Search Results (1,724)

Pyranose oxidases (POXs, EC 1.1.3.10) are a class of fungal FAD-dependent oxidoreductases with potential for lignocellulosic bioconversion because they generate H₂O₂ during sugar oxidation. Despite their known catalytic properties, the role of these enzymes in promoting lignocellulose enzymatic saccharification remains largely unexplored. In this study, POXs from Phanerochaete chrysosporium (PcPOX) and Trametes versicolor (TvPOX) were comparatively evaluated through biochemical characterization, kinetic analysis, molecular simulation, and supplementation for lignocellulose hydrolysis. PcPOX exhibited a broader substrate spectrum and a slightly higher optimum temperature, whereas TvPOX demonstrated greater stability under acidic and hydrolysis-relevant conditions and a longer half-life at 50 °C. TvPOX also showed a numerically lower apparent K_m toward D-glucose, while the apparent catalytic efficiencies were comparable between the two enzymes. Molecular simulation results suggested more stable glucose binding in TvPOX. Accordingly, TvPOX was selected for hydrolysis experiments and was shown to increase the measured glucan conversion of phosphoric acid-swollen cellulose, Avicel, and corncob residue. Mixture design analysis further indicated that this positive effect depended on balanced peroxide regulation, with low catalase supplementation providing better performance. These results identify TvPOX as a promising auxiliary enzyme for cellulase-based lignocellulosic saccharification. Full article

Inorganic phosphate (Pi) is an essential nutrient required for energy metabolism, macromolecule biosynthesis, and signal transduction. Protozoan parasites are exposed to pronounced fluctuations in nutrient availability throughout their life cycles and therefore rely on adaptive strategies to secure phosphate from diverse host and environmental niches. This review summarizes current knowledge on phosphate acquisition mechanisms in protozoan parasites, with emphasis on membrane transport systems and extracellular phosphate-releasing enzymatic activities. Phosphate transport systems energized by proton or sodium gradients have been functionally characterized in several protozoan species, and in many cases phosphate uptake capacity is modulated by extracellular Pi availability. In addition, ectophosphatases expressed at the parasite surface contribute to phosphate acquisition by hydrolyzing extracellular phosphorylated substrates and releasing inorganic phosphate that can be subsequently internalized and metabolically utilized. Although phosphate-dependent regulation of ectophosphatase activity has been demonstrated in a more limited number of species, available evidence supports a functional interplay between extracellular phosphate scavenging and transmembrane transport, particularly under phosphate-limiting conditions. Despite these advances, the molecular mechanisms underlying phosphate sensing and regulatory coordination in protozoan parasites remain poorly understood. This review provides a comparative overview of phosphate transport systems and extracellular phosphate-scavenging enzymes in protozoan parasites, highlighting current evidence and remaining knowledge gaps. Full article

The transition towards a circular bioeconomy is essential to mitigate the environmental pressures caused by the increasing global demand for food and energy. Agro-food waste (AFW) is a plentiful, inexpensive feedstock, exploitable in biorefineries to produce valuable molecules. The aim of this study was to isolate native non-conventional yeasts (NCY) from various AFW and to evaluate their potential for the ‘natural’ synthesis of aroma compounds via fermentation. Ten strains were isolated and identified as belonging to Saccharomyces cerevisiae, Pichia kluyveri, Pichia californica and Wickerhamomyces anomalus species. The fermentative performance and production of aroma volatile compounds were tested using different household wastes as substrates. Figs containing substrate, which is the richest in fermentable sugars, allowed for the fastest microbial adaptation and highest yields of volatile compounds. HS-SPME-GC/MS analysis revealed that the most prominent compounds were isoamyl alcohol, ethyl acetate and isoamyl acetate with the highest production levels showed by W. anomalus YDSCYP4 and P. kluyveri YDSCYP5. Enzymatic profiling revealed significant arylamidase and esterase activities in the selected strains, related to their role in the hydrolysis of aroma precursors. These findings demonstrate the efficiency of these autochthonous yeasts for the sustainable production of aroma compounds, supporting the development of eco-friendly biotechnological processes. Full article

