Search Results (1,597)

Collagenases are enzymes with broad biotechnological applications in medicine. This study describes the production and characterization of a collagenase from Aspergillus serratalhadensis URM 7866, isolated from the Caatinga biome. Solid-state fermentations were conducted using wheat bran under varying conditions of pH (6, 7, 8), moisture content (50%, 60%, 70%), and substrate concentration (2.5 g, 5 g, 10 g). The optimal condition—10 g of wheat bran at pH 8 and 70% moisture—yielded the highest collagenolytic activity (177.96 U/mL) and a specific activity of 50.55 U/mg. The enzyme was purified via multiple chromatography, with pre-purification and final purification factors of 18.09 and 20.21, respectively, reaching a specific activity of 1021.86 U/mg. The enzyme showed optimal activity at 50 °C and pH 8, with stability from 20 to 40 °C and pH 7–9. PMSF caused >80% inhibition; EDTA caused ~34% inhibition. Activity increased with Na⁺ and Ca²⁺ and was inhibited by Zn²⁺. The enzyme retained full activity in anionic and non-ionic surfactants (1–10%). FTIR confirmed characteristic amide bands, and kinetic analysis revealed a Km of 1.72 mg/mL and Vmax of 6.89 mg/mL/min. These findings support its potential for alkaline and surfactant-rich industrial processes. Full article

Equisetum telmateia Ehrh. (great horsetail) belongs to the Equisetaceae family and its aerial parts have been traditionally used for skin conditions and to achieve healthy and resilient skin, nails, and hair. This study aimed to evaluate the inhibition of skin-related enzymes by, the antioxidant capacity of, and the phytochemical composition of E. telmateia. Additionally, a novel emulgel was formulated from the main methanolic extract and characterized in terms of pH, viscosity, determination of content quantification, textural profile analysis, and spreadability. After the characterization studies, in vitro release and ex vivo permeation and penetration studies were performed. Firstly, the dried aerial parts of E. telmateia were macerated in methanol, followed by partitioning with solvents of increasing polarity: n-hexane, chloroform, ethyl acetate, and n-butanol. Antioxidant activity was assessed using DPPH, FRAP, CUPRAC, and TOAC assays, while enzyme inhibition was analyzed for collagenase, elastase, hyaluronidase, and tyrosinase. LC-MS/MS analysis identified 53 phytochemical compounds. Protocatechuic acid, the main phenolic compound, was quantitatively analyzed in each subfraction by HPTLC. The in vitro release studies showed sustained release of the reference substance (protocatechuic acid) and the kinetic modeling of the release was fitted to the Higuchi model. The ex vivo permeation and penetration studies showed that the formulation exhibited a retention of 3.06 ± 0.21 µg.cm⁻² after 24 h, whereas the suspended extract demonstrated a skin retention of 1.28 ± 0.47 µg.cm⁻². Both the extracts and the formulated emulgel exhibited inhibitory effects on skin-related enzymes. Our finding suggested that E. telmateia might be a valuable ingredient for wrinkle care and skin-regenerating cosmetics. Full article

In some cases, the catalytic processes involve the formation of self-organized supramolecular structures due to H-bonds and other non-covalent interactions. It has been suggested that the construction of self-assembled catalytic systems is a promising strategy to mimic enzyme catalysis at the model level. As a rule, the real catalysts are not the primary catalytic complexes, but rather, those that are formed during the catalytic process. In our earlier works, we have established that the effective catalysts M(II)_xL¹_y(L¹_ox)_z(L²)_n(H₂O)_m (M = Ni, Fe, L¹ = acac⁻, L² = activating electron-donating ligand) for the selective oxidation of ethylbenzene to α-phenyl ethyl hydroperoxide are the result of the transformation of primary (Ni(Fe)L¹)_x(L²)_y complexes during the oxidation of ethylbenzene. In addition, the mechanism of the transformation to active complexes is similar to the mechanism of action of NiFeARD (NiFe-acireductone dioxygenase). Based on kinetic and spectrophotometric data, we hypothesized that the high stability of effective catalytically active complexes may be associated with the formation of stable supramolecular structures due to intermolecular hydrogen bonds and possibly other non-covalent bonds. We confirmed this assumption using AFM. In this work, using AFM, we studied the possibility of forming supramolecular structures based on iron complexes with L²-crown ethers and quaternary ammonium salts, which are catalysts for the oxidation of ethylbenzene and are models of FeARD (Fe-acireductone dioxygenase). The formation of supramolecular structures based on complexes of natural Hemin with PhOH and L-histidine or Hemin with L-tyrosine and L-histidine, which are models of heme-dependent tyrosine hydroxylase and cytochrome P450-dependent monooxygenases (AFM method), may indicate the importance of outer-sphere regulatory interactions with the participation of Tyrosine and Histidine in the mechanism of action of these enzymes. Full article

