Search Results (1,761)

Bacterial inclusion bodies (IBs) are still generally considered to be waste products of recombinant protein production, despite various studies that have challenged this conventional view in the last two decades, and have been proposed for use as immobilized enzymes in vivo for biocatalysis. Current advances in genetic and molecular biology make it possible to perform multienzymatic reactions or enzymatic cascades to synthesize valuable products. When cascades need cofactor regener tion, it is difficult to use “cheap” whole cells or their lysates, and “expensive” enzyme purification is required. The capture of enzymatic activity into active IBs (aIBs), well-separable protein aggregates from cell lysate, could represent a usable compromise between purified enzymes and cell lysates. It is shown here that the combination of two polyphosphate kinases (PPKs) in the form of aIBs leads to almost 10-fold ATP regeneration and 100% UTP utilization without degradation into adenosine or uridine. PPKs have been combined with N-acetylhexosamine 1-kinase and N-acetylglucosamine-1-phosphate uridyltransferase to produce valuable UDP-N-acetylglucosamine, but the described approach could be used in various multienzymatic syntheses to avoid enzyme purification and ensure nucleotide triphosphate regeneration. Full article

Fungal laccases are promising oxidative enzymes for bioremediation applications, particularly in the degradation of synthetic dyes present in industrial effluents. Here, we evaluated the inhibitory effects of sodium chloride (NaCl) and sodium sulfate (Na₂SO₄) on the activity of Trametes versicolor laccase and its ability to decolorize Congo Red (CR), Malachite Green (MG), and Remazol Brilliant Blue R (RBBR). Enzyme assays revealed concentration-dependent inhibition, with IC₅₀ values of 0.22 ± 0.04 M for NaCl and 1.00 ± 0.09 M for Na₂SO₄, indicating stronger inhibition by chloride. Kinetic modeling showed mixed-type inhibition for both salts. Despite this effect, the enzyme maintained significant activity: after 12 h, decolorization efficiencies reached 95 ± 4.0% for MG, 88 ± 3.0% for RBBR, and 75 ± 3.0% for CR, even in the presence of 0.5 M salts. When applied to a mixture of the three dyes, decolorization decreased only slightly in saline medium (94.04 ± 4.0% to 83.43 ± 5.1%). FTIR spectra revealed minor structural changes, but toxicity assays confirmed marked detoxification, with radicle length in lettuce seeds increasing from 20–38 mm (untreated dyes) to 41–48 mm after enzymatic treatment. Fungal growth assays corroborated reduced toxicity of treated dyes. These findings demonstrate that T. versicolor laccase retains functional robustness under ionic stress, supporting its potential application in saline textile wastewater remediation. Full article

The utilization of encapsulated biopharmaceuticals, including peptides and proteins, has grown substantially in recent years. In this study, the influence of aliphatic polyester physicochemical properties, specifically crystallinity and hydrophobicity, on the development of protein-loaded microparticles was investigated. A series of polyesters, namely amorphous PDLLA and semicrystalline PLLA, PCL, and PPDL, were synthesized via chemical and enzymatic ring-opening polymerization. Bovine serum albumin (BSA)-loaded microparticles were fabricated using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method. The size of microparticles obtained was determined by scanning electron microscopy and dynamic light scattering methods. The enzymatic degradation of the polymer microparticles was assessed through incubation in a lipase-containing buffer solution. BSA and α-chymotrypsin (ACHT) were used as model proteins for the preparation of encapsulated polymer microspheres and comparison of their characteristics and properties. Protein encapsulation efficacy, release rate, and enzyme activity retained after encapsulation were evaluated and compared for selected aliphatic polyesters. The release profiles were processed with the use of various mathematical models to reveal the possible mechanism(s) of protein release. Full article