Municipal wastewater represents an underutilized secondary biomass resource rich in organic carbon and nutrients that can be valorized through biotechnological conversion. In this study, we developed an integrated multi-microbial biorefinery platform to transform municipal wastewater into value-added biofuel via sequential bacterial treatment, microalgal biomass generation, and fungal-assisted harvesting. Wastewater was first pretreated with Bacillus subtilis to enzymatically hydrolyze complex organic substrates and enrich the medium with bioactive metabolites, including auxins and gibberellins. The conditioned wastewater was subsequently used to cultivate Chlorella vulgaris, followed by biomass recovery using Trichoderma viride pellets as a sustainable bioflocculant. The integrated consortium significantly enhanced nutrient removal efficiency and promoted algal biomass accumulation, lipid enrichment, and biodiesel productivity compared to monoculture controls. Elevated hydrolytic enzyme activities (cellulase, protease, and amylases) facilitated organic matter conversion into bioavailable substrates, while increased phytohormone levels stimulated algal growth and lipid biosynthesis. Additionally, fungal bioflocculation substantially improved biomass recovery efficiency, reducing the need for energy-intensive harvesting technologies. This work highlights the potential of a biotechnology-driven approach for integrating wastewater remediation with biofuel production. By integrating microbial metabolism, enzymatic transformation, and sustainable separation processes, the proposed biorefinery system suggests a potentially low-carbon approach for simultaneous environmental remediation and biomass valorization, although further life cycle and energy balance analyses are required to validate this aspect. Full article

DNA damage requires repair via the endonuclease IV-mediated base excision repair (BER) pathway, which corrects apurinic/apyrimidinic (AP) sites. Chlamydophila pneumoniae AP endonuclease IV (CpEndoIV), the sole AP endonuclease in this pathogen, is crucial for genomic integrity. As humans lack a homologous protein, it represents a potential therapeutic target. In this study, we report the first crystal structure of CpEndoIV at 1.97 Å resolution. The structure reveals two Zn²⁺, one Mg²⁺, and a malonate molecule bound in the active site, marking the first observation of Mg²⁺ coordination in the EndoIV family. Compared to the three-Zn²⁺ model with a narrow, deep pocket for precise AP-site cleavage, the Zn²⁺/Mg²⁺-bound state has a wider, shallower pocket that might promote diverse catalytic activities. Combined with enzymatic assays, we suggest that the mixed Zn²⁺/Mg²⁺ model is better adapted for CpEndoIV to operate under host oxidative stress. Malonate binds to the metal ions, occupying the positions normally coordinated by water molecules. This binding mode may mimic the coordination of the substrate to the metal ions, and the protein conformation resembles that of the enzyme upon substrate binding at the active site. This study provides a structural basis for the functional characterization of CpEndoIV and offers a reference for the development of targeted inhibitors against diseases caused by Chlamydophila pneumoniae. Full article

The enzymatic saccharification of cellulose remains a key step in biomass conversion processes, often influenced by enzyme stability, distribution, and accessibility at solid–liquid interfaces. Immobilization of cellulolytic enzymes on nanostructured supports has been proposed as a strategy to modulate catalytic behavior; however, the relationship between enzyme loading and catalytic response remains insufficiently understood. In this study, CuO-based nanobiocatalysts were prepared through controlled cellulase immobilization and systematically evaluated under defined experimental conditions. Structural and physicochemical characterization was performed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and integrated thermal analysis (TGA–DTG–DSC), enabling a comparative assessment of the analyzed systems. SEM analysis showed that the average particle diameter increased from 39.5 ± 14.8 nm (CuO nanoparticles) to 95.6 ± 21.8 nm (NPI10), 106.6 ± 27.7 nm (NPI15), and 113.5 ± 23.1 nm (NPI20), indicating progressive variations in particle organization with increasing enzyme loading. Catalytic performance was evaluated through enzymatic hydrolysis of cellulose filter paper as a model substrate, with products quantified by HPLC at a representative reaction time. The system prepared at lower enzyme loading (NPI10) exhibited product formation comparable to that of the free enzyme, with apparent average glucose formation values of 1.054 and 1.047 mg·mL⁻¹·h⁻¹, respectively. In contrast, higher immobilization levels were associated with reduced catalytic output. Across all systems, glucose was the predominant product, with negligible accumulation of intermediate oligomers under the evaluated conditions. These results indicate that increasing enzyme loading does not correspond to proportional increases in product formation and highlight the influence of enzyme distribution and accessibility within the system. The combined structural and catalytic observations provide a controlled framework for evaluating how immobilization conditions influence system behavior in nanobiocatalytic systems. Full article