Industrial applications of xylanases in high-temperature settings are limited by enzyme instability. This study evaluated glycerol and phenolic compounds as modulators of the catalytic and structural properties of a recombinant Myceliophthora heterothallica endoxylanase (rMhXyn) expressed in Komagataella phaffii. Glycerol (20% v/v) significantly improved thermostability (5-fold increase in half-life at 55 °C), decreased the activation energy for catalysis, and enhanced structural rigidity as evidenced by molecular dynamics simulations (reduced RMSD and Rg). In contrast, phenolic acids provided only short-term stabilization at moderate temperatures and did not confer structural benefits. Enzyme kinetics revealed that glycerol enhanced catalytic turnover (↑V_max), while phenolic compounds modified both K′ and cooperativity (Hill coefficient). Thermodynamic analysis supported glycerol’s stabilizing effect, with increased ∆H_(D) and a positive shift in ∆S_(D). These results suggest glycerol as a superior stabilizer for rMhXyn in high-temperature bioprocesses such as lignocellulosic biomass hydrolysis. These findings highlight the potential of targeted additives to improve enzyme performance for biotechnological applications. Full article

Hydrogels with spatially programmable mechanical properties hold great potential for use in biomedical applications. Inspired by the architecture of the cytoskeleton, we present a strategy for constructing tensegrity-structured hydrogels (TS-Gels) through enzyme-triggered crystal growth to enable precise mechanospatial manipulation. Specifically, alkaline phosphatase (ALP) was covalently anchored to a polyacrylamide (PAAm) hydrogel matrix to catalyze the in situ dephosphorylation of phosphotyrosine precursors, leading to the formation of rigid tyrosine crystals. These crystals functioned as compressive sticks, establishing tensegrity structures within the hydrogel network. By tuning the crystallization kinetics, both the structural morphology and mechanical reinforcement could be precisely controlled. The resulting TS-Gels exhibited significantly enhanced local tensile strength and stiffness, allowing for spatial–mechanical patterning via photo-initiated printing, mold-assisted shaping, and laser engraving. Furthermore, the unique mechanospatial tunability of TS-Gels was demonstrated in tribological surface engineering, underscoring their potential for use in tissue engineering and responsive biomaterials. Full article

The SARS-CoV-2 spike (S1) protein facilitates viral entry through binding to angiotensin-converting enzyme 2 (ACE2), but it also contains an Arg–Gly–Asp (RGD) motif that may enable interactions with RGD-binding integrins on ACE2-negative cells. Here, we provide quantitative evidence for this alternative binding pathway using a live-cell, label-free resonant waveguide grating (RWG) biosensor. RWG technology allowed us to monitor real-time adhesion kinetics of live cells to RGD-displaying substrates, as well as cell adhesion to S1-coated surfaces. To characterize the strength of the integrin–S1 interaction, we determined the dissociation constant using two complementary approaches. First, we performed a live-cell competitive binding assay on RGD-displaying surfaces, where varying concentrations of soluble S1 were added to cell suspensions. Second, we recorded the adhesion kinetics of cells on S1-coated surfaces and fitted the data using a kinetic model based on coupled ordinary differential equations. By comparing the results from both methods, we estimate that approximately 33% of the S1 molecules immobilized on the Nb₂O₅ biosensor surface are capable of initiating integrin-mediated adhesion. These findings support the existence of an alternative integrin-dependent entry route for SARS-CoV-2 and highlight the effectiveness of label-free RWG biosensing for quantitatively probing virus–host interactions under physiologically relevant conditions without the need of the isolation of the interaction partners from the cells. Full article