Mucosal drug delivery is gaining attention for its ability to provide localized treatment with reduced systemic side effects. The vaginal route has been proven effective for managing gynecological conditions, though it poses certain limitations. Biopolymers can help overcome these challenges by enhancing therapeutic efficiency and offering beneficial properties. This study aimed to develop and evaluate hydrogels and their freeze-dried forms (wafers) based on collagen, hydroxypropyl methylcellulose, and gellan gum. Initially, a collagen gel was obtained by extraction from calfskin, which was brought to a concentration of 1% and a physiological pH with 1 M sodium hydroxide solution. This gel was combined with either 2% hydroxypropyl methylcellulose gel, 1.2% gellan gum gel, or both, in different proportions. Thus, five mixed hydrogels were obtained, which, along with the three individual gels (controls), were lyophilized to obtain wafers. Furthermore, the hydrogels were assessed for rheological behavior, while the collagen structural integrity in the presence of the other biopolymers was evaluated using circular dichroism and FT-IR spectroscopy. The wafers were characterized for morphology, wettability, swelling capacity, enzymatic degradation resistance, and in vitro biocompatibility. All hydrogels exhibited non-Newtonian, pseudoplastic behavior and showed collagen structure preservation. The wafers’ characterization showed that gellan gum enhanced the hydrophilicity and enzymatic stability of the samples. In addition, the extracts from the tested samples maintained cell viability and did not affect actin cytoskeleton morphology, indicating a lack of cytotoxic effects. This study emphasizes the importance of evaluating both the physicochemical properties and biocompatibility of biopolymeric supports as a key preliminary step in the development of vaginal drug delivery platforms with biomedical applications in the management of gynecological conditions. Full article

Highly Hazardous Pesticides (HHPs) pose severe risks to human health and the environment, making it essential to understand their molecular stability and degradation pathways. In this study, the Quantum Theory of Atoms in Molecules (QTAIM) was applied to four representative organophosphate pesticides, allowing the identification of electronically weak bonds as intrinsic sites of lability. These findings are consistent with reported hydrolytic, oxidative, enzymatic, and microbial degradation routes. Importantly, QTAIM descriptors proved largely insensitive to solvation, confirming their intrinsic character within the molecular electronic structure. To complement QTAIM, conceptual DFT (Density Functional Theory) reactivity indices were analyzed, revealing that solvent effects induce more noticeable variations in global and local descriptors than in topological parameters. In addition, a Topological Analysis of the Fukui Function (TAFF) was performed, which mapped nucleophilic, electrophilic, and radical susceptibilities directly onto QTAIM basins. The TAFF analysis confirmed that bonds identified as weak by QTAIM (notably P–O, P–S, and P–N linkages) also coincide with the most reactive sites, thereby reinforcing their mechanistic role in degradation pathways. This integrated framework highlights the robustness of QTAIM, the sensitivity of global and local reactivity descriptors to solvation revealed by conceptual DFT, and the complementary insights provided by TAFF, contributing to risk assessment, remediation strategies, and the rational design of safer pesticides. Full article

This study investigated the effects of inulin concentration and ultrasonic homogenization on the particle size distribution and microstructure of oil-in-water emulsion gels stabilized with psyllium husk. These gels were then incorporated into meal purees formulated for individuals with dysphagia. Under ultrasound treatment, an increase in inulin from 0% to 20% reduced the average droplet size from 14.98 μm to 1.58 μm, indicating a synergistic effect between ultrasound treatment and inulin in reducing and stabilizing droplet size. The optimal formulation under ultrasound was 20% (w/w) inulin. Scanning electron and polarized light microscopy confirmed that ultrasonic homogenization improved emulsion integrity by minimizing droplet size and promoting encapsulation. Inulin-rich emulsion gels, when added to purees, reduced structural voids, improved matrix cohesion, and lowered expressible fluid content. Enzymatic assays showed enhanced inhibition of α-amylase and α-glucosidase, indicating increased resistance to oral enzymatic degradation. Importantly, substituting emulsion gels at 10% (w/w) did not compromise puree firmness. All formulations met International Dysphagia Diet Standardization Initiative (IDDSI) Level 4 requirements, confirming their suitability for individuals with oropharyngeal dysphagia (OD). These findings demonstrate the potential of psyllium husk-stabilized emulsion gels as innovative texture-modifying agents for dysphagia-friendly food development. Full article