Brewer’s spent grain (BSG) is an abundant lignocellulosic by-product whose valorization can support circular bioeconomy strategies. This study evaluated BSG bioconversion by Aspergillus oryzae ATCC 10124 under solid-state fermentation (SSF) to produce lignocellulolytic enzymes and release second-generation (2G) sugars relevant to biorefinery applications. SSF was monitored over 0–10 days, and FPase, endo-cellulase, β-glucosidase, xylanase, mannanase, amylase, and ligninolytic enzyme activities were quantified. Enzymatic crude extracts were further assessed in SDS-PAGE analysis. Glucose, cellobiose, xylose and arabinose release and consumption were tracked throughout fermentation, and substrate transformation was supported by FTIR. The secretome exhibited a predominantly hydrolytic profile, with maximal hemicellulolytic and cellulolytic activity around days 2–4, as well as sustained amylase activity. Ligninolytic activity was not detected. Sugar profiles indicated rapid early hydrolysis of glucose, followed by progressive pentose release. The stabilization and decline were consistent with fungal uptake. Changes in the carbohydrate fingerprint and SDS–PAGE banding supported structural polysaccharide remodeling and hydrolytic protein secretion. Thus, this SSF platform confirmed certain potential for low-cost cellulolytic and hemicellulolytic enzyme generation. However, because sugar accumulation was temporary and followed by consumption, this system is best interpreted as a biological pretreatment and enzyme-generation step that supports subsequent downstream valorization. Full article

Cannabinoids are high-value bioactive compounds whose sustainable production remains challenging, prompting interest in biocatalytic and microbial platforms as alternatives to plant extraction. In this study, we investigated the heterologous expression and functionality of two key cannabinoid-related enzymes in the photosynthetic microalga Chlamydomonas reinhardtii: the aromatic prenyltransferase, NphB_G286S/Y288A from Streptomyces sp., and the plant-derived cannabidiolic acid synthase (CBDAS) from Cannabis sativa. Codon-optimized genes were introduced into the nuclear genome of C. reinhardtii using several construct configurations and promoters, and stable transformants were generated and characterized for genomic integration, transcript accumulation, protein production, enzymatic activity, and cannabinoid-related metabolite formation. While NphB protein accumulation was achieved under the PSAD promoter control, CBDAS was not detected at the protein level under any condition tested. In vitro enzymatic assays using soluble algal protein extracts from NphB-expressing lines confirmed catalytic activity, yielding cannabigerolic acid (CBGA), reaching up to 633 ± 58 µg L⁻¹. However, no CBGA production was detected in vivo, despite substrate supplementation. These results indicate that, although bacterial prenyltransferase can be functionally expressed in C. reinhardtii, efficient metabolic conversion in vivo is limited by cellular and biochemical constraints, including substrate availability, intracellular compartmentalization, and potential competition with endogenous pathways. In contrast, the absence of detectable CBDAS highlights the challenges associated with expressing complex plant oxidocyclases in this photosynthetic host. Overall, this work provides mechanistic insights into enzyme compatibility and metabolic bottlenecks in microalgal systems and outlines key considerations for the future development of photosynthetic platforms for cannabinoid biocatalysis. Full article