Background: Obesity is a well-documented risk factor for cardiometabolic diseases associated with insulin resistance. However, research on its relationship with alcohol intake and stress markers, such as cortisol and α-amylase, remains limited, particularly among young adults in the general population. Objective: This study investigated the relationship between adiposity measures, alcohol intake, and biological stress biomarkers among college students. Methods: Participants (n = 189) completed the NIH Diet History Questionnaire II. Body composition was measured via bioelectrical impedance analysis. Salivary α-amylase (sAA) activity and cortisol (sCort) were assessed using the Salimetrics α-amylase kinetic enzyme assay and enzyme immunoassay kits, respectively. Multivariable linear regression models were used to determine the association between alcohol consumption and adiposity on biological stress biomarkers. Results: Among students who were overweight and obese, higher alcohol consumption increased sAA activity (β = 1.52, p = 0.030), with a greater effect in females (β = 2.24, p = 0.012). Body fat percentage showed similar patterns with sAA activity (β = 2.20, p = 0.015), with no significant effect in males. There was no significant interaction between BMI or body fat and alcohol consumption on sCort levels. However, significant main effects were observed for African Americans (β = 0.22, p = 0.020) and overweight and obese status (β = −0.19, p = 0.025) on male students’ sCort levels. African Americans (β = 0.21, p = 0.026) and young male adults within the underfat category (β = 0.35, p = 0.022) also exhibited increased sCort levels. Conclusion: Sex-specific patterns in physiological responses between males and females revealed stronger associations in females for sAA activity and distinct patterns in sCort levels among African American males. Full article

Background: The Citrobacter genus harbors class C (AmpC) and class A β-lactamases. Citrobacter freundii produces an inducible AmpC β-lactamase controlled by the LysR-type transcriptional regulator AmpR and cytosolic amidase AmpD. Citrobacter sedlakii produces the class A β-lactamase Sed-1, whose expression is believed to be regulated by the transcriptional regulator SedR and AmpD. Objectives:C. sedlakii NR2807, isolated in Japan, is resistant to third-generation cephalosporins and displays extended-spectrum β-lactamase characteristics. Here, we sought to understand the mechanism for successful resistance to third-generation cephalosporins by investigating the regulators controlling Sed-1 production. Methods: Plasmids containing bla_Sed-1 and sedR (pCR2807) or truncated sedR (pCR2807ΔSedR) were constructed and introduced into Escherichia coli. Antibiotic-resistant mutants of NR2807 were obtained, and enzyme kinetics were assessed. Results: The AmpD mutant (pCR2807/ML4953) showed an 8-fold increase in cefotaxime MIC and an 8.46-fold increase in Sed-1 activity compared to the wild-type (pCR2807/ML4947). However, induction of pCR2807/ML4947 also led to a 1.32-fold higher Sed-1 activity, indicating semi-inducibility. Deletion of sedR (pCR2807ΔSedR/ML4947) led to a 4-fold decrease in cefotaxime MIC and 1.93-fold lower Sed-1 activity, confirming SedR as an activator. While wild-type C. sedlakii ATCC51115 is susceptible to third-generation cephalosporins, the AmpD mutation in NR2807 led to Sed-1 overproduction and resistance to this class of antibiotics. Finally, mutagenesis revealed that amino acid substitution in Sed-1 conferred resistance to ceftazidime and extended-spectrum β-lactamase characteristics. Conclusions: Sed-1 producers, though usually susceptible to third-generation cephalosporins, may develop extended-spectrum β-lactamase traits due to AmpD or Sed-1 mutations, thereby requiring careful monitoring. Full article

Aiming to address the key challenges of poor enzyme stability, difficult recovery, and difficult synergistic optimization of catalytic efficiency in high-value conversion of biomass, this study utilizes mineralization self-assembly technology to combine laccase with Fe₃O₄@SiO₂-PMIDA-Cu²⁺ composite, constructing magnetic laccase nanoflower (MLac-NFs) materials with a porous structure and superparamagnetism. This synthetic material can efficiently catalyze the selective oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA). The characterization results indicated that MLac-NFs exhibit optimal catalytic activity (63.4 U mg⁻¹) under conditions of pH 6.0 and 40 °C, with significantly enhanced storage stability (retaining 94.26% of activity after 30 days of storage at 4 °C). Apparent kinetic analysis reveals that the substrate affinity and maximum reaction rate of MLac-NFs were increased by 38.3% and 439.6%, respectively. In the laccase–mediator system (LMS), MLac-NFs mediated by 30 mM TEMPO could achieve complete conversion of HMF to FDCA within 24 h. Moreover, due to the introduction of magnetic nanoparticles, the MLac-NFs could be recovered and reused via an external magnetic field, maintaining 53.26% of the initial FDCA yield after six cycles. Full article

