Hydrolysis Of Glycosidic Bonds Research Articles

A novel mechanocatalytic process by vibratory disk mill for efficient hydrolysis of cellulose, chitin and xylan

Polysaccharides are primary sources to manufacture chemical products with low carbon footprints, owing to their carbons from atmospheric carbon dioxide fixed through photosynthesis. Breaking down such polysaccharides into water-soluble saccharides through the hydrolysis of glycosidic bonds is one of key steps to produce valuable chemicals; however, previous chemical and enzymatic processes have been suffered from their fundamental drawbacks (e.g. not environmentally benign, high production cost) and are not regarded as feasible approaches. Recently, mechanocatalytic process, which proceeds hydrolysis of polysaccharides by simply milling with solid acids, attracts attentions owing to its cost-effectiveness and environmental beingness. Herein, we propose the novel mechanocatalytic system to efficiently proceed the hydrolysis of glycosidic bonds in polysaccharides by using “vibratory disk mill”, which can produce strong shear and impact force. By milling polysaccharides (cellulose, chitin and xylan) with solid acid kaolin, the polysaccharides can be efficiently hydrolysed to water-soluble saccharides with a rate twice higher than the previous milling devices, achieving quantitative conversion of polysaccharide to water soluble products (solubility: >99 %). We elucidated the reaction mechanism of polysaccharides with kaolin by X-ray diffractograms: forces produced by the vibratory disk mill can efficiently delaminate layered aluminosilicate nanosheets in kaolin, leading to better accessibility of polysaccharides on the acidic surface of the nanosheets. This novel mechanocatalytic system can also be applicable to produce oligosaccharides directly from crude biomass samples (birchwood chips and shrimp shells) without any chemical purification steps, demonstrating that this system can be used in industrial biomass processing.

Developing Catalysts for the Hydrolysis of Glycosidic Bonds in Oligosaccharides Using a Spectrophotometric Screening Assay.

In a proof-of-concept study, a method for the empirical design of polyacrylate gel catalysts with the ability to cleave 1→4 α-glycosidic bonds in di- and trisaccharides was elaborated. The study included the synthesis of a 300-gel member library based on two different cross-linkers and 10 acrylate monomers, identification of monomodal gels by dynamic light scattering, and a 96-well plate spectrophotometric screening assay to monitor the hydrolysis of chromophore-free maltose into glucose units. The composition of the matrix of the most efficient catalysts in the library was found to enable CH-π, hydrophobic, and H-bond accepting interactions during the hydrolysis as typically seen in glycosylases. The same gel catalysts allowed the hydrolysis of the trisaccharide maltotriose with a catalytic proficiency of 2 × 106 indicating transition state stabilization during the hydrolysis of 5 × 10-7. The results place the developed gels among the most efficient catalysts developed for the hydrolysis of natural saccharides. The elaborated strategy may lead to catalysts that can transform polysaccharides into valuable synthons in the near future.

Development of a Zn-Based Single-Atom Nanozyme for Efficient Hydrolysis of Glycosidic Bonds.

Hydrolytic enzymes are essential components in second-generation biofuel technology and food fermentation processes. Nanozymes show promise for large-scale industrial applications as replacements for natural enzymes due to their distinct advantages. However, there remains a research gap concerning glycosidase nanozymes. In this study, a Zn-based single-atom nanozyme (ZnN4-900) is developed for efficient glycosidic bond hydrolysis in an aqueous solution. The planar structure of the class-porphyrin N4 material approximatively mimicked the catalytic centers of natural enzymes, facilitating oxidase-like (OXD-like)activity and promoting glycosidic bond cleavage. Theoretical calculations show that the Zn site can act as Lewis acids, attacking the C─O bond in glycosidic bonds. Additionally, ZnN4-900 has the ability to degrade starch and produce reducing sugars that increased yeast cell biomass by 32.86% and ethanol production by 14.56%. This catalyst held promising potential for enhancing processes in ethanol brewing and starch degradation industries.

Frequent Acquisition of Glycoside Hydrolase Family 32 (GH32) Genes from Bacteria via Horizontal Gene Transfer Drives Adaptation of Invertebrates to Diverse Sources of Food and Living Habitats.