Sustainable manure recycling in cold climates faces low efficiency and nutrient loss. This study evaluated housefly larva-pretreated manure (HL) for winter swine manure composting in East China, comparing it to sawdust-conditioned (CK2) and untreated manure (CK1). Larval pretreatment converted 12.71% of manure weight into biomass, assimilating 10.69% C, 30.55% N, 8.54% P, and 11.53% K. Harvested larvae contained 53.35% crude protein, with amino acids matching/exceeding fishmeal and soybean meal, while heavy metals were below safety limits. Theoretical annual larval protein yield per unit area (29,530 kg·mu⁻¹·year⁻¹) was 206.5 times higher than soybean crops. During composting, the HL treatment promoted early protease and catalase activation. This enzymatic synergy accelerated organic matter degradation and maturation, achieving a germination index of 147.67% by day 51. Coordinated nitrate and nitrite reductase activity in HL facilitated efficient denitrification, minimizing NO₂⁻ accumulation and N₂O emissions. Consequently, HL composting achieved faster stabilization, enhanced nutrient retention, and greater protein recovery compared to controls. These findings demonstrate that housefly larval pretreatment offers a climate-resilient and scalable strategy for winter manure management and protein valorization, with strong potential for applications in cold and resource-limited agricultural systems worldwide. Full article

Challenges significantly hinder the sustainable cultivation of tea chrysanthemum, leading to imbalances in soil nutrients, the accumulation of allelopathic phenolic acids, reduced enzymatic activity, and disruptions in rhizosphere microbial communities. To explore potential mitigation strategies, this study systematically evaluated the integrative effects of exogenous methyl jasmonate (MeJA, 0–400 (μmol L⁻¹)) on both soil environmental parameters and plant growth performance under continuous cropping conditions. The results revealed that treatment with 100 (μmol L⁻¹) MeJA significantly enhanced plant height, canopy width, flower number, and fresh flower weight. Concurrently, it improved soil organic matter content, the available nitrogen levels, and redox stability while increasing the activity of key enzymes, including polyphenol oxidase, urease, and catalase. Notably, this treatment markedly reduced the accumulation of allelopathic phenolic acids, such as p-hydroxybenzoic acid and vanillic acid. High-throughput sequencing further demonstrated that 100 (μmol L⁻¹) MeJA optimized the composition of soil microbial communities, increasing the abundance of beneficial taxa, such as nitrogen-fixing and phosphate-solubilizing bacteria, while suppressing pathogenic fungi. Metabolomic analysis showed that this concentration of MeJA activated stress-resistance metabolic pathways involving flavonoids and terpenoids while downregulating degradation-related processes, thereby supporting enhanced plant resilience at the metabolic level. Collectively, these findings demonstrate that an appropriate concentration of exogenous MeJA can effectively alleviate continuous cropping obstacles in Chrysanthemum morifolium, providing both theoretical insights and practical guidance for its eco-friendly and efficient cultivation. Full article

Ochratoxin A (OTA) is a widespread foodborne mycotoxin that poses significant risks to both human and animal health. Upon ingestion, the gastrointestinal tract (GIT) becomes the main site of exposure, where OTA interacts directly with the intestinal epithelium and resident microbiota. Research indicates that OTA disrupts the integrity of the intestinal barrier and alters its permeability. Moreover, OTA undergoes transport and partial metabolism within the intestine before being excreted. Detoxification pathways for OTA include enzymatic degradation and adsorption by microorganisms. Notably, OTA has profound toxic effects on the gut ecosystem; it can alter the relative abundance of bacterial taxa by reducing beneficial populations and promoting opportunistic or pathogenic strains. These changes contribute to an imbalance in the microbiota, impairing host metabolic and immune functions. This dysbiosis is characterized by disrupted microbial homeostasis and impaired communication between the host and its gut microbiome. This review highlights the dual role of the intestine as both a target and a modulator of OTA toxicity. It emphasizes the importance of gut microbiota in mediating the toxicological outcomes of OTA and explores microbiome-based strategies as potential avenues for detoxification. Full article

