Structural Characterization Research Articles

Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose.Key points• β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield.• Thermotoga maritima β-glucosidase has high activity and high thermostability.• Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.Graphical

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.

Novel Aluminum Alloy Tailored for Additive Manufacturing: Structural Characterization and Qualification Perspectives

The recent advances achieved in additive manufacturing (AM) technology demonstrate the potential to realize customized metal components, ensuring weight reduction opportunities. These benefits make AM attractive for high-cost aerospace applications, especially where high geometric complexity is required. In the context of an EU research scenario, the H2020 MANUELA (Additive Manufacturing Using Metal Pilot Line) project promotes the development of new technologies for design optimization by enabling the application of novel materials in AM. This paper illustrates recent advances in a new aluminum alloy (Al-HS1) with high strength emphasizing all of the characterization steps at the coupon level. This material has been employed in the re-engineering of a conventional hydraulic manifold using a powder bed fusion-laser beam (PBF-LB) process. Both the simulations and structural tests allowed for proving its compliance and technological maturity with industrial standards and applicable airworthiness requirements.

Preparation and characterization of tea tree essential oil microcapsule-coated packaging paper with bacteriostatic effect

We prepared tea tree essential oil microcapsules, and the microcapsules and pullulan were coated on kraft paper to prepare an antibacterial paper. The antibacterial activity, structural characterization, and thermal stability of the prepared microcapsules and packaging paper were then tested. We found that the retention rate of microcapsules reached 87.1% after a 70 min of high-temperature treatment. The minimum inhibitory concentrations of microcapsules to S. aureus and E. coli were 112 mg/mL and 224 mg/mL, and the bacteriostatic zones of the packaging paper to E. coli and S. aureus were 17.49 mm and 22.75 mm, respectively. The prepared microcapsules were irregular. The paper coating was formed via hydrogen bonding, which filled the pores of paper fibers. When compared with the base paper, the roughness of the paper was reduced to 7.16 nm (Rq) and 5.61 nm (Ra), and no thermal decomposition occurred at <288 °C, which together implies a good application prospect.

Mechanical characterization and constitutive modeling of additively-manufactured polymeric materials and lattice structures

Additively manufactured polymeric lattice structures are being extensively studied, primarily because their mechanical properties can be tailored by controlling the unit cell geometry, giving them higher designability than stochastic materials. However, the inherent layer-wise additive manufacturing process affects the base material properties related to the printing direction, which in turn affects the macroscopic responses of the entire lattice materials. A robust understanding and modeling of lattice structures' elastic and plastic yield behavior in a homogenized approach are essential to enhance their design and analysis efficiency in engineering applications. In pursuit of this goal, a unified printing angle-dependent constitutive model of base materials is proposed in line with the tensile experimental data. The elastic material properties (elastic modulus, shear modulus, and Poisson's ratio), obtained through numerical simulations of one unit-cell with periodic boundary conditions, exhibit anisotropic properties, with the degree of anisotropy determined by the angle of the constituent members and base materials. Furthermore, both experimental and numerical results of lattices demonstrate anisotropic mechanical response under horizontal and vertical compression. Virtual multiaxial experiments are conducted through multi-cell numerical simulations, enabling the determination of initial yielding points of two different lattice structures (Kelvin and Simple cubic and body-centered cubic hybrid structures) under various loading conditions using a dissipation energy-based criterion. Overall, the multiaxial yield surface of the investigated lattices under various stress states, except for the isotropic principal stress plane, can be properly depicted by the Extended-Hill anisotropic yield criterion.

Condensation or Desublimation: Nanolevel Structural Look on Two Frost Formation Pathways on Surfaces with Different Wettabilities.

Processes of water condensation and desublimation on solid surfaces are ubiquitous in nature and essential for various industrial applications, which are crucial for their performance. Despite their significance, these processes are not well understood due to the lack of methods that can provide insight at the nanolevel into the very first stages of phase transitions. Taking advantage of synchrotron grazing-incidence wide-angle X-ray scattering (GIWAXS) and environmental scanning electron microscopy (ESEM), two pathways of the frosting process from supersaturated vapors were studied in real time for substrates with different wettabilities ranging from highly hydrophilic to superhydrophobic. Within GIWAXS, a fully quantitative structural and orientational characterization of the undergoing phase transition reveals the information on degree of crystallinity of the new phase and determines the ordering at the surfaces and inside the films at the initial stages of water/ice nucleation from vapor onto the substrates. The diversity of frosting scenarios, including direct desublimation from the vapor and two-stage condensation-freezing processes, was observed by both GIWAXS and ESEM for different combinations of substrate wettability and vapor supersaturations. The classical nucleation theory straightforwardly predicts the pathway of the phase transition for hydrophobic and superhydrophobic substrates. The case of hydrophilic substrates is more intricate because the barriers in Gibbs free energy for nucleating both liquid and solid embryos are close to each other and comparable to thermal energy kBT. At that end, classical nucleation theory allows concluding a relation between contact angles for ice and water embryos on the basis of the observed frosting pathway.