Glycoside hydrolases (GHs, also called glycosidases) catalyze the hydrolysis of glycosidic bonds in polysaccharides. Numerous GH genes have been identified from various organisms and are classified into 188 families, abbreviated GH1 to GH188. Enzymes in the GH32 family hydrolyze fructans, which are present in approximately 15% of flowering plants and are widespread across microorganisms. GH32 genes are rarely found in animals, as fructans are not a typical carbohydrate source utilized in animals. Here, we report the discovery of 242 GH32 genes identified in 84 animal species, ranging from nematodes to crabs. Genetic analyses of these genes indicated that the GH32 genes in various animals were derived from different bacteria via multiple, independent horizontal gene transfer events. The GH32 genes in animals appear functional based on the highly conserved catalytic blades and triads in the active center despite the overall low (35-60%) sequence similarities among the predicted proteins. The acquisition of GH32 genes by animals may have a profound impact on sugar metabolism for the recipient organisms. Our results together with previous reports suggest that the acquired GH32 enzymes may not only serve as digestive enzymes, but also may serve as effectors for manipulating host plants, and as metabolic enzymes in the non-digestive tissues of certain animals. Our results provide a foundation for future studies on the significance of horizontally transferred GH32 genes in animals. The information reported here enriches our knowledge of horizontal gene transfer, GH32 functions, and animal-plant interactions, which may result in practical applications. For example, developing crops via targeted engineering that inhibits GH32 enzymes could aid in the plant's resistance to animal pests.

Horizontally transferred glycoside hydrolase 26 may aid hemipteran insects in plant tissue digestion

Glycoside hydrolases are enzymes that break down complex carbohydrates into simple sugars by catalyzing the hydrolysis of glycosidic bonds. There have been multiple instances of adaptive horizontal gene transfer of genes belonging to various glycoside hydrolase families from microbes to insects, as glycoside hydrolases can metabolize constituents of the carbohydrate-rich plant cell wall. In this study, we characterize the horizontal transfer of a gene from the glycoside hydrolase family 26 (GH26) from bacteria to insects of the order Hemiptera. Our phylogenies trace the horizontal gene transfer to the common ancestor of the superfamilies Pentatomoidea and Lygaeoidea, which include stink bugs and seed bugs. After horizontal transfer, the gene was assimilated into the insect genome as indicated by the gain of an intron, and a eukaryotic signal peptide. Subsequently, the gene has undergone independent losses and expansions in copy number in multiple lineages, suggesting an adaptive role of GH26s in some insects. Finally, we measured tissue-level gene expression of multiple stink bugs and the large milkweed bug using publicly available RNA-seq datasets. We found that the GH26 genes are highly expressed in tissues associated with plant digestion, especially in the principal salivary glands of the stink bugs. Our results are consistent with the hypothesis that this horizontally transferred GH26 was co-opted by the insect to aid in plant tissue digestion and that this HGT event was likely adaptive.

Enhancing Xylanase Production from Aspergillus tamarii Kita and Its Application in the Bioconversion of Agro-Industrial Residues into Fermentable Sugars Using Factorial Design

The endo-1,4-β-xylanases (EC 3.2.1.8) are the largest group of hydrolytic enzymes that degrade xylan, the major component of hemicelluloses, by catalyzing the hydrolysis of glycosidic bonds β-1,4 in this polymer, releasing xylooligosaccharides of different sizes. Xylanases have considerable potential in producing bread, animal feed, food, beverages, xylitol, and bioethanol. The fungus Aspergillus tamarii Kita produced xylanases in Adams’ media supplemented with barley bagasse (brewer’s spent grains), a by-product from brewery industries. The culture extract exhibited two xylanase activities in the zymogram, identified by mass spectrometry as glycosyl hydrolase (GH) families 10 and 11 (GH 10 and GH 11). The central composite design (CCD) showed excellent predictive capacity for xylanase production (23.083 U mL−1). Additionally, other enzyme activities took place during the submerged fermentation. Moreover, enzymatic saccharification based on a mixture design (MD) of three different lignocellulosic residues was helpful in the production of fermentable sugars by the A. tamarii Kita crude extract.