Despite the widespread use of dithiocarbamate fungicides such as maneb, mancozeb, metiram, propineb, thiram, and ziram detected, according to EU legislation, via common degradation product carbon disulfide (CS₂), recent and comprehensive reviews on analytical methods for their determination in plant-based foods are lacking. Given the well-documented toxicity shown by the experimental model for these pesticides, including neurotoxicity and endocrine disruption, harmonized and reliable analytical protocols are crucial for food safety monitoring and regulatory compliance. Dithiocarbamates, beyond CS₂ release, have been associated with immunotoxicity, thyroid dysfunction, and potential carcinogenicity, raising further concern regarding chronic dietary exposure. Their metabolites may disrupt enzymatic activity and oxidative balance, enhancing systemic toxicity. Early methods, had limited sensitivity, poor reproducibility, and relied on hazardous solvents, reducing practical value. Although later advancements improved detection limits, modern procedures, including those proposed by the European Union Reference Laboratory (EURL), still show limitations. The EURL-recommended protocol involves acid hydrolysis using concentrated HCl, extraction with isooctane, heating to 85 °C, and rapid ice-bath cooling, which poses environmental concerns. Recovery efficiency remains inconsistent in some cases, and reproducibility within commodity groups is poor. This review discusses the status of methods for determining dithiocarbamates as individual compounds and via CS₂ moiety. Full article

Novel therapies to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of respiratory coronavirus disease 2019 (COVID-19), would be of great clinical value to combat the current and future pandemics. Two viral proteases, papain-like protease (PL^pro) and the main protease 3-chymotrypsin-like protease (3CL^pro), are vital in processing the SARS-CoV-2 polyproteins (pp1a and pp1ab) and in releasing 16 nonstructural proteins, making them attractive antiviral drug targets. In this study, we investigated the degradation of the SARS-CoV-2 main protease 3CL^pro by matrix metalloproteinase-14 (MMP14). MMP14 is known to recognize over 10 distinct substrate cleavage sequences. Through sequence analysis, we identified 17 and 10 putative MMP14 cleavage motifs within the SARS-CoV-2 3CL^pro and PL^pro proteases, respectively. Despite the presence of potential sites in both proteins, our in vitro proteolysis assays demonstrated that MMP14 selectively binds to and degrades 3CL^pro, but not PL^pro. This selective proteolysis by MMP14 results in the complete loss of 3CL^pro enzymatic activity. In addition, SARS-CoV-2 pseudovirus replication was inhibited in 293 T cells when either full-length MMP14 or its catalytic domain (cat-MMP14) were overexpressed, presumably due to 3CL^pro degradation by MMP14. Finally, to prevent MMP14 from degrading off-target proteins, we propose a new recombinant pro-PL-MMP14 construct that can be activated only by another SARS-CoV-2 protease, PL^pro. These findings could open the potential of an alternative therapeutic strategy against SARS-CoV-2 infection. Full article

Micro- and nanoplastics (MPs and NPs) are recognized as emerging contaminants posing potential risks to human health. Recent evidence highlights the potential of food-grade microbial strains to bind these particles and facilitate their removal, suggesting a promising probiotic-based strategy for mitigating their adverse health effects. This study investigated the adsorption and biodegradation capabilities of Bacillus subtilis DCP04, a strain isolated from Korean fermented soybean paste, cheonggukjang, on low-density polyethylene (LDPE) particles. Biofilm formation assays and morphological observations confirmed the strain’s ability to adhere to the surface of LDPE. Subsequent experiments demonstrated that DCP04 effectively adsorbed LDPE particles in a size-, time-, and concentration-dependent manner. This interaction induced significant morphological changes and increased hydrophilicity on the polymer surface. Furthermore, a positive correlation was observed between the activities of laccase and manganese peroxidase and a measurable weight loss in LDPE films, suggesting direct enzymatic involvement in polymer degradation. Crucially, the DCP04 strain also met key safety and functional criteria for use as a probiotic. These findings highlight the potential of DCP04 strain as a functional probiotic agent for mitigating the accumulation of MPs and NPs within the human body. Full article

