Search Results (1,767)

Pancreatic lipase (PL) inhibition is a promising dietary strategy for obesity management. In this study, the inhibitory mechanisms and structural basis of polyphenols extracted from different sugarcane fractions were investigated using in vitro enzyme assays, spectroscopy, and molecular docking analyses. PL inhibitory activity was evaluated using p-nitrophenyl laurate (pNPL) as the substrate, with all assays performed in triplicate and results statistically analyzed. Among the extracts, sugarcane peel polyphenols (SP) exhibited the strongest inhibition, with a half-maximal inhibitory concentration (IC₅₀) of 31.56 mg/mL, significantly lower than that of sugarcane juice polyphenols (SJ, 55.86 mg/mL) and sugarcane bagasse polyphenols (SB, 65.31 mg/mL). Enzyme kinetic analyses revealed a reversible mixed-type inhibition mechanism. In contrast to crude extracts, individual phenolic monomers showed substantially lower IC₅₀ values (0.13–1.33 mg/mL), highlighting the intrinsic dilution. Compositional analysis identified ferulic acid, gallic acid, chlorogenic acid, and schaftoside as key contributors to PL inhibition. Fourier transform infrared (FTIR) and fluorescence spectroscopy demonstrated that polyphenols altered PL secondary structure by modulating α-helix and β-sheet contents and perturbed the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. Molecular docking further indicated that these compounds bind within or near the substrate-binding channel via hydrogen bonding and hydrophobic interactions, engaging critical residues including Ser152, His263, and Phe77, and potentially influencing conformational elements involved in active-site accessibility. Collectively, these results suggest that sugarcane, particularly its peel, represents a valuable natural source of PL inhibitors. Despite the relatively high IC₅₀ values of crude extracts, their inhibitory activity arises from multicomponent contributions and supports their potential application as dietary modulators of fat digestion rather than as pharmaceutical lipase inhibitors. Full article

Background/Objectives: Synthetic stimulants represent the most prevalent subclass on the new psychoactive substances (NPSs) market. However, the toxicokinetic properties of M-ALPHA, a regioisomer of MDMA and N-methyl-cyclazodone a pemoline derivative, are not yet characterized. Methods: Therefore, this study investigated the metabolism of both NPSs in pooled liver S9 fraction and rat urine, characterized cytochrome P450 (CYP) kinetics and plasma protein binding (PPB), and assessed the CYP inhibition potential of M-ALPHA, using high-performance liquid chromatography coupled to high resolution tandem mass spectrometry (HPLC-HRMS/MS). Results: Four metabolites of M-ALPHA were detected including one phase I and three phase II metabolites, resulting from demethylenation followed by subsequent methylation or glucuronidation. For N-methyl-cyclazodone, one phase I metabolite formed via N-demethylation was identified. The primary enzymes involved in M-ALPHA metabolism were CYP2B6 and CYP2D6. Notably, M-ALPHA inhibited these enzymes to a strong or moderate extent, respectively. In contrast, the metabolism of N-methyl-cyclazodone was primarily mediated by CYP2A6. PPB studies indicated low-to-moderate binding for both compounds, suggesting that significant protein-binding interactions are unlikely. Conclusions: As M-ALPHA only formed metabolites that overlapped with those of MDMA, differing only by minor retention time shifts, reliable HPLC-HRMS/MS-based identification may be challenging in clinical and forensic toxicology settings as well as doping analysis. Furthermore, drug–drug interactions following polydrug use cannot be excluded for either NPS, particularly when co-ingested with other CYP substrates metabolized by the same isoforms. Full article

Trans-anethole oxygenase (TAO) exhibits broad arylpropene substrate specificity but has low activity in converting isoeugenol to high-value vanillin. Herein, we employed computer-aided rational design to engineer TAO from Pseudomonas putida (PpTAO) for enhanced catalytic efficiency toward isoeugenol. Structural modeling and AlphaFold 3 docking identified two key catalytic residues, Arg86 and His118. Through substrate channel engineering and computation-guided mutagenesis, a series of targeted variants were constructed. Three variants, H93A, Q207R/G249C, and I59T/F62T, showed significant improvements in whole-cell performance, with activity increases of 1.8-, 2.13-, and 4.83-fold over the wild type (WT), respectively. Purified enzyme kinetics corroborated these findings, as reflected in k_cat/K_m values that reached 1.6, 2.1, and 4.7 times that of the WT. Mechanistic molecular dynamics simulations revealed that H93A enhances activity by widening the access tunnel, whereas Q207R/G249C exerts beneficial distal effects. Notably, the I59T/F62T variant significantly increases substrate affinity by optimizing hydrophobic interactions within the binding pocket. These results validate the efficacy of computational modeling in enzyme redesign and provide a robust biocatalyst for the sustainable biosynthesis of vanillin. Full article