Laccase plays an important role in the detection and degradation of phenolic compounds, but it is limited by its cost and stability. In this study, the laccase-like property of copper peptide (GHK-Cu) has been revealed. In terms of enzymatic reaction kinetics, GHK-Cu has a V_max of 1.735 × 10⁻⁴ mM·s⁻¹ and a K_m of 0.061 mM, demonstrating good substrate affinity and excellent catalytic efficiency. Then, a colorimetry was developed for rapid detection of epinephrine (EP) and 2-aminophenol (2-AP). The linear response range of EP is 20–240 μM, with a limit of detection (LOD) of 9.5 μM. The linear response ranges of 2-AP are 14–100 μM (in ultrapure water) and 2–120 μM (in seawater), with LODs of 2.56 μM and 1.65 μM. In addition, combined with a smartphone platform, a cotton-based sensor has been developed for the detection of 2-AP in seawater. The linear response ranges are 0–0.2 mM and 0.2–1 mM, with LOD of 0.033 mM. The structure of GHK-Cu provides a reference for the development of novel laccase mimetic enzymes. The constructed colorimetry offers an option for the rapid detection of phenolic compounds, and the developed cotton-based sensor enabled rapid and portable detection of 2-AP. Full article

The growing production volume of the meat industry has increased the need for revalorization of meat by-products to reduce economic and environmental impacts. Enzymatic hydrolysis of protein-rich meat by-products is an effective strategy for producing hydrolyzates with bioactive potential. Combining sequential enzymatic hydrolysis with γ-glutamyl transpeptidase activity can promote the formation of γ-glutamyl peptides associated with koku perception, a sensory attribute that increases taste intensity, continuity, and palatability. This study aimed to develop porcine liver hydrolyzates enriched in koku-related peptides through enzymatic hydrolysis followed by post-treatment with the transpeptidase Protana Uboost. Substrate specificity assays showed that a 0.2 U/mL enzyme concentration maximized γ-glutamyl dipeptide formation. Sequential hydrolysis using Alcalase and Protana Prime followed by Protana Uboost post-treatment generated the highest levels of koku-related peptides. Moreover, post-treatment significantly enhanced antioxidant capacity in the resulting hydrolyzates, supporting their potential as a functional ingredient. Full article

Using density functional theory (DFT) and the Gaussian 09 program, the study calculated Gibbs free energy to understand how easily each NP can transform. Results showed that only 2,6-dinitrophenol (2,6-DNP) and 2-chloro-6-nitrophenol (2-Cl-6-NP) had Gibbs free energies above 0 kJ/mol. The study also evaluated the toxicity of the NPs, leading to the identification of trinitrophenol (TNP), 2-chloro-4-nitrophenol (2-Cl-4-NP), and 2-nitrophenol (2-NP) with the highest risk scores. In the present study, binding energies were used only as comparative indicators of enzyme–substrate interaction favorability within a screening framework, rather than direct measures of catalytic degradation efficiency. The enzyme 1,2-dioxygenase from Acinetobacter baylyi ADP1 showed strong degradation effects on catechol, with significant binding energies for 2-NP, 2-Cl-4-NP, and TNP. The PS-AOP changed the degradation environment, which reduced enzymatic efficiency. The study also modified specific amino acids in enzymes to improve their performance. For example, the enzyme 1DLT-6 had a degradation increase of nearly 27% compared to the reference enzyme. Finally, we tried to measure the impact of different forces on the breakdown of nitrophenols by enzymes. We used a two-dimensional amino acid map based on enzyme–ligand interactions and a visualization of non-covalent interactions. Our findings show that van der Waals forces and electrostatic forces are the main factors affecting how well the material breaks down. From a sustainability perspective, the study highlights a promising strategy for mitigating secondary pollution, improving the environmental compatibility of PS-AOP-based remediation, and supporting safer and more sustainable restoration of petroleum hydrocarbon-contaminated soil and groundwater. These findings help strengthen the theoretical basis for developing greener post-oxidation remediation pathways. Full article

