Search Results (214)

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The puff adder (Bitis arietans) and saw-scaled viper (Echis romani) have cytotoxic venoms that cause permanent injury via dermonecrosis around the bite site. Identifying the cytotoxic toxins within these venoms will allow for the development of targeted treatments to prevent snakebite morbidity. In this study, venoms from both species were fractionated using gel filtration chromatography, and a combination of cytotoxicity approaches, SDS-PAGE gel electrophoresis, and enzymatic assays were applied to identify the venom cytotoxins in the resulting fractions. Our results indicate that snake venom metalloproteinase (SVMP) toxins are responsible for causing cytotoxic effects across both venoms. The PI subclass of SVMPs is likely the main driver of cytotoxicity following envenoming by B. arietans, while the structurally distinct PIII subclass of SVMPs is mostly responsible for conveying this effect in E. romani venom. Identifying distinct SVMPs as cytotoxicity-causing toxins in these two African viper venoms will facilitate the future design and development of novel therapeutics targeting these medically important venoms, which in turn could help to mitigate the severe life- and limb-threatening consequences of tropical snakebites. Full article

Olive mill wastewater (OMW), a byproduct of the olive oil industry, is a potential source of natural bioactive phenolic polymers. In this work, a column chromatography technique was used for the isolation of a new complex polymer (named OMW-2000XAD) from OMW via fractionation on Amberlite^® XAD16 resin. The developed procedure was simple and proved to be reproducible using OMW from two different sources. OMW-2000XAD was further characterized by elemental, glycosidic, and amino acid composition analysis, as well as spectroscopic techniques. The polymer’s molecular size, which was estimated via gel filtration chromatography, was 1960 kDa, which is significantly larger than other high-molecular weight fractions previously isolated from OMW or other agro-industrial wastes. OMW-2000XAD was mainly composed of phenolic compounds (89.8%). It also contained polysaccharides (16.1%) and proteins (10.3%), with glucose (12.25%) and cysteine (1.71%) being the most abundant sugar and amino acid, respectively, as well as metals (1.29%, primarily potassium). However, due to its low solubility, complexity, and heterogeneous composition, it was not possible to identify all phenolic compounds or elucidate a definitive structure via MS, FTIR, and NMR. OMW-2000XAD exhibited strong radical scavenging antioxidant capacity (ABTS^•+, DPPH^• and peroxyl radicals), with results up to 7415 µmol Trolox equivalent/mol (ORAC method), but showed no antiproliferative effects, highlighting the need for further research. Full article

SGT1 (the suppressor of the G2 allele of Skp1) functions as an adaptor protein that positively regulates plant defense and developmental processes. It comprises three functional domains: the tetratricopeptide repeat (TPR) domain, Chord SGT1 motif (CS), and SGT1-specific motif (SGS). In this study, we resolved the crystal structure of the Oryza sativa OsSGT1-TPR domain at 1.53 Å resolution. Structural analysis showed that the TPR domain adopts a homo-dimeric architecture stabilized by salt bridges (mediated by K52/R79/R109) and hydrophobic interactions (involving F17). Functional validation through gel filtration chromatography revealed that the disruption of the dimerization interface via F17A/K52A/R79A mutations caused complete dissociation into monomers, establishing the essential role of TPR-mediated oligomerization in maintaining the structural stability of full-length OsSGT1. Yeast two-hybrid assays showed that the dimerization disruption of SGT1 mutants retained the interaction with OsHSP81-2 (an HSP90 ortholog) and OsRAR1, indicating that SGT1 oligomerization serves primarily as a structural stabilizer rather than a prerequisite for partner interaction. Evolutionary analysis through the sequence alignment of plant SGT1 proteins revealed the conservation of the dimerization interface residues. This study provides structural insights into the conserved molecular features of SGT1 proteins and highlights the functional significance of their oligomerization state. Full article

Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (srfAA, ituC, fenD, bmyB, and bacA), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management. Full article

Aminosteroid derivative RM-581 stands out as an anticancer agent, supported by positive in vitro and in vivo studies on resistant cancers of the breast, prostate, and pancreas. A synthetic route has already been developed to obtain aminosteroid RM-581 in small quantities (scale of milligrams to a few grams). However, this route has significant limitations in view of its transposition to scaling up to larger quantities to support late preclinical and clinical trials. Among the problems are the use of toxic reagents, the moderate overall yield, and the need for multiple purifications through chromatographic columns. The development of a new synthetic route has therefore been explored. Starting from commercially available estrone, 2,4-dibromo-estrone was rapidly formed, followed by the regioselective introduction of a nitro group at the C2 position and by the methylation of phenol at the C3 position. The 4-bromo-2-nitro-3-O-methylestrone was then reduced to 2-amino-3-O-methylestrone and the primary amine was used to form the piperazine ring. Once the cyclization step was carried out, the last two steps were identical to the first synthetic route previously reported, i.e., introducing an ethynyl group at the C-17α position and then adding the quinoline-proline side chain with an N-acylation, assisted by a peptide coupling reagent. Importantly, no purification by chromatography was necessary during the whole sequence of reactions and only a final silica gel filtration, followed by recrystallization, led to RM-581 at a very high level of purity. The structure was also fully characterized by 2D NMR analysis. Full article