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

The search for new anticancer agents with improved efficacy and reduced toxicity has intensified interest in metal-based compounds. In this study, two novel palladium(II) complexes, synthesized from Schiff base ligands derived from 5-chloro-salicylaldehyde and p-hydroxybenzylamine or tyramine, were chemically characterized and biologically evaluated. Both complexes exhibited significant cytotoxic activity against the MCF-7 breast cancer cell line in a dose- and time-dependent manner, with Pd2 showing slightly higher potency. Morphological analysis of treated cells indicated that apoptosis is the predominant mechanism of cell death. To gain deeper insight into the potential mechanisms underlying the observed anticancer activity, several biologically relevant targets were investigated. Enzyme kinetics revealed that the complexes act as uncompetitive inhibitors of liver catalase, suggesting a possible role in the induction of oxidative stress. Fluorescence studies demonstrated that Pd2 interacts with CT-DNA through combined intercalative and minor groove binding modes and exhibits significant binding affinity toward human serum albumin, predominantly at Sudlow’s site I. Molecular docking analysis further supported favorable interactions with catalase, estrogen receptor α, and B-form DNA, providing structural insight into the experimentally observed biological effects. Overall, the study explores multiple potential mechanisms of anticancer action, underscoring the promising therapeutic potential of these palladium(II) complexes, while antitumor activity has been initially assessed using a MCF-7 cell line as a preliminary model. Full article

Alzheimer’s disease (AD) develops as a progressive dementia condition through the step-by-step breakdown of nerve cells. Neurodegeneration in this context primarily results from metal ions, including copper, iron, zinc, and aluminum, building up in the system. The aggregation of amyloid-beta (Aβ) peptides and oxidative stress generation stem from metal ion involvement acting as defining characteristics of Alzheimer’s disease pathology. We developed a comprehensive mathematical model based on 24 coupled ordinary differential equations (ODEs) to represent the interactions between metal ions, Aβ peptides, reactive oxygen species (ROS), antioxidant defenses, and tau protein phosphorylation. The mathematical model monitors how metal ion concentrations change over time and examines their competitive binding effects, which trigger a series of reactions, resulting in oxidative stress and subsequent tau protein damage. The model uses analytical and numerical mathematical methods to expose nonlinear behaviors and threshold effects while offering mechanistic insights into the course of disease development. This model functions as a quantitative framework for assessing how therapeutic interventions that target metal dyshomeostasis and oxidative stress can potentially affect outcomes. Full article

The exponential growth of the fruit-processing industry generates significant quantities of organic by-products, such as peels, seeds, and pomace, which represent a rich but underutilized source of bioactive polyphenols. Valorizing these residues is critical for the transition toward a circular bioeconomy, yet conventional extraction methods remain solvent-intensive and kinetically inefficient. This review provides a comprehensive analysis of emerging green extraction technologies, specifically Ultrasound-Assisted (UAE), Microwave-Assisted (MAE), Enzyme-Assisted (EAE), Pressurized Liquid (PLE), and Supercritical Fluid Extraction (SFE), and Pulsed Electric Field (PEF), applied to key industrial matrices including apple, citrus, grape, olive, and coffee. Comparative data demonstrate that intensification technologies significantly outperform conventional maceration, with UAE and MAE reducing processing times by up to 90% while enhancing polyphenol yields by 20–55% through mechanisms such as acoustic cavitation and dipole rotation. Furthermore, high-pressure methods exhibit tunable selectivity, enabling the specific recovery of heat-sensitive anthocyanins and bound phenolics without the use of toxic organic solvents. The study concludes that the future of industrial valorization lies in the adoption of hybrid technologies and sequential biorefinery strategies to achieve high-purity isolates with minimal environmental impact. Full article