Catalase is a pivotal antioxidant enzyme that decomposes hydrogen peroxide and reduces oxidative stress. However, its low thermal and operational stability limits applications in challenging environments, particularly those contaminated with emerging pollutants such as polystyrene-based microplastics (PS-MPs). In this study, cryogels composed of Poly(2-hydroxyethyl methacrylate-co-allyl glycidyl ether) [Poly(HEMA-co-AGE)] were synthesized and evaluated as immobilization matrices to enhance catalase stability. Cryogels containing varying AGE concentrations were characterized using FT-IR, SEM, TEM, TGA, and BET analyses. The formulation with 250 µL AGE exhibited optimal physicochemical properties, including improved water retention, increased surface area, and high immobilization capacity (356.3 mg·g⁻¹). Immobilized catalase maintained superior activity under PS-MP-induced stress across a range of concentrations (0–1.0 mg·mL⁻¹), temperatures (4–60 °C), and exposure times (up to 5 h). Kinetic modeling revealed a significant improvement in substrate affinity, with Km decreasing from 54.9 to 17.1 mM, while Vmax decreased moderately. Long-term stability tests showed that immobilized catalase retained ~80% activity after 70 days at 4 °C and 55% after 15 reuse cycles. Desorption studies confirmed the reusability of the cryogel system. These findings suggest that Poly(HEMA-co-AGE) cryogels provide a robust and reusable platform for catalase stabilization, offering potential for applications such as wastewater treatment and biosensing in microplastic-contaminated systems. Full article

Lipoxygenases (LOXs) are a family of metalloenzymes that oxidize polyunsaturated fatty acids producing cell-signaling hydroperoxides. Fungal LOXs have drawn interest because of their roles in plant and animal pathogenesis. A new subfamily of annotated fungal LOXs has been predicted. One of its unique structural features is the presence of a cysteine amino acid encoded at the invariant leucine clamp. Herein, we isolate three representatives of this LOX subfamily from recombinant expressions in both yeast and bacterial cultures. Metal analysis indicates that the proteins accommodate a mononuclear manganese ion center, similar to other eukaryotic LOXs, but have nominal LOX activity. The functional consequence of the non-conservative mutation is further explored using a Leu-to-Cys (L546C) variant of soybean lipoxygenase, a model plant orthologue. While this L546C variant has comparable structural integrity and metal content to the native enzyme, the variant is associated with a 50-fold decrease in the first-order rate constant. The presence of cysteine at 546, compared to leucine, alanine, or serine, also results in a distinctive kinetic lag phase and product inhibition. The collective data highlight that Cys encoded at the Leu clamp is detrimental to LOX activity. Potential biological functions of these annotated fungal LOXs are discussed. Full article

This study evaluated the effects of replacing SBM with CWYWEP on in vitro rumen fermentation, nutrient degradability, and gas production kinetics. Citric waste was co-fermented with yeast waste and a multi-enzyme complex for 14 days, then sun-dried and pelleted. The final CWYWEP product contained 50.4% crude protein (DM basis). A completely randomized design tested seven diets in which SBM was replaced by CWYWEP or non-enzymatic citric waste–yeast waste pellets (CWYWP) at 0%, 33%, 66%, or 100% inclusion. Replacing SBM with CWYWEP significantly increased cumulative gas production at 96 h, with the 100% CWYWEP group achieving 93.7 mL/0.5 g DM—a 14% increase over the control (p < 0.01). Microbial lag time was reduced to 0.17 h vs. 0.28 h in the control (p < 0.05), suggesting faster microbial colonization. The highest in vitro DM degradability (IVDMD) at 48 h was observed in the 100% CWYWEP group (64.5%), outperforming both the SBM control and all CWYWP treatments (p < 0.01). Notably, CWYWEP increased total volatile fatty acids by 5% at 4 h and propionate by 9% at 2 h, while reducing methane production by 5% (p < 0.05). Other parameters, including pH, ammonia nitrogen, organic matter digestibility, and protozoal counts, were unaffected (p > 0.05). In contrast, CWYWP without enzymes showed minimal improvement. These findings indicate that CWYWEP is a promising high-protein alternative to SBM, enhancing fermentation efficiency and reducing methane under in vitro conditions. Further in vivo studies are warranted to validate these effects. Full article

Soil organic matter (SOM) decomposition is a critical biogeochemical process that regulates the carbon cycle, nutrient availability, and agricultural sustainability of cropland systems. Recent progress in multi-omics and microbial network analyses has provided us with a better understanding of the decomposition process at different spatial and temporal scales. Climate factors, such as temperature and seasonal variations in moisture, play a critical role in microbial activity and enzyme kinetics, and their impacts are mediated by soil physical and chemical properties. Soil mineralogy, texture, and structure create different soil microenvironments, affecting the connectivity of microbial habitats, substrate availability, and protective mechanisms of organic matter. Moreover, different microbial groups (bacteria, fungi, and archaea) contribute differently to the decomposition of plant residues and SOM. Recent findings suggest the paramount importance of living microbial communities as well as necromass in forming soil organic carbon pools. Microbial functional traits such as carbon use efficiency, dormancy, and stress tolerance are essential drivers of decomposition in the soil. Furthermore, the role of microbial necromass, alongside live microbial communities, in the formation and stabilization of persistent SOM fractions is increasingly recognized. Based on this microbial perspective, feedback between local microbial processes and landscape-scale carbon dynamics illustrates the cross-scale interactions that drive agricultural productivity and regulate soil climate. Understanding these dynamics also highlights the potential for incorporating microbial functioning into sustainable agricultural management, which offers promising avenues for increasing carbon sequestration without jeopardizing soil nutrient cycling. This review explores current developments in intricate relationships between climate, soil characteristics, and microbial communities determining SOM decomposition, serving as a promising resource in organic fertilization and regenerative agriculture. Specifically, we examine how nutrient availability, pH, and oxygen levels critically influence these microbial contributions to SOM stability and turnover. Full article