Hexagonal Mo Bronze: Single Crystal Structures, Electrocatalytic Hydrogen Evolution, and Proton Conductivity.

Molybdenum trioxide (MoO3) is a well-known transition metal oxide that has drawn much attention as a functional material having numerous applications. However, a vast majority of studies have primarily focused on α-MoO3, the thermodynamically stable polymorph of MoO3. This present work encompasses the synthesis of single crystals of two metastable hexagonal MoO3 described by the formulas {Mn0.03Na0.01}@[Mo0.93VIMo0.07VO3] (1) and {Cu0.01Na0.01}@[Mo0.97VIMo0.03VO3] (2), their comprehensive structural characterization by single-crystal X-ray crystallography, and routine spectral and microscopic studies. Interestingly, compound 1 acts as an efficient electrocatalyst for the hydrogen evolution reaction (HER) as well as an effective proton conductor in comparison to the performance of compound 2. The HER activity of compound 1 is characterized by an overpotential of 340 mV@1 mA cm-2 and a low Tafel slope of 75 mV/decade. The catalyst (compound 1) displays a Faradaic efficiency of 88% with a turnover frequency of 2.9 s-1. The proton conductivity value of this compound (1) is determined to be 4.9 × 10-3 S cm-1 at 55 °C under 98% relative humidity; the relevant proton conduction is operated by the Grotthuss mechanism with an activation energy of 0.17 eV.

Inhibition of Phase Transition in Amorphous Niobium Oxide by Covalent Carbon Reinforcement Enables Fast‐Charge and Long‐Duration Lithium Storage

AbstractNiobium oxides are potential anode materials for ultrafast and safe lithium‐ion batteries due to their high ionic conductivity and relatively high operation voltage. However, the electrochemically induced phase transformations that involve multi‐electron redox reactions in a wide voltage window cause rapid capacity degradation and poor rate capability. Here, a novel carbon‐covalent amorphous niobium oxide anode is reported to greatly suppress the phase transition during cycling via formation of strong Nb─O─C bonds, achieving high‐capacity, fast‐charge and long‐duration lithium storage. This amorphous structure and forming covalent carbon contribute to good volume accommodation and high electron conductivity. The carbon‐covalent amorphous Nb2O5 displays a high reversible capacity of 361.5 mAh g−1 at 0.1 A g−1 and excellent cycling stability with a capacity of 189.8 mAh g−1 at a high rate of 10 A g−1 after 9000 cycles. Structure characterizations reveal that the well‐preserved amorphous structure without phase transition during repeated Li+ insertion/desertion is responsible for the superior performance. This work opens a new avenue on rational design of high‐performance amorphous electrode materials for next‐generation batteries.

Monosaccharide composition analysis by 2D quantitative gsHSQCi

The physicochemical properties and biological activities of polysaccharides depend on their structures. Monosaccharide composition analysis is indispensable for the structural characterization of polysaccharides and is helpful in the quality control of polysaccharide preparation. Here, using a model mixture and tamarind seed polysaccharide as examples, we demonstrated that a quantitative 2D NMR method, gsHSQCi (three gradient-selective Heteronuclear Single Quantum Coherence spectra acquired with incremented repetition times, i = 1, 2, 3) can directly quantify a variety of monosaccharides in solution with adequate precision and accuracy, requiring no derivatization, postprocessing steps and column separation. Both anomeric and non-anomeric signals of monosaccharides can be utilized for content determination. More accurate quantification of fructose in a mixture containing nine monosaccharides is obtained, which is difficult to achieve by quantitative 1D 1HNMR and the common PMP-HPLC method (high-performance liquid chromatography through pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone) due to the peak overlapping and the poor derivatization efficiency, respectively. The results also revealed that Na[Fe(EDTA)] can serve as a proper relaxation-enhancing agent for saccharide samples to save experimental time. We expect that this approach can be applied as an alternative to analyzing the monosaccharide composition and be helpful in interpreting the structure of polysaccharides.

