largely unexplored on account of their intrinsic constitutional and configurational complexity, not to mention their inherent conformational flexibility. Here, we report the bottom-up assembly of two classes of hierarchical superstructures that are formed from a highly flexible cyclo-oligosaccharide─namely, cyclofructan-6 (CF-6). The formation of coordinative bonds between the oxygen atoms of CF-6 and alkali metal cations (i) locks a myriad of flexible conformations of CF-6 into a few rigid conformations, (ii) bridges adjacent CF-6 ligands, and (iii) gives rise to the multiple-level assembly of three extended frameworks. The hierarchical superstructures present in these frameworks have been shown to modulate their nanomechanical properties. This research highlights the unique opportunities of constructing convoluted superstructures from carbohydrates and should encourage future endeavors in this underinvestigated field of science.

Sweet Janus Particles: Multifunctional Inhibitors of Carbohydrate-Based Bacterial Adhesion.

Escherichia coli and other bacteria use adhesion receptors, such as FimH, to attach to carbohydrates on the cell surface as the first step of colonization and infection. Efficient inhibitors that block these interactions for infection treatment are multivalent carbohydrate-functionalized scaffolds. However, these multivalent systems often lead to the formation of large clusters of bacteria, which may pose problems for clearing bacteria from the infected site. Here, we present Man-containing Janus particles (JPs) decorated on one side with glycomacromolecules to target Man-specific adhesion receptors of E. coli. On the other side, poly(N-isopropylacrylamide) is attached to the particle hemisphere, providing temperature-dependent sterical shielding against binding and cluster formation. While homogeneously functionalized particles cluster with multiple bacteria to form large aggregates, glycofunctionalized JPs are able to form aggregates only with individual bacteria. The formation of large aggregates from the JP-decorated single bacteria can still be induced in a second step by increasing the temperature and making use of the collapse of the PNIPAM hemisphere. This is the first time that carbohydrate-functionalized JPs have been derived and used as inhibitors of bacterial adhesion. Furthermore, the developed JPs offer well-controlled single bacterial inhibition in combination with cluster formation upon an external stimulus, which is not achievable with conventional carbohydrate-functionalized particles.

Degradation selectivity for bamboo fiber and parenchyma lignin-carbohydrates complexes (LCC) esters.

The degradation of lignin-carbohydrate complex (LCC) esters has been proven to be crucial for the selective separation of lignocellulosic components. This study utilized Raman microspectroscopy to image the preferential degradation of lignin and LCC esters from the bamboo wall during successive NaOH (0.2 to 5.0 % w/w), H2SO4 (1 to 8 % v/v), and NaClO2 (5 to 20 min) treatments. Raman imaging showed that lignin and LCC esters were selectively removed from the middle lamella of fibers and the secondary wall of parenchyma during NaOH and NaClO2 treatments. In contrast, H2SO4 primarily caused the simultaneous removal of lignin and LCC esters from the fiber wall under harsh conditions (8 %), while the middle lamella of parenchyma was less affected, both morphologically and topochemically. Raman spectral analysis indicated that the band intensity at 1605 cm-1 for lignin and at 1173 cm-1 for LCC esters decreased by >87.0 % in the highly lignified parenchyma secondary wall after a 5.0 % NaOH treatment, while the decrease was <67 % in the fiber wall. Interestingly, a strong linear correlation was observed between LCC esters and carbohydrates in the parenchyma (R2 > 0.912). These findings provide important insights into the graded and classified utilization of bamboo resources.

Direct Formation of Amide-Linked C-Glycosyl Amino Acids and Peptides via Photoredox/Nickel Dual Catalysis.

Glycoproteins account for numerous biological processes including those associated with diseases and infections. The advancement of glycopeptides has emerged as a promising strategy for unraveling biological pathways and discovering novel medicines. In this arena, a key challenge arises from the absence of efficient synthetic strategies to access glycopeptides and glycoproteins. Here, we present a highly concise approach to bridging saccharides with amino acids and peptides through an amide linkage. Our amide-linked C-glycosyl amino acids and peptides are synthesized through cooperative Ni-catalyzed and photoredox processes. The catalytic process generates a glycosyl radical and an amide carbonyl radical, which subsequently combine to yield the C-glycosyl products. The saccharide reaction partners encompass mono-, di-, and trisaccharides. All 20 natural amino acids, peptides, and their derivatives can efficiently undergo glycosylations with yields ranging from acceptable to high, demonstrating excellent stereoselectivities. As a substantial expansion of applications, we have shown that simple C-glycosyl amino acids can function as versatile building units for constructing C-glycopeptides with intricate spatial complexities.

Glycomimetic inhibitors of tandem-repeat galectins: Simple and efficient.

The binding of human galectins by glycomimetic inhibitors is a promising therapeutic approach. The structurally distinct group of tandem-repeat galectins has scarcely been studied so far, and there is hardly any knowledge on their ligand specificity or their inhibitory potential, particularly concerning non-natural carbohydrates. Here, we present the synthesis of a library of seven 3-O-disubstituted thiodigalactoside-derived glycomimetics and their affinity to two tandem-repeat galectins, Gal-8 and Gal-9. The straightforward synthesis of these glycomimetics involved dibutyltin oxide-catalyzed 3,3́-O-disubstitution of commercially available unprotected thiodigalactoside, and conjugation of various aryl substituents by copper-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC). The inhibitory potential of the prepared glycomimetics for Gal-8 and Gal-9 was assessed, and compared with the established galectins Gal-1 and Gal-3. The introduction of C-3 substituents resulted in an over 40-fold increase in affinity compared with unmodified TDG. The structure-affinity relations within the studied series were discussed using molecular modeling. Furthermore, the prepared glycomimetics were shown to scavenge Gal-8 and Gal-9 from the surface of cancer cells. This pioneering study on the synthetic inhibitors especially of Gal-9 identified lead compounds that may be used in further biomedical research.