α-Glucosidase is an essential enzyme widely used in the food and biotechnology industries for the production of isomaltose oligosaccharides (IMOs). In this study, Aspergillus niger ASP004 was subjected to two rounds of nitrosoguanidine (NTG) mutagenesis, yielding a high-producing strain, NTG-II-3-64, with a 3.7-fold increase in enzyme activity under optimized fermentation conditions. The enzyme was purified through ultrafiltration, ethanol precipitation, DEAE ion-exchange, and gel filtration chromatography, resulting in an electrophoretically pure heterodimeric protein with a native molecular mass of approximately 230 kDa, composed of two subunits. The purified α-glucosidase exhibited optimal activity at 60 °C and pH 4.5, maintaining over 90% activity within a pH range of 3.0–6.0. Kinetic analysis using p-nitrophenyl-α-D-glucopyranoside as the substrate showed a K_m of 0.17 mM and a Vmax of 18.7 µmol min⁻¹ mg⁻¹. Additionally, the enzyme displayed transglucosidase activity, converting maltose into isomaltose oligosaccharides (IMOs), including isomaltose, panose, and isomaltriose. These findings highlight the effectiveness of NTG mutagenesis for enhancing α-glucosidase production and support its industrial application. Full article

A novel protease was isolated from the fruiting bodies of the straw mushroom Volvariella volvacea. The protease was purified 13.48-fold using a series of techniques, including ammonium sulfate precipitation, ultrafiltration, diethylaminoethyl fast-flow (DEAE FF) ion-exchange chromatography, and Superdex 75 gel filtration chromatography, resulting in a specific enzyme activity of 286.82 U/mg toward casein as a substrate. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the purified protease had a molecular weight of 24 kDa. The enzyme exhibited optimal activity at pH 7 and 50 °C, showing sensitivity to alkaline conditions and instability at elevated temperatures. The presence of Ca²⁺ significantly enhanced enzyme activity, whereas Ni²⁺ and Cu²⁺ exerted strong inhibitory effects, with other metal ions showing weak inhibition. β-mercaptoethanol, Tween-80, and Triton X-100 had more pronounced inhibitory effects, whereas PMSF, EDTA, and CTAB had weaker inhibitory effects. The Michaelis constant (Km) and maximum velocity (Vm) of the protease were determined to be 1.34 g/L and 3.45 μg/(mL·min), respectively. The protease exhibited a greater degree of enzymatic degradation of soybean-isolate protein (7.58%) compared to trypsin (5.24%), with the enzyme product containing a high percentage of medicinal amino acids (73.54%), particularly phenylalanine (Phe) and arginine (Arg), suggesting their presence at the enzyme’s active site. These findings suggest that the protease from V. volvacea holds promising potential for applications in the food industry, particularly in protein hydrolysate production and flavor enhancement. Full article

Efficient and sustainable catalytic processes are crucial for advancing green chemical manufacturing. Here, we describe the synthesis of novel silver artificial metalloenzymes in colloidal form in aqueous media and room temperature. The strategy is based on the in situ generation of silver nanoparticles by a genetically modified Geobacillus thermocatenulatus lipase (GTL) in the active site as an inducer and scaffold protein, producing an enzyme–Ag bioconjugate. Using a structural analysis of the formation of silver nanoparticles by XRD and UV spectra, we found the formation of Ag₂O species with nanoparticles of around 11 nm average diameter size. Gel filtration chromatography demonstrated the presence of single protein molecules in the bioconjugates, although silver nanoparticles were initially formed by cysteine coordination in the active site but later were formed in other parts of the protein (five AgNPs per molecules, which is in concordance with the UV size). The enzyme structure was altered after nanoparticle formation and Ag-S interaction, which was observed in fluorescence analysis. This new enzyme showed reductive activity against p-nitrophenol to p-amino and a high conversion > 99% in the reduction of acetophenone to phenylethanol, although the enantioselective was quite moderate but higher in water that in the presence of co-solvents. Finally, oxidase-like activity was evaluated in the direct oxidation of phenylethanol to acetophenone in water, obtained at around a 23% yield of ketone after 60 h. Full article