Lignin, the most abundant aromatic biopolymer, represents a key renewable feedstock for sustainable biorefineries, yet its structural complexity poses a formidable challenge for enzymatic degradation. While ligninolytic enzymes such as laccases (LACs), lignin peroxidases (LiPs), and manganese peroxidases (MnPs) exhibit remarkable catalytic versatility, the molecular mechanisms underlying their ability to balance substrate specificity and structural flexibility remain unresolved. Here, we employed all-atom molecular dynamics (MD) simulations and virtual mutagenesis to dissect the dynamic interactions between these enzymes and lignin model compound (β-O-4-linked H-type dimers). Our simulations revealed a dual recognition mechanism in which polar residues (such as Asp, Glu, Arg and His) formed hydrogen bonds with hydroxyl and keto groups near catalytic cleavage sites, ensuring precise alignment for bond scission, while aromatic residues stabilized diverse lignin conformations via hydrophobic interactions with conserved aromatic rings. Conformational dynamics of active-site residues enabled adaptive adjustments to substrate heterogeneity, reconciling enzymatic specificity with structural promiscuity. Virtual mutation experiments further demonstrated that aromatic residues were indispensable for binding stability, whereas polar residues dictated cleavage-site selectivity. These findings provide atomic-scale insights into the catalytic mechanism of ligninolytic enzymes, with implications in the rational design of superior biocatalyst for lignin biorefineries. Full article

Alpine wetlands, key carbon sinks and biodiversity hubs, remain understudied, especially under climate change pressures. Hence, the present study was conducted to assess the variability in soil enzyme activity (SEA) and the carbon management index (CMI) and to utilize principal component analysis (PCA) to explore the variation and correlation between SEA and CMI as influenced by altitudinal gradients in alpine wetlands. This information is essential for exploring the impacts of soil degradation and guiding restoration efforts. The study was designed in blocks (catchments) with six altitudinal variations (from 2500 to 3155 m a.s.l), equivalent to alpine wetlands from three catchments (Senqunyane, Khubelu and Sani) as follows: Khorong and Tenesolo in Senqunyane; Khamoqana and Khalong-la-Lichelete in Sani; and Lets’eng-la-Likhama and Koting-Sa-ha Ramosetsana in Khubelu. The soil samples were collected in February 2025 (autumn season, i.e., wet season) at depths of 0–15 and 15–30 cm and analyzed for bulk density, texture, pH, electrical conductivity (EC), soil organic carbon (SOC), SEA, and carbon pools, and the CMI was computed following standard procedures. The results demonstrated that the soil was loam to sandy loam and was slightly acidic and non-saline in nature in the 0–15 cm layer across the wetlands. The significant decreases in SEA were 45.33%, 32.20% and 15.11% (p < 0.05) for dehydrogenase, fluorescein di-acetate and β-Galactosidase activities, respectively, in KSHM compared with those in Khorong (lower elevated site). The passive carbon pool (C_PSV) was dominant over the active carbon pool (C_ACT) and contributed 76–79% of the SOC to the total organic carbon, with a higher C_PSV (79%) observed at KSHM. The CMI was also greater (91.05 and 75.88) under KSHM at the 0–15 cm and 15–30 cm soil depths, respectively, than in all the other alpine wetlands, suggesting better carbon management at higher altitudinal gradients and less enzymatic activity. These trends shape climate change outcomes by affecting soil carbon storage, with high-altitude regions serving as significant, though relatively less active, carbon reservoirs. The PCA-Biplot graph revealed a negative correlation between the CMI and SEA, and these variables drove more variation across sites, highlighting a complex interaction influenced by higher altitude with its multiple ecological drivers, such as temperature variation, nutrient dynamics, and shifts in microbial communities. Further studies on metagenomics in alpine soils are needed to uncover altitude-driven microbial adaptations and their role in carbon dynamics. Full article

This study examined the effects of Bacillus probiotic strains on red abalone Haliotis rufescens reproductive performance. We supplemented plant- and fish-based feeds and compared them to fresh giant kelp Macrocystis pyrifera as a control diet. Over 180 days, abalone fed the plant–probiotic diet reached higher female gonadal maturation, with 56% of females attaining the maximum Visual Gonad Index (VGI 3). Additionally, plant-based treatment showed a female-biased sex ratio (1.5:1 female-to-male ratio, F:M) compared with the kelp control treatment (0.8:1 F:M). These results suggest that probiotics can improve nutrient utilization from soybean meal and may enhance the bioavailability of phytoestrogens and other bioactive compounds, contributing to reproductive outcomes. Although the mechanisms remain to be confirmed, this approach provides a promising strategy to reduce reliance on fishmeal and wild macroalgae while supporting faster reproductive cycles in abalone aquaculture. Future research should focus on biochemical validation, molecular pathways, and multigenerational trials to ensure the long-term safety and sustainability of probiotic–plant-based feeds. Full article