Surface Encapsulation of Layered Oxide Cathode Material with NiTiO3 for Enhanced Cycling Stability of Na-Ion Batteries

Abstract In Na-ion batteries, O3-type layered oxide cathode materials encounter challenges such as particle cracking, oxygen loss, electrolyte side reactions, and multi-phase transitions during the charge/discharge process. This study focuses on surface coating with NiTiO3 achieved via secondary heat treatment using a coating precursor and the surface material. Through in-situ X-ray diffraction (XRD) and differential electrochemical mass spectrometry (DEMS), along with crystal structure characterizations of post-cycling materials, it was determined that the NiTiO3 coating layer facilitates the formation of a stable lattice structure, effectively inhibiting lattice oxygen loss and reducing side reaction with the electrolyte. This enhancement in cycling stability is evidenced by a capacity retention of approximately 74% over 300 cycles at 1C, marking a significant 30% improvement over the initial sample. Furthermore, notable advancements in rate performance were observed. Experimental results indicate that a stable and robust surface structure substantially enhances the overall stability of the bulk phase, presenting a novel approach for designing layered oxide cathodes with higher energy density.

Effects of Micropores on the Vapor Sorption Isotherm and Structural Parameters Characterization of Kaolinite

The vapor sorption isotherm (VSI) of soil serves as a basic tool in geotechnical engineering, widely employed for characterizing the structural parameters of soil. Micropores are commonly found in soil, significantly altering the VSI and impacting the characterization of structural parameters. Nonetheless, these micropores are often overlooked in relevant analyses. Moreover, the characterization process relies on the utilization of theoretical models to fit the VSI. However, the commonly used models are not designed for VSI, raising concerns about their reliability. To investigate the influence of micropores on VSI and assess the reliability of various characterization models, this study used molecular simulation techniques to generate VSIs for kaolinite samples with different pore sizes. The simulation results reveal the effects of pore size on VSI shape, shed light on the influence of soil surfaces on pore water density and orientation, and highlight the important role of hydrogen bonds in water retention within the kaolinite pores. Furthermore, the reliability of three structural parameter characterization models, namely BET, DR, and as plot, was evaluated with the aid of the explicit geometric dimensions in molecular models, and the mechanisms responsible for their reliability were elucidated. This study deepens the understanding of VSI and provides crucial references for the characterization of soil structural parameters.

Tuning Lubrication Performance of Phase‐changing Emulsion‐filled Gels by Sugar alcohols

Lubrication by hydrogels is proving to be an emerging area in colloid science. Although oil‐driven friction reduction is predominant in literature, emulsion gel‐based lubrication has attracted very little attention to date. We hypothesize that an interplay of viscolelasticity and oil volume fraction may modulate lubricity of emulsion‐filled gels. Herein, phase‐change gelatin‐based emulsion‐filled gels with different sugar alcohol and oil droplet concentrations (0‐30 wt%) were tested for their lubrication performance. The rheological and tribological tests were analysed in conjunction with spectroscopic and structural characterizations in order to reveal structure‐function relationship. Structural characterizations demonstrated that hydrogen bonding was enhanced in pure gelatin gels with the increase of sugar alcohol (glycerol)‐replacement time, which enhanced the storage modulus (G') of the gel networks. Emulsified droplets served as active fillers further strengthening the G’ and strikingly the presence of glycerol reduced the thermo‐responsiveness of the emulsion‐filled gels. Emulsion‐filled gels containing sugar alcohols, irrespective of their type, can greatly reduce the friction coefficients (μ) between hydrophobic surfaces in the boundary and mixed regimes versus the systems without sugar alcohols. In the hydrodynamic regime, the friction coefficient of the system was proportional to the second plateau shear viscosity, regulated by the oil content.This article is protected by copyright. All rights reserved.

Nitrogen, sulfur co-coordinated iron single-atom catalysts with the optimized electronic structure for highly efficient oxygen reduction in Zn-air battery and fuel cell

Atomically dispersed iron–nitrogen-carbon (FesbndNsbndC) materials have been considered ideal catalysts for the oxygen reduction. Unfortunately, designing and adjusting the electronic structure of single-atom Fe sites to boost the kinetics and activity still faces grand challenges. In this work, the coordination environment engineering is developed to synthesize the FeSA/NSC catalyst with the tailored N, S co-coordinated Fe atomic site (Fe-N3S site). The structural characterizations and theoretical calculations demonstrate that the incorporation of sulfur can optimize the charge distribution of Fe atoms to weaken the adsorption of OH* and facilitate the desorption of OH*, thus leading to enhanced kinetics process and intrinsic activity. As a result, the S-modified FeSA/NSC exhibits outstanding catalytic activity with the half-wave potentials (E1/2) of 0.915 V and 0.797 V, as well as good stability, in alkaline and acidic electrolytes, respectively. Impressively, the excellent performance of FeSA/NSC is further confirmed in Zn-air batteries (ZABs) and fuel cells, with high peak power densities (146 mW cm−2 and 0.259 W cm−2).