Fucanases Related to the GH107 Family from Members of the PVC Superphylum

The glycoside hydrolase 107 (GH107) family includes fucanase enzymes from only two bacterial phyla, Bacteroidota and Pseudomonadota. The goal of this work was to explore the diversity of putative fucanase enzymes related to this family in organisms of the PVC superphylum (Planctomycetota, Verrucomicrobiota, Chlamydiota), in order to expand our knowledge of the fucoidan-degrading potential in this ecologically and biotechnologically relevant group. Using hidden Markov model- and peptide-based annotation tools, 26 GH107 homolog sequences were identified in metagenome and genome datasets. The sequences formed two distinct clusters in a phylogenetic analysis, only one including members of the GH107 family. The endo-acting fucoidan degrading activity was confirmed in an enzyme included in the most divergent cluster. The fucanase, which probably originated in an uncultured planctomycete from the sampled subantarctic sediments, was cloned and expressed in Escherichia coli. The enzyme catalyzed the rapid hydrolysis of internal glycosidic bonds of fucoidan from Macrocystis pyrifera, a macroalgae species abundant at the site. It was active in a wide range of temperatures (5–45 °C), salinities (9.5–861 mM NaCl), and pH values (4.5–9), mainly producing sulfated α-(1,3)-linked fuco-oligosaccharides of various lengths. The PVC superphylum represents a promising source of fucanase enzymes with various biotechnological applications.

A Broad-Spectrum α-Glucosidase of Glycoside Hydrolase Family 13 from Marinovum sp., a Member of the Roseobacter Clade.

Glycoside hydrolases (GHs) are a diverse group of enzymes that catalyze the hydrolysis of glycosidic bonds. The Carbohydrate-Active enZymes (CAZy) classification organizes GHs into families based on sequence data and function, with fewer than 1% of the predicted proteins characterized biochemically. Consideration of genomic context can provide clues to infer possible enzyme activities for proteins of unknown function. We used the MultiGeneBLAST tool to discover a gene cluster in Marinovum sp., a member of the marine Roseobacter clade, that encodes homologues of enzymes belonging to the sulfoquinovose monooxygenase pathway for sulfosugar catabolism. This cluster lacks a gene encoding a classical family GH31 sulfoquinovosidase candidate, but which instead includes an uncharacterized family GH13 protein (MsGH13) that we hypothesized could be a non-classical sulfoquinovosidase. Surprisingly, recombinant MsGH13 lacks sulfoquinovosidase activity and is a broad-spectrum α-glucosidase that is active on a diverse array of α-linked disaccharides, including maltose, sucrose, nigerose, trehalose, isomaltose, and kojibiose. Using AlphaFold, a 3D model for the MsGH13 enzyme was constructed that predicted its active site shared close similarity with an α-glucosidase from Halomonas sp. H11 of the same GH13 subfamily that shows narrower substrate specificity.

A chalcone-based ESIPT and AIE fluorophore for β-gal imaging in living cells.

β-Galactosidase (β-gal), which is responsible for the hydrolysis of the glycosidic bond of lactose to galactose, has been recognized as an important biomarker of cell or organism status, especially cell senescence and primary ovarian cancer. Extensive efforts have been devoted to develop probes for detecting and visualizing β-gal in cells. Herein, a fluorescent probe gal-HCA which possesses both excited-state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE) properties was prepared to monitor β-gal in living cells. The probe consists of 2-hydroxy-4'-dimethylamino-chalcone (HCA) capped with a D-galactose group. The cleavage of the glycosidic bond in gal-HCA triggered by β-gal releases HCA, which results in a significant bathochromic shift in fluorescence from 532 to 615 nm. The probe exhibited high selectivity and sensitivity toward β-gal with a detection limit as low as 0.0122 U mL-1. The confocal imaging investigation demonstrated the potential of gal-HCA in monitoring the endocellular overexpressed β-gal in senescent cells and ovarian cancer cells. This study provides a straightforward approach for the development of fluorescent probes to monitor β-gal and detection of β-gal-associated diseases.

Design strategies and biological applications of β-galactosidase fluorescent sensor in ovarian cancer research and beyond.