Nitrogen (N) availability significantly influences plant metabolism and productivity. The aim of this study was to assess the effects of low N stress and subsequent N supplementation on key enzymes of nitrogen metabolism, nitrogen metabolism-related substances, and chlorophyll a fluorescence kinetic parameters in rice genotypes with different nitrogen utilization efficiencies. We used the Jijing 88 (low-N tolerant) and Xinong 999 (low-N sensitive) as test materials. During the seedling, tillering, and booting stages, the 1/2N and 1/4N treatments were restored to the 1N treatment level. Nine treatments were used in this experiment: CK (1N), A1 (1/2N), A2 (1/2N restored to 1N during the seedling stage), A3 (1/2N restored to 1N during the tillering stage), A4 (1/2N restored to 1N during the booting stage), B1 (1/4N), B2 (1/4N restored to 1N during the seedling stage), B3 (1/4N restored to 1N during the tillering stage), and B4 (1/4N restored to 1N during the booting stage). Key physiological responses, nitrogen compounds, enzymes activities, and chlorophyll a fluorescence kinetics were analyzed. Under low nitrogen conditions, the growth and nitrogen assimilation of rice were inhibited. Compared to XN 999, JJ 88 maintains higher levels of dry matter, nitrate reductase activity (NR), glutamine synthetase activity (GS), glutamate oxaloacetate transaminase activity (GOT), glutamate pyruvate transaminase activity (GPT), as well as nitrate (NO₃⁻) and ammonium (NH₄⁺) nitrogen contents. After N supplementation during the early growth stage, both JJ 88 and XN 999 exhibit recovery capabilities. However, in the late growth stage, JJ 88 demonstrates superior recovery capabilities. In addition to enhancing nitrogen metabolism levels, there is also an increase in the content of osmotic regulation substances such as soluble sugars, free amino acids, and proline, along with responses in chlorophyll fluorescence kinetic parameters. This was primarily manifested in the enhancement of performance index (PI_ABS, PI_total), and quantum yield (φ_EO, φ_RO, ψ_EO), which maintain photosynthetic performance and electron transport efficiency. The research findings indicated that reducing N supply during the early growth stage and restoring N levels in the later stage are beneficial for the recovery of low-nitrogen-tolerant rice varieties. Therefore, in the context of sustainable agricultural production, the breeding of low-nitrogen-tolerant rice varieties and the optimization of N fertilizer management are crucial. Full article

Plastic waste, particularly poly(ethylene terephthalate) (PET), negatively impacts the environment and human health. Biotechnology could become an alternative to managing PET waste if enzymes ensure the recovery of terephthalic acid with efficiencies comparable to those of chemical treatments. Recent research has highlighted the potential of fungal cutinases, such as wild-type ANCUT1 (ANCUT1^wt) from Aspergillus nidulans, in achieving PET depolymerization. Fungal cutinases’ structures differ from those of bacterial cutinases, while their PET depolymerization mechanism has not been well studied. Here, a reliable model of the ANCUT1^wt was obtained using AlphaFold 2.0. Computational chemistry revealed potential cation-binding sites, which had not been described regarding enzymatic activation in fungal cutinases. Moreover, it allowed the prediction of residues with the ability to interact with a PET trimer that were mutation candidates to engineer the substrate binding cleft, seeking enhancements of PET hydrolysis. Enzyme kinetics revealed that both ANCUT1^wt and ANCUT1^N73V/L171Q (DM) were activated by MgCl₂, increasing the dissociation constant of the substrate and maximal reaction rate. We found that in the presence of MgCl₂, DM hydrolyzed different PET samples and released 9.1-fold more products than ANCUT1^wt. Scanning Electron Microscopy revealed a different hydrolysis mode of these enzymes, influenced by the polymer’s crystallinity and structure. Full article