Zwitterionic polymers have garnered significant attention for their distinctive properties, such as biocompatibility, antifouling capabilities, and resistance to protein adsorption, making them promising candidates for a wide range of applications, including drug delivery, oil production inhibitors, and water purification membranes. This study reports the synthesis and characterization of zwitterionic monomers and polymers through the modification of linear, vinyl, and aromatic heterocyclic functional groups via reaction with 1,3-propanesultone. Four zwitterionic polymers with varying molecular structures—ranging from linear to five and six membered ring systems—were synthesized: poly(sulfobetaine methacrylamide) (pSBMAm), poly(sulfobetaine-1-vinylimidazole) (pSB1VI), poly(sulfobetaine-2-vinylpyridine) (pSB2VP), and poly(sulfobetaine-4-vinylpyridine) (pSB4VP). Their molecular weights, thermal behavior, and self-assembly properties were analyzed using gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and zeta potential measurements. The glass transition temperatures (Tg) ranged from 276.52 °C for pSBMAm to 313.69 °C for pSB4VP, while decomposition temperatures exhibited a similar trend, with pSBMAm degrading at 301.03 °C and pSB4VP at 387.14 °C. The polymers’ self-assembly behavior was strongly dependent on pH and their surface charge, particularly under varying pH conditions: spherical micelles were observed at neutral pH, while fractal aggregates formed at basic pH. These results demonstrate that precise modifications of the chemical structure, specifically in the linear, imidazole, and pyridine moieties, enable fine control over the thermal properties and self-assembly behavior of polyzwitterions. Such insights are essential for tailoring polymer properties for targeted applications in filtration membranes, drug delivery systems, and solid polymer electrolytes, where thermal stability and self-assembly play crucial roles. Full article

The large-scale production of soybean proteins results in the generation of a significant volume of wastewater, containing a substantial amount of valuable β-amylase. The β-amylase enzyme was purified from the soybean whey wastewater using a three-step process, including alcohol precipitation, ion-exchange chromatography, and gel filtration chromatography. The specific activity of the purified β-amylase was 29,700 U/mg, with an enzyme activity recovery of 17.3% and purification fold of 16.5. The β-amylase had a molecular mass of around 56 kDa and an isoelectric point (pI) value of 4.8. The β-amylase exhibited optimal activity at 55 °C and reasonable stability between 30 °C and 40 °C. The enzyme demonstrated an optimum pH of 6.0 and relative stability in the pH range of 5.0–8.0. Furthermore, the β-amylase activity was stimulated by PMSF, Tween-20, Tween-40, Tween-60, Tween-80, and Triton X-100. In terms of substrate preference, the enzyme hydrolyzed potato starch worked most effectively, followed by amylose, amylopectin, soluble starch, maltose, and pullulan. The purified β-amylase showed K_m and V_max values of 3.62 μM and 1.04 μM/ (g protein min), respectively. The purification process was simple and yielded high purification and recovery. The β-amylase from soybean whey wastewater has potential applications in the food and beverage industries. Full article

Haliotis discus, an abalone, is a marine gastropod mollusk that has been cultivated globally owing to its nutritional value and high market demand. However, the visceral parts of H. discus are typically discarded as by-products, highlighting the need to explore their potential value in developing cosmeceuticals and pharmaceuticals. This study investigated the potential moisturizing effects of H. discus visceral tissues. Various hydrolysates from H. discus viscera tissue were evaluated for proximate composition, radical scavenging, and hyaluronidase inhibition activities. Alcalase hydrolysate was isolated using gel filtration chromatography (GFC), and its moisturizing effects were tested on human dermal fibroblasts (HDF), HaCaT keratinocytes, and reconstructed human skin tissue. The Alcalase hydrolysate showed the highest extraction yield, radical scavenging, and hyaluronidase inhibition activities. The Alcalase hydrolysate GFC fraction 1 increased collagen synthesis-related molecules, including procollagen type 1 in HDF and hyaluronic acid-related molecules in HaCaT cells. These moisturizing effects were confirmed in reconstructed human skin tissues by increased levels of aquaporin 3 and filaggrin. Fraction 1 consisted of two main peptides: DNPLLPGPPF and SADNPLLPGPPF. In conclusion, H. discus Alcalase hydrolysate and its fractions have potential moisturizing properties and can be used as cosmeceuticals. Full article