Beta-galactosidase (β-galactosidase), a lysosomal hydrolytic enzyme, plays a critical role in the catalytic hydrolysis of glycosidic bonds, leading to the conversion of lactose into galactose. This hydrolytic enzyme is used as a biomarker in various applications, including enzyme-linked immunosorbent assays (ELISAs), gene expression studies, tuberculosis classification, and in situ hybridization. β-Galactosidase abnormalities are linked to various diseases, such as ganglioside deposition, primary ovarian cancer, and cell senescence. Thus, effective detection of β-galactosidase activity may aid disease diagnoses and treatment. Activatable optical probes with high sensitivity, specificity, and spatiotemporal resolution imaging capabilities have become powerful tools for visualization and real time tracking in vivo in the past decade. This manuscript reviews the sensing mechanism, molecular design strategies, and advances of fluorescence probes in the biological application of β-galactosidase, particularly in the field of ovarian cancer research. Current challenges in tracking β-galactosidase and future directions are also discussed.

Glycosidic bond protection of cellulose during solvent dissolution by coordination interaction competition strategy

Exploiting new solvents on efficiently dissolving cellulose is imperative to promote the utilization of cellulosic resources. The process of cellulose dissolution typically necessitates extreme conditions, such as high-temperature treatment, utilization of potent acidic or basic solvents, or the catalytic action of Lewis acids. As a result, the structure of the cellulose is invariably compromised, subsequently obstructing the creation of high-performance materials. In this study, we address this challenge through a simple process, introducing polyethylene glycol (PEG) as glycosidic bond protecting agent, to preserve the polymerization degree of cellulose during its room-temperature dissolution in ZnCl2-phosporic acid eutectic solvent. The PEG units preferentially coordinate with Zn2+ to weaken the hydrolysis of glycosidic bond of cellulose through ether bond competition. The polymerization degree of regenerated cellulose is thus greatly improved, reaching up to seven times that of unprotected cellulose. Overall, this study offers an easy and cost-effective approach to develop cellulose solvents and provides a significant drive towards the fabrication of practical materials through cellulose dissolution.

Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae.

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes thecleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F.evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Å resolution, a single-domain organization comprising a (β/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

Glycoside hydrolases from (hyper)thermophilic archaea: structure, function, and applications.

(Hyper)thermophilic archaeal glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds to break down complex sugars and polysaccharides at high temperatures. These enzymes have an unique structure that allows them to remain stable and functional in extreme environments such as hot springs and hydrothermal vents. This review provides an overview of the current knowledge and milestones on the structures and functions of (hyper)thermophilic archaeal glycosidases and their potential applications in various fields. In particular, this review focuses on the structural characteristics of these enzymes and how these features relate to their catalytic activity by discussing different types of (hyper)thermophilic archaeal glycosidases, including β-glucosidases, chitinase, cellulases and α-amylases, describing their molecular structures, active sites, and mechanisms of action, including their role in the hydrolysis of carbohydrates. By providing a comprehensive overview of (hyper)thermophilic archaeal glycosidases, this review aims to stimulate further research into these fascinating enzymes.

Incubation of protonated NADH or NADPH with ortho-aminobenzaldehyde generates a novel fluorescent nicotinamide dihydroquinazoline condensate

Incubation of reduced nicotinamide adenine dinucleotide (NADH) but not oxidized NAD+ with ortho-aminobenzaldehyde (oABA) generated an uncharacterized chromophore with an absorption peak characteristic of a dihydroquinazoline condensate. This chromophore is responsible for a non-specific signal in a diamine oxidase (DAO) activity assay based on the generation of fluorescent dihydroquinazoline structures directly from DAO substrates.Herein we show that at pH values below 3.0 the glycosidic bond of NADH/NADPH is broken releasing double protonated dihydro-nicotinamide (dihydro-NAM), which consequently condensates with oABA to a novel dihydroquinazoline chromophore and fluorophore, namely the 6- or 8-carbamoyl-5H,7H,8H,9H-10λ⁵-pyrido[2,1-b]quinazolin-10-ylium isomer (CMPQ). The second protonation event closely correlates with the pKa of the N1 nitrogen of C5-protonated dihydro-NAM and fluorophore stability. The fusion partner of oABA is likely the iminium of the primary acid product of dihydro-NAM after glycosidic bond hydrolysis and before irreversible cyclization.Trapping of protonated dihydro-NAM from NADH or NADPH with oABA allows quantification of these dinucleotides. Despite almost a century of research studying acid-catalyzed molecular rearrangements of NADH and NADPH, new and surprising details can be discovered.