The global burden of hypertension continues to rise, highlighting an urgent need for effective therapeutic strategies. Angiotensin-converting enzyme (ACE) is central to blood pressure regulation, but commonly used synthetic ACE inhibitors often have adverse side effects, spurring the search for safer natural alternatives. The aim of this study was to investigate Yanbian cattle hemoglobin as a novel precursor for ACE inhibitory peptides. The <1 kDa fraction was identified as exhibiting the highest inhibitory activity through the systematic screening of hydrolysates across multiple molecular weight ranges. LC-MS/MS analysis identified 1980 peptides, of which four were selected for further experiments. Solid-phase synthesis confirmed that NFGYDL exhibited the strongest ACE inhibition (IC₅₀ = 54.95 μM). Inhibition kinetics showed FHDYL acted as a mixed-type inhibitor, DLGHF and NFGYDL as competitive inhibitors and GFHLD as a non-competitive inhibitor. Molecular dynamics simulations validated the stable binding of these bovine blood-derived peptides to the ACE complex. HUVEC functional assays demonstrated that four peptides significantly increased angiotensin II-induced nitric oxide production and endothelin-1 levels, suggesting their potential antihypertensive activity. These findings suggested that bovine blood is a promising natural source of ACE-inhibitory peptides and holds potential for application as a functional component in functional foods targeting hypertension management. Full article

Polyamines, putrescine, spermidine and spermine, are essential polycationic metabolites present in all eukaryotic cells, where they regulate fundamental processes including nucleic acid stabilization, translation, and stress responses. Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3Δ mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with K_half values of 65.5 ± 7.11 µM for putrescine and 66.9 ± 2.09 µM for dc-SAM, and V_max values of 7.1 ± 0.57 and 7.9 ± 0.12 nmol·µg⁻¹·min⁻¹, respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase. Full article

Background/Objectives: Chronic metabolic acidosis (CMA) is a mild, persistent acid–base imbalance characterized by low serum bicarbonate and urinary pH and is common in chronic illness, aging, and metabolic disorders such as type 2 diabetes (T2D). This review highlights the critical, yet often overlooked, role of CMA in T2D (CMAD) and its contribution to disease pathophysiology. Methods: We conducted a comprehensive review of the systemic impacts of CMA, from molecular mechanisms to organ-specific dysfunction. The analysis covers physiological pH dynamics in intracellular (IC) and extracellular (EC) fluids and explores their effects on cellular processes, including the cell cycle and apoptosis. Results: At the molecular level, acidosis significantly alters enzyme kinetics, macromolecule metabolism, and ion conductance. Cell-level analysis shows that pH shifts impact proliferation and programmed cell death. Systemically, the manifestations of CMA align closely with T2D features in vital organs, including the pancreas, liver, skeletal muscle, adipose tissue, and the renal, nervous, and immune systems. Our findings indicate that the pathophysiological landscape of T2D largely mirrors the biological effects of chronic acidosis. Conclusions: The alignment between the effects of CMA and the clinical features of T2D suggests that T2D pathophysiology is worth revisiting through the lens of CMAD. This perspective is further supported by therapeutic interventions showing preliminary efficacy signals in limited studies of acid-neutralization in managing T2D symptoms and progression. Full article

In selenium-rich foods, selenium speciation significantly impacts food quality. This study cultivated selenium-enriched bean sprouts using various selenium sources and systematically examined the dynamic changes in major selenium-containing compounds during thermal processing and storage. The results showed that selenomethionine (SeMet) is the predominant species in selenium-rich bean sprouts, and its stability is closely related to the redox enzymes in the bean sprout system. During processing or storage, SeMet is readily oxidized by reactive oxygen species into selenomethionine selenium oxides (SeMetO). However, SeMetO can be thermally reduced back to SeMet upon heating, revealing the reason for the stability of SeMet after processing. Additionally, the study elucidated the reduction kinetic equation of SeMetO, calculated its activation energy as 120.73 kJ·mol⁻¹, and clarified the impact of coexisting proteins, cellulose, and other components on the reduction process. This research provides an important theoretical basis for the processing of SeMet in selenium-rich foods. Full article