Unravelling carbohydrate binding module 21 (CBM21) dynamics of interaction with amylose.

The carbohydrate binding module 21 (CBM21) from Rhizopus oryzae is a dual-site CBM proposed to disrupt polysaccharide structures. Additionally, it serves as a purification tag in industry. CBM21 crystal structure features a Glc residue in an unusual 1S3 conformation, whose relevance for the CBM mechanism of action is unclear. In this context, we seek to contribute for the understanding of CBM21 mechanism of action by: i) investigating the role of the 1S3 conformation on carbohydrate recognition, and ii) characterize the protein-carbohydrate binding dynamics using molecular dynamics and metadynamics simulations at MM and QM/MM levels. Results indicate the 1S3 Glc conformation is unlikely to occur under biological conditions, being originated from the crystallographic environment. CBM21 binding to small ligands appears transient and unstable, while protein dimerization and polysaccharide chain size influence complex stability. In interactions with amylose, CBM21 exhibits a repeated unbinding followed by re-binding, while simultaneously alternating between binding sites I and II. These results suggest that CBM21 acts through transient interactions, directing carbohydrates to the catalytic center rather than forming strong and long-lasting bonds with carbohydrates. Accordingly, we expect such atomistic depiction of CBM21 mechanism could aid in CBM design targeting biotechnological applications.

Preparation of Mucin Glycopeptides by Organic Synthesis.

Mucins are sugar-rich glycoproteins. Glycoprotein sugar moieties are structurally diverse, making it difficult to obtain naturally pure glycoproteins. Chemical synthesis is a powerful tool for obtaining target or designed compounds. Automated peptide synthesizers are commercially available, and many use the solid-phase peptide synthesis (SPPS) method. In addition, some of these synthesizers apply microwave irradiation to obtain higher reaction yields, thereby enabling the synthesis of 40 to 50 amino acid residual glycopeptides. Theoretically, glycopeptides can be synthesized using methods similar to those used for peptide synthesis, but glycosylated amino acid synthons are less stable than amino acid synthons and are also very expensive. Therefore, they are not suitable for use in large excess amounts. Many of oligosaccharide-linked amino acid synthons are not commercially available, so they must be specially prepared, and they also require careful handling that demands specific organic synthesis experience and techniques. However, monosaccharide-linked amino acid synthons are commercially available and are relatively easy to handle. Here, as an entry into glycopeptide synthesis, we describe a typical glycopeptide synthesis procedure for a 27 amino acid residual MUC1 repeating unit with monosaccharides.

Proximate biomass characterization of the high productivity marine microalga Picochlorum celeri TG2.

Microalgae are compelling renewable resources with applications including biofuels, bioplastics, nutrient supplements, and cosmetic products. Picochlorum celeri is an alga with high industrial interest due to exemplary outdoor areal biomass productivities in seawater. Detailed proximate analysis is needed in multiple environmental conditions to understand the dynamic biomass compositions of P. celeri, and how these compositions might be leveraged in biotechnological applications. In this study, biomass characterization of P. celeri was performed under nutrient-replete, nitrogen-restricted, and hyper-saline conditions. Nutrient-replete cultivation of P. celeri resulted in protein-rich biomass (∼50% ash-free dry weight) with smaller carbohydrate (∼12% ash-free dry weight) and lipid (∼11% ash-free dry weight) partitions. Gradual nitrogen depletion elicited a shift from proteins to carbohydrates (∼50% ash-free dry weight, day 3) as cells transitioned into the production of storage metabolites. Importantly, dilutions in nitrogen-restricted 40 parts per million (1.43 mM nitrogen) media generated high-carbohydrate (∼50% ash-free dry weight) biomass without substantially compromising biomass productivity (36 g ash-free dry weight m-2 day-1) despite decreased chlorophyll (∼2% ash-free dry weight) content. This strategy for increasing carbohydrate content allowed for the targeted production of polysaccharides, which could potentially be utilized to produce fuels, oligosaccharides, and bioplastics. Cultivation at 2X sea salts resulted in a shift towards carbohydrates from protein, with significantly increased levels of the amino acid proline, which putatively acts as an osmolyte. A detailed understanding of the biomass composition of P. celeri in nutrient-replete, nitrogen-restricted, and hyper saline conditions informs how this strain can be useful in the production of biotechnological products.

Enhancing Bioethanol Production from Rice Straw through Environmentally Friendly Delignification Using Versatile Peroxidase.

Rice straw (RS), an agricultural residue rich in carbohydrates, has substantial potential for bioethanol production. However, the presence of lignin impedes access to these carbohydrates, hindering efficient carbohydrate-to-bioethanol conversion. Here, we expressed versatile peroxidase (VP), a lignin-degrading enzyme, in Pichia pastoris and used it to delignify RS at 30 °C using a membrane bioreactor that continuously discarded the degraded lignin. Klason lignin analysis revealed that VP-treatment led to 35% delignification of RS. We then investigated the delignified RS by SEC, FTIR, and SEM. The results revealed the changes of RS caused by VP-mediated delignification. Additionally, we compared the saccharification and fermentation yields between RSs treated with and without VP, VP-RS, and Ctrl-RS, respectively. This examination unveiled an improvement in glucose and bioethanol production, VP-RS exhibiting up to 1.5-fold and 1.4-fold production, respectively. These findings underscore the potential of VP for delignifying RS and enhancing bioethanol production through an eco-friendly approach.