Distinctive Formation of a DNA-Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations.

Reactive oxygen species damage DNA and result in health issues. The major damage product, 8-oxo-7,8-dihydroguanine (8oG), is repaired by human adenine DNA glycosylase homologue (MUTYH). Although MUTYH misfunction is associated with a genetic disorder called MUTYH-associated polyposis (MAP) and MUTYH is a potential target for cancer drugs, the catalytic mechanism required to develop disease treatments is debated in the literature. This study uses molecular dynamics simulations and quantum mechanics/molecular mechanics techniques initiated from DNA-protein complexes that represent different stages of the repair pathway to map the catalytic mechanism of the wild-type MUTYH bacterial homologue (MutY). This multipronged computational approach characterizes a DNA-protein cross-linking mechanism that is consistent with all previous experimental data and is a distinct pathway across the broad class of monofunctional glycosylase repair enzymes. In addition to clarifying how the cross-link is formed, accommodated by the enzyme, and hydrolyzed for product release, our calculations rationalize why cross-link formation is favored over immediate glycosidic bond hydrolysis, the accepted mechanism for all other monofunctional DNA glycosylases to date. Calculations on the Y126F mutant MutY highlight critical roles for active site residues throughout the reaction, while investigation of the N146S mutant rationalizes the connection between the analogous N224S MUTYH mutation and MAP. In addition to furthering our knowledge of the chemistry associated with a devastating disorder, the structural information gained about the distinctive MutY mechanism compared to other repair enzymes represents an important step for the development of specific and potent small-molecule inhibitors as cancer therapeutics.

Design and application of prodrug fluorescent probes for the detection of ovarian cancer cells and release of anticancer drug

Ovarian cancer is a gynecologic malignancy with high mortality. The main reason is that it is detected at an advanced stage due to a lack of early diagnosis and treatment. Therefore, it is of great interest to develop a chemical tool that can visualize ovarian cancer cells in real-time and eliminate them. Unfortunately, probes that can simultaneously monitor both modes of action for the diagnosis and treatment of ovarian cancer have not been developed. Here, we designed a novel prodrug fluorescent probe (YW-OAc) that not only visually tracks cancer cells but also enables the on-demand delivery of chemotherapeutic agents. By β-Gal-mediated glycosidic bond hydrolysis, the fluorescent signal changed from blue to green (signal 1), enabling visual tracking of ovarian cancer cells. Subsequently, the identified cancer cells were subjected to precise light irradiation to induce anticancer drug release accompanied by a fluorescence transition from green to blue (signal 2), enabling real-time information on drug release. Thus, the prodrug fluorescent probe YW-OAc provides comprehensive two-step monitoring during cancer cell recognition and clearance. Notably, YW-OAc exhibited high affinity (Km = 3.74 μM), high selectivity, and low detection limit for β-Gal (0.0035 U/mL). We also demonstrated that YW-OAc can visually trace endogenous β-Gal in different cells and exhibit high phototoxicity in ovarian cancer cells. We hope that the prodrug fluorescent probe YW-OAc, can be used as an effective tool for biomedical diagnosis and treatment.

Nanogel Catalysts for the Hydrolysis of Underivatized Disaccharides Identified by a Fast Screening Assay

Applying high-throughput synthetic and screening methods for the development of catalytic micro- and nanogels remains challenging due to the nature of the workflow. Typical procedures require the preparation of stable miniemulsions, followed by radical polymerization, purification of the obtained gels by extensive extraction or dialysis, and subsequent characterization of physical properties and examination of desired functions. To streamline the process, we altered the gel synthesis procedure and developed a screening protocol to identify gel compositions with the desired catalytic function. The assays identify five out of 50 synthesized gels from eight different cross-linkers with high potential to hydrolyze carbohydrates. A catalytic proficiency (kcat/KM × knon) of up to 1.4 × 106 and gel activity of 0.8 μmol h–1 mg–1 were observed placing the synthesized gels among the most proficient catalysts known for the hydrolysis of nonactivated glycosidic bonds.