The rapid detection of organophosphorus (OP) compounds is crucial for safeguarding human health and ensuring food safety. This study presents a novel wearable electrochemical biosensor that integrates miniaturized screen-printed electrodes with wearable devices to achieve real-time, on-site OP detection. The biosensor was fabricated by constructing a screen-printed carbon electrode (SPCE) on a thermoplastic polyurethane (TPU) substrate, sequentially modified with graphene (GR), gold nanoparticles (AuNPs), and organophosphorus hydrolase (OPH), and finally encapsulated with Nafion. This SPCE/GR/AuNPs/OPH/Nafion configuration yields a highly flexible and portable device. The detection principle relies on the enzymatic hydrolysis of methyl paraoxon (MPOX) by OPH, generating p-nitrophenol (PNP), which is quantitatively measured via square wave voltammetry (SWV). The sensor exhibits a broad linear detection range (30–400 μM) with a strong linear correlation (R² = 0.995) and a low detection limit (0.321 μM). It demonstrates excellent selectivity against common interfering substances, including urea, sucrose, and various metal ions. Application to real-world samples such as cabbage and tap water yielded high recoveries (107.2% for cabbage and 101.2% for tap water), with relative standard deviations (RSDs) below 8%. Furthermore, the biosensor maintains robust flexibility and mechanical resilience, with less than 5% signal loss after 100 bending cycles, confirming its suitability for wearable applications and reliable operation under mechanical stress. This innovative, flexible electrochemical biosensor provides a powerful and reliable platform for rapid OP detection, particularly in complex testing environments. Full article

Corn bran is a major by-product of corn starch processing. Due to its high cellulose and hemicellulose contents and relatively low lignin abundance, it represents a promising feedstock for biorefineries. However, efficiently deconstructing corn bran cell wall to maximize fermentable sugar yield while minimizing inhibitor formation remains a challenge due to the complex cross-linked structure of its lignocellulosic matrix that hinders substrate accessibility and prone to side reactions during deconstruction. This study systematically evaluated various pretreatment strategies and identified dilute sulfuric acid as the optimal method to maximize hemicellulose dissolution and total sugar recovery while maintaining low levels of refractory phenolic inhibitors (1.03 g/L, far lower than alkaline and sulfite-based pretreatment). Under optimal conditions (0.80% v/v sulfuric acid, 129 °C, and 23 min), the hemicellulose dissolution rate reached 99.58%, with a pentose yield of 0.38 g/g corn bran and hexose yield of 0.16 g/g corn bran. Subsequent enzymatic hydrolysis of the solid residue (20 FPU/g initial dry weight cellulase) further released hexose-rich sugars. The integrated process achieved a significant total reducing sugar yield of 0.79 g/g corn bran. These findings demonstrate an effective pathway for the high-value utilization of corn bran and provide a scalable process strategy applicable to other lignocellulosic agricultural wastes for sustainable bioenergy production. Full article

Biosurfactant-producing microorganisms play an important ecological role in soils impacted by hydrophobic contaminants by enhancing substrate bioavailability and influencing microbial interactions. In this study, we critically evaluated the reliability of commonly used screening methods for biosurfactant detection. A total of 71 microbial isolates (16 bacteria and 55 fungi) were obtained from vegetable oil-contaminated soil and screened using a multi-step approach combining enzymatic assays (lipolytic and hemolytic activity) and physicochemical methods, including drop-collapse, oil spreading, emulsification index (E₂₄), and surface tension reduction. Although 21 isolates exhibited lipolytic activity and 9 showed hemolysis, inconsistent responses among assays revealed significant limitations of individual screening methods. Only two bacterial isolates consistently tested positive across all criteria. When cultivated in mineral salt medium supplemented with hydrophobic substrates, both isolates produced stable emulsions and significantly reduced surface tension (from 54.26 mN/m to 31.46 mN/m). Substrate-dependent variation was observed for isolate C3, which showed reduced surface tension (39.63 mN/m) when grown with biodiesel. These findings highlight the risk of relying on single assays and emphasize the need for integrated screening strategies to ensure reliable detection of biosurfactant-producing microorganisms. Full article

Selective protein terminal labeling has become essential for system-wide studies of proteolytic mechanisms in disease. These methods enable precise tracking of cleavage dynamics, protease interactions, and cellular networks, offering transformative potential for proteolytic event analysis. This review explores recent advances in N-/C-terminal modification strategies, specifically for the applications in terminomics—the field focused on protein termini characterization. While protein termini provide valuable insights into functional proteome states, their low abundance in complex samples demands highly selective labeling approaches. We evaluate modern chemical and chemoenzymatic methods that leverage engineered chemical reactivity thresholds or enzymatic precision for site-specific modifications. Emerging strategies show enhanced substrate adaptability, reaction efficiency, and workflow compatibility, enabling broader applications in terminome studies. Full article