Adhesive formulations derived from sustainable feedstocks, like waste-activated sludge and biosolids from wastewater treatment plants, are developed due to protein-based adhesives receiving attention for their low-cost, resourcefulness, and minimal ecological footprint. The protein composition and associated dynamic changes of the adhesive formulations were studied via gel permeation chromatography, which detailed a molecular size distribution of 8.72 × 10⁵ g/mol for the adhesive formulation and 6.89 × 10³ g/mol for the dewatered biosolid base fraction, which confirms the formation of multiple protein functional groups combining to form the larger adhesive molecules. Further analysis determined the types of proteins present in the dewatered biosolids as glutelin, prolamin, globulin, and albumin proteins, with the glutelin proteins as the most prevalent, as thus likely responsible for adhesive formation. The rheological properties of the novel protein adhesive were also studied to interpret the structure of the adhesives, which detailed the findings of viscoelastic properties and flow behaviors of each adhesive in relation to the wastewater treatment plant sample location, which yielded higher flow points, storage moduli, and loss moduli for the dewatered biosolids in comparison to the waste-activated sludge and biosolid adhesives, which correlates with the higher solids content of the dewatered biosolids and potentially cell rupturing when exposed to filtration stress. Full article

The interest in microbial pectinases is increasing due to their use in the biotechnological and food industries in response to global demand. In this study, among the 22 halophilic bacterial strains screened, 20 showed pectinase activity. The highest activity was measured for Virgibacillus salarius strain 434. As pectinase production meets many parameter optimization constraints, optimal production conditions for maximizing the yield of the pectinase synthesized using the investigated strain were followed. Enzyme purification was accomplished at up to 11.5-fold via ultrafiltration and gel filtration chromatography, until the enzyme’s specific activity had increased 10-fold (104.3 U/mg). The enzyme’s molecular weight was determined to be 68 kDa. Km and Vmax values of 0.38 mg/mL and 120 U/mg, respectively, were determined. The purified pectinase from Virgibacillus salarius strain 434 showed good thermal stability. A half-life of 20 min at 70 °C and high catalytic activity under haloalkaline conditions (pH 9 and 70 g L⁻¹ NaCl) were observed. Its storage stability at −20 °C exceeded 2 months. This demonstrates its potential for application in industries where these extremes are present, as the harsh conditions in industrial processes require novel pectinases with unique properties instead of the use of hazardous chemicals. Full article

Body temperature is primarily regulated by the hypothalamus, ensuring proper metabolic function. Envenomation by Phoneutria nigriventer can cause symptoms such as hypothermia, hyperthermia, sweating, and shivering, all related to thermoregulation. This study aims to analyze and identify components of the venom that affect thermoregulation and to evaluate possible mechanisms. Rats were used for thermoregulation analysis, venom fractionation by gel filtration and reverse-phase chromatography (C18), and sequencing by Edman degradation. The venom exhibited hypothermic effects in rats, while its fractions demonstrated both hypothermic (pool II) and hyperthermic (pool III) effects. Further separations of the pools with C18 identified specific peaks responsible for these effects. However, as the peaks were further purified, their effects became less significant. Tests on U87 human glioblastoma cells showed no toxicity. Sequencing of the most active peaks revealed masses similar to those of the Tachykinin and Ctenotoxin families, both known to act on the nervous system. The study concludes that molecules derived from venom can act synergistically or antagonistically. Additionally, toxins that affect thermoregulation are poorly studied and require further characterization. These toxins could potentially serve as sources for the development of new thermoregulatory drugs. Full article

To address the public health challenges posed by high-salt diets, this study utilized pepsin and flavourzyme for the continuous enzymatic hydrolysis of a soy protein isolate (SPI). The separation, purification, and identification of salt-containing peptides in SPI hydrolysate were conducted using ultrafiltration (UF), gel filtration chromatography (GFC), and Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC-MS/MS). Subsequently, a molecular docking model was constructed between salt receptor protein transmembrane channel 4 (TMC4) and the identified peptides. Basic bioinformatics screening was performed to obtain non-toxic, non-allergenic, and stable salt peptides. After the enzymatic hydrolysis, separation, and purification of SPI, a component with a sensory evaluation score of 7 and an electronic tongue score of 10.36 was obtained. LC-MS/MS sequencing identified a total of 1697 peptides in the above component, including 84 potential salt-containing peptides. A molecular docking analysis identified seven peptides (FPPP, GGPW, IPHF, IPKF, IPRR, LPRR, and LPHF) with a strong theoretical salty taste. Furthermore, residues Glu531, Asp491, Val495, Ala401, and Phe405 of the peptides bound to the TMC4 receptor through hydrogen bonds, hydrophobic interactions, and electrostatic interactions, thereby imparting a significant salty taste. A basic bioinformatics analysis further revealed that IPHF, LPHF, GGPW, and IPKF were non-toxic, non-allergenic, and stable salt-containing peptides. This study not only provides a new sodium reduction strategy for the food industry, but also opens up new avenues for improving the public’s healthy eating habits. Full article