The impact of thermal extraction on the quality of Phyllanthus emblica Linn. fruit: A systematic study based on compositional changes

Phyllanthus emblica (PE) is a well-known tropical crop with a distinct flavor and several health advantages. From the standpoint of the transformation of volatile and nonvolatile components, the purpose of this paper is to investigate the impact of heat extraction on the flavor attributes and biological activity of PE. According to studies, thermal extraction can greatly enhance the amount of the molecule 4,5-epoxy-(E)-2-decanal with a green odor while removing offensive scents like 2-isobutyl-3-methoxy pyrazine. Temporal dominant description evaluation found that with the extension of thermal extraction time, the five flavors of PE are enhanced. According to high-resolution mass spectrometry studies, the primary reaction processes during thermal extraction were the hydrolysis and condensation of tannin and flavonoid glycosidic bonds. The main core groups of compound transformation were galloyl and hexahydroxydibenzoyl (HHDP), which led to the final product gallic acid and ellagic acid were significantly increased. Finally, it was found that thermal extraction can significantly enhance the antioxidant and antibacterial activities of PE.

Designing robust xylan/chitosan composite shells around drug-loaded MSNs: Stability in upper GIT and degradation in the colon microbiota

Long residence times, near-neutral pH values, and release triggered by the enzymatic action of the resident microbiota offer unique opportunities for improved drug delivery in the colon. The fact that a delivery agent must also pass through the complete GI tract without degradation presents a challenge due to widely changing pH conditions. In this study, a promising colon-targeted drug delivery system was composed of a xylan/chitosan composite shell formed on curcumin-loaded mesoporous silica nanoparticles (MSNs). A novel synthesis approach was employed to facilitate precipitation of negatively charged xylan on negatively charged MSNs by concurrent chitosan polymerization. Curcumin-loaded xylan/chitosan-coated MSNs (C-MSNs) were determined to contain nearly 42% xylan by the inclusion of chitosan in a one-to-one ratio with xylan. The xylan/chitosan composite shell demonstrated excellent stability in the acidic upper GI tract. The hydrolysis of glycosidic bonds by resident microbiota was the triggering mechanism for xylan degradation and curcumin release in the colon. The presence of xylan has the further benefit of increasing the number of beneficial bacteria and improving short-chain fatty acid production for improved colon health.

Proteomic analysis response of rice (Oryza sativa) leaves to ultraviolet-B radiation stress.

Rice (Oryza sativa) is a human staple food and serves as a model organism for genetic and molecular studies. Few studies have been conducted to determine the effects of ultraviolet-B (UV-B) stress on rice. UV-B stress triggers morphological and physiological changes in plants. However, the underlying mechanisms governing these integrated responses are unknown. In this study, we conducted a proteomic response of rice leaves to UV-B stress using two-dimensional gel electrophoresis and identified the selected proteins by mass spectrometry analysis. Four levels of daily biologically effective UV-B radiation intensities were imposed to determine changes in protein accumulation in response to UV-B stress: 0 (control), 5, 10, and 15 kJ m−2 d−1in two cultivars, i.e., IR6 and REX. To mimic the natural environment, we conducted this experiment in Sunlit Soil-Plant-Atmosphere-Research (SPAR) chambers. Among the identified proteins, 11% of differentially expressed proteins were found in both cultivars. In the Rex cultivar, only 45% of proteins are differentially expressed, while only 27.5% were expressed in IR6. The results indicate that REX is more affected by UV-B stress than IR6 cultivars. The identified protein TSJT1 (spot 16) in both cultivars plays a crucial role in plant growth and development during stress treatment. Additionally, we found that UV-B stress altered many antioxidant enzymes associated with redox homeostasis and cell defense response. Another enzyme, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been identified as spot 15, which plays an essential role in glycolysis and cellular energy production. Another vital protein identified is glycosyl hydrolase (GH) as spot 9, which catalyzes the hydrolysis of glycosidic bonds in cell wall polymers and significantly affects cell wall architecture. Some identified proteins are related to photosynthesis, protein biosynthesis, signal transduction, and stress response. The findings of our study provide new insights into understanding how rice plants are tailored to UV-B stress via modulating the expression of UV-B responsive proteins, which will help develop superior rice breeds in the future to combat UV-B stress. Data are available via ProteomeXchange with identifier PXD032163.