Structural characterization and attempted displacement interpretation of the Baishuihe landslide using integrated geophysical methods

The Baishuihe landslide, a typical large-scale deposit landslide in the Three Gorges Reservoir area, has attracted considerable societal and scholarly attention. Since 2003, 11 displacement monitoring points have been established on the landslide mass for monitoring displacement data. Displacement monitoring data from 14 years have been widely applied in landslide displacement prediction research. However, there is a lack of research involving interpretations of displacement data. To interpret landslide displacement from geological and hydrodynamic perspectives, we applied integrated geophysical methods, including electrical resistivity tomography (ERT), self-potential (SP), and surface nuclear magnetic resonance (SNMR), to model the landslide structure and investigate groundwater activity. The results indicate that the landslide deposit is predominantly located at the foot of the landslide, with a total volume of approximately 7.7 × 106 cubic meters. The thickness gradually increases in the eastern direction of the landslide along the sliding direction, while in the western direction, it initially increases and then decreases. The SNMR results reveal that groundwater is primarily situated in the gravel soil layer, demonstrating a volumetric water content of approximately 2.8% per unit volume. The SP results suggest that below an elevation of 190 m, the groundwater flow field is turbulent and irregular. Above an elevation of 190 m, the general direction of the groundwater flow field tends to align roughly with the sliding direction. Through the integration of GPS displacement monitoring data and comprehensive geophysical analysis, we discerned that the thickness of the deposit layer, the morphology of the sliding surface, and the interaction with groundwater recharge collectively exert significant influences on the displacement of the Baishuihe landslide. Generally, the thickness of the deposit layer and the morphology of the sliding surface determine whether the landslide will exhibit significant displacement. At a more detailed level, the weight of the accumulated mass and the influence of groundwater play a significant role in determining the displacement velocity.

Structural characterization and bioactivities of maize bran feruloylated arabinoxylan and oligosaccharides obtained by a combined hydrothermal and enzymatic pre-treatment

Feruloylated arabinoxylans (F-AX) are gaining importance due to their immense health benefits as nutraceuticals. In the current study, we extracted F-AX from maize bran by a combination of hydrothermal pre-treatment and xylanase and cellulase double enzymatic digestion and studied them in detail. The F-AX composition included carbohydrate (533.74 ± 7.59 mg/g), and phenolic acid (153.71 ± 3.55 mg/g) with an arabinoxylan percentage of 74.17% and degree of substitution of 0.99. Ferulic acid was the major phenolic acid found esterified to arabinose. The GC, FTIR, UPLC, and methylation analysis revealed the typical structure of arabinoxylan with bound ferulic acid and a highly substituted xylopyranose backbone. The LC-MS/MS analysis revealed the structure of F-AXOS, which were the predominant oligosaccharides in the purified fraction. The F-AX showed potent antioxidant activity (DPPH, ABTS, and FRAP) and could effectively inhibit the formation of advanced glycation end products (argpyrimidine, pentosidine, and vesperlysine) and protein oxidation products (dityrosine and N′-formylkynurenine). F-AX also showed potent antiproliferative activity against the human colon cancer cells - Colo 205 and HCT 116. The current study revealed that the combined extraction method has yielded a unique blend of F-AX with bioactivities and these molecules have the potential to be used in the development of innovative functional foods.

Structural and Biochemical Characterization of the Nucleosome Containing Variants H3.3 and H2A.Z.

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA's flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.

Preparation and structural characterization of disordered Na-gallosilicate zeolite with natrolite framework and its K+ and NH4+ exchanged analogues

Preparation and structural characterization of disordered Na-gallosilicate zeolite with natrolite framework and its K+ and NH4+ exchanged analogues

Dimensional Reduction of Metal-Organic Frameworks for Photocatalytic Synthesis of Fused Tetracyclic Heterocycles.