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) efficiency constrains carbon fixation, making it a high-value target in biotechnology. The core task of this work is a supervised regression and ranking problem on Rubisco: given a numerical representation of a protein sequence (a PLM embedding), we predict continuous phenotypic scores such as an enzyme kinetic proxy or fitness value. The predictions then guide which variants to test next. Engineering Rubisco is a point of focus but remains challenging due to selection forces in vivo and the combinatorial space of potential mutants for ex vivo uses. We combine protein language model (PLM) embeddings with tabular learning to model Rubisco variant landscapes in two regimes. First, we analyze deep mutational scanning data providing inferred kinetic proxies, including K_m for CO₂ and V_max. Second, we model a cyanobacterial screening dataset measuring mutant fitness under differing oxygen and nitrogen regimes, enabling an oxygen tolerance objective. Across tasks, a tabular foundation (TabPFN-2.5) model outperforms gradient-boosted trees on rank-based criteria for variant prioritization, including Spearman correlation and top 5% hit recovery. We then simulate active-learning campaigns initialized with 200 measured variants and iteratively acquiring batches of 48. Model-guided selection recovers more top-performing mutants than random sampling at fixed experimental budgets, even with a conservative XGBoost surrogate. We also demonstrate that Rubisco large-subunit embeddings predict cyanobacterial doubling time and cross-species kinetic parameters, suggesting that Rubisco representation remains meaningful across organisms even with multi-objective cellular constraints. Together, these results support a practical, data-efficient workflow for enzyme engineering and motivate objective-aware design strategies that complement directed evolution. Full article

The urea cycle (UC) is usually described as the hepatic metabolic pathway responsible for ammonia detoxification, but its role extends far beyond nitrogen (N) elimination to include cellular biosynthesis and metabolic signalling. In cancer cells, the UC is reconfigured/reorchestrated to support high anabolic demand, often involving the dysregulation of key enzymes such as ASS1, ASL, OTC and CPS1. While these changes support biomass production and stress resistance, they also generate measurable biochemical signatures through kinetic and thermodynamic isotope effects (¹⁴N/¹⁵N). In this review, we summarise UC biochemistry and recall key enzymatic mechanisms. Together, these elements provide a mechanistic framework to interpret changes in ¹⁵N abundance observed in tumour tissues and cells. We discuss how the redirection of N flux toward nucleotide and polyamine synthesis, coupled with partial excretion of ¹⁵N-depleted urea, may shape the isotopic composition of cancer cells. By integrating molecular oncology with stable isotope analysis, this review highlights the potential of natural isotope abundance as a functional readout of tumour metabolism and supports further investigation of its translational relevance in cancer phenotyping and monitoring. 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

Ionizing radiation affects enzymes, which are essential for most cellular functions, by inducing chemical alterations in their molecular structures, often resulting in the inhibition of their activities. Unraveling the molecular and kinetic mechanisms driving these effects requires irradiation protocols that ensure accurate dose delivery, spatial homogeneity, and reproducibility. In this study, we established a systematic experimental framework that adapts a medical linear accelerator (LINAC) as a precision source for biochemical irradiation experiments. A rigorous protocol was developed that allows enzyme solutions to be irradiated under strictly defined and verifiable dosimetric conditions. Using this approach, we quantified the radiation-induced modulation of enzyme activity in two representative enzymes: invertase (β-fructofuranosidase) and collagenase. For invertase, a pronounced nonlinear decrease in enzyme activity was observed, with the enzyme retaining approximately only 2.2% of its initial activity at 50 Gy. Conversely, collagenase activity exhibited an exponential dose–response behavior over the dose range 0.1–200 Gy, yielding a global inactivation constant of $K = 0.015 {\mathrm{G}\mathrm{y}}^{-1}$. Complementary SDS–PAGE analysis revealed no detectable radiation-induced protein fragmentation or aggregation under the investigated conditions. These results confirm enzyme-specific radiation sensitivity and demonstrate the efficacy of this LINAC-based methodology for quantitative dose–effect studies. Overall, this work provides a versatile experimental tool for applied radiation research, bridging the gap between clinical medical physics and fundamental biochemistry. Full article