Heterogeneous palladium catalysts with high efficiency, high Pd atom utilization, simplified separation, and recycle have attracted considerable attention in the field of synthetic chemistry. Herein, we reported a zirconium-based two-dimensional metal-organic framework (2D-MOF)-based Pd(II) photocatalyst (Zr-Ir-Pd) by merging the Ir photosensitizers and Pd(II) species into the skeletons of the 2D-MOF for the Pd(II)-catalyzed oxidation reaction. Morphological and structural characterization identified that Zr-Ir-Pd with a specific nanoflower-like structure consists of ultrathin 2D-MOF nanosheets (3.85 nm). Due to its excellent visible-light response and absorption capability, faster transfer and separation of photogenerated carriers, more accessible Pd active sites, and low mass transfer resistance, Zr-Ir-Pd exhibited boosted photocatalytic activity in catalyzing sterically hindered isocyanide insertion of diarylalkynes for the construction of fused tetracyclic heterocycles, with up to 12 times the Pd catalyst turnover number than the existing catalytic systems. In addition, Zr-Ir-Pd inhibited the competitive agglomeration of Pd(0) species and could be reused at least five times, owing to the stabilization of 2D-MOF on the single-site Pd and Ir sites. Finally, a possible mechanism of the photocatalytic synthesis of fused tetracyclic heterocycles catalyzed by Zr-Ir-Pd was proposed.

Novel azo- and bisazo-5-pyrazolone dyes and their application as a colorimetric chemosensor for naked eye recognition of Cu2+

In this study, novel azo- and bisazo-5-pyrazolones (1/2a, b, and 1/2c, d, respectively) were synthesized. The structures of the new compounds were characterized using FTIR, UV–Vis., 1H NMR, 13C NMR-APT, and HRMS spectroscopic methods. 1H-NMR study revealed that the compounds are in hydrazone- form in solution. Among fourteen metal nitrate salts, the azo-5-pyrazolones showed a colorimetric response only to the Cu2+ ion, whereas the new bisazo-5-pyrazolones did not cause a specific color change to only one cation. The produced ligand changed from orangey-yellow to very light yellow, making it possible to identify Cu2+ without the need for spectroscopic equipment. Compound 1b [4-((2,4-dimethylphenyl)diazenyl)-2-(naphthalene-1-yl)-5-phenyl-2,4-dihydro-3H-pyrazole-3-one)] was taken as a representative example of the new azo-5-pyrazolones for the chemosensor research. The Cu(1b)2 complex was also synthesized and characterized. The binding ratio between the 1b sensor and Cu2+ was found to be 2:1 (ligand:metal) from the structural characterization of Cu(1b)2 and using Job's plot method. The pH study showed that even if the pH changes, the Cu2+ ion can still bind to the 1b receptor. In the reversibility experiment, when EDTA was added to the (1b+Cu2+) solution, the spectrum returned to the 1b spectrum. The interference study showed the selectivity of 1b with Cu2+ even in the presence of other cations. A study of the detection limit (LOD) revealed considerable sensitivity towards Cu2+metal cation using absorption titration method and LOD value was found to be 4.31 μM. The association constant (Ka) of (1b+Cu2+) was determined to be 0.79×105M−1 using the Benesi-Hildebrand plot.

Effect of low-frequency ultrasound-assisted acid extraction on gel properties and structural characterization of sheep's hoof gelatin

In this study, we investigated the effects of various low-frequency ultrasound-assisted extraction processes, including ultrasound-assisted acid-soaked water bath extraction (UAW), ultrasound-assisted water bath extraction after acid soaking (AUW), acid-soaked water bath extraction followed by ultrasonics (AWU), and acid-soaked water bath extraction without ultrasound (CON), on the structural properties, thermal stability, gel properties, and microstructure of sheep's hoof gelatin. The results revealed that the primary components of sheep's hoof gelatin consisted of α1-chain, α2-chain (100–135 kDa), and β-chain. In addition, it was observed that among the three sonication groups, sheep's hoof gelatin extracted in the AUW group exhibited the highest yield (27.16 ± 0.41 %), the best gel strength (378.55 ± 7.34 g), and higher viscosity at the same shear rate. The gelling temperature (25.38 ± 0.45 °C) and melting temperature (32.28 ± 0.52 °C) of sheep's hoof gelatin in the AUW group were significantly higher than those in the other groups (p > 0.05). Moreover, our experiments revealed that the sequence of low-frequency ultrasonic pretreatment processes was a crucial factor influencing the gel properties and structural characteristics of sheep's hoof gelatin. Specifically, the acid treatment followed by the ultrasound-assisted approach in the AUW group yielded high-quality and high-yield sheep's hoof gelatin.