Search Results (32)

Seed priming has become a promising technique in agriculture and crop-stress management. Several authors have shown that the positive effects of seed priming are associated with various metabolic, physiological, and biochemical modifications (enzyme activation, membrane repair, initiation of DNA/RNA, and protein synthesis) that enhance the speed, uniformity, and vigor of germination. However, the mechanisms underlying seed priming are not yet well understood. The aim of our work was to study the quantitative and qualitative metabolic changes in the embryonic axes (radicle and plumule) and cotyledons of Vigna unguiculata (L.) Walp. Seeds were subjected to osmopriming with polyethylene glycol (PEG), simple hydropriming, and double hydropriming (a novel treatment). Results indicated that all types of priming, particularly double hydropriming, strongly stimulated the hydrolysis of protein and carbohydrate reserves. This resulted in a decrease in soluble proteins and starch contents and an increase in amino acids and soluble sugars contents. Moreover, the priming promoted the biosynthesis of osmolytes such as proline and induced qualitative changes in the composition of amino acids and soluble sugars. These biochemical changes depend on the organ and treatment method applied to the seeds. It is worth noting that double hydropriming induces metabolic modifications to a greater extent than single hydropriming. Full article

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples. Full article

Pea protein yogurt (PPY), as an alternative to traditional dairy yoghurt, has the advantages of being a green raw material, lactose cholesterol-free, and adaptable to the needs of lactose-intolerant people. PPY was prepared by fermenting a mixture of pea protein and water (1:10, w/v) supplemented with 5% fructose for 10 h after heat sterilisation. During fermentation, lactic acid bacteria metabolise pea protein to produce aldehydes and other aromatic compounds, imparting a unique sweet–sour balance and mellow flavour. However, issues such as weak gel formation and prominent soybean-like off-flavours severely restrict the development and consumer acceptance of PPY. In this study, five fermentation systems were systematically investigated to elucidate the fermentation mechanisms of pea yoghurt and explore effective methods for eliminating undesirable soy flavours. The results indicated that hydrophobic interactions and disulfide bonds are the predominant forces driving gel formation in PPY. Additionally, the protein content increased by 0.81 g/100 g following fermentation. A total of 43 volatile flavour compounds—including aldehydes, alcohols, acids, ketones, and furans—were identified, among which the concentrations of hexanal and 2-pentylfuran, known markers for soybean off-flavour, significantly decreased. Furthermore, high-temperature and high-pressure treatments (121 °C, 3 min) demonstrated superior effectiveness in reducing soybean-like flavours. Although the high-temperature and high-pressure treatment, double-enzyme hydrolysis, and flavour-masking methods operate through distinct mechanisms, their flavour profiles converged, displaying substantial deodorisation effects and synergistic interactions. These findings provide a theoretical basis and processing parameters for flavour modulation in PPY; however, further formulation optimisation is required to enhance its nutritional and textural properties. PPY shows promise as a potential alternative to conventional dairy products in the future. Full article

Background: Insulin-degrading enzyme (IDE) has become an essential target for the clinical treatment of various important diseases, including type 2 diabetes, Alzheimer’s disease, and breast cancer, owing to its diverse substrate specificity. Particularly in cancer therapy, IDE inhibitors have received significant attention. Methods: We evaluated the in vitro inhibitory activity (IC₅₀) of ethyl 2-(3,5-dioxo-2-p-tolyl-1,2,4-thiadiazolidin-4-yl) acetate (1) against wild-type IDE. The mechanism of action was investigated using Lineweaver–Burk double reciprocal plots and molecular docking analyses. Additionally, we examined the structure–activity relationship, cytotoxicity, selectivity, and effects on cell migration to assess its potential druggability. Based on molecular docking results, we prepared the mutant protein T142A and compared its inhibitory effects with those of the wild-type and mutant proteins. Results: Compound 1 exhibited an inhibitory effect on IDE (IC₅₀ = 3.60 μM). This compound exerts its inhibitory effect through competitive binding to the catalytic site of IDE. Compound 1 demonstrated selective cytotoxicity toward cancer cells compared to normal cells, effectively inhibiting IDE at concentrations ≤ 10 μM. At a concentration of 3.6 μM, the inhibitory effect of the compound on cancer cell migration was significantly stronger than that observed in normal cells. Although the T142A mutant retained catalytic hydrolysis activity with a similar K_m value, its reaction rate was markedly lower than that of the wild-type enzyme. Conclusions: Compound 1 exhibits a competitive inhibitory effect on IDE, selectively targeting IDE with greater toxicity toward cancer cells compared to normal cells. It also inhibits cancer cell migration. Notably, 1 demonstrates significantly stronger inhibitory activity against the T142A mutant than the wild-type IDE, indicating that the Thr142 residue plays a crucial role in the interaction between the IDE hydrophobic pocket and 1. These findings suggest that 1 holds potential as a chemotherapeutic agent for treating IDE-related cancers, including breast, prostate, and pancreatic cancers. Full article

In this study, critical melting followed by freeze–thaw (CMFT) pretreatment was employed as an effective strategy to partially weaken and modify the surface structure of starch, enhancing enzymatic hydrolysis (EH) for porous starch preparation. Compared with EH alone, the CMFT + EH treatment synergistically facilitated porous structure formation while preserving structural integrity. Partial structural weakening and surface modifications induced by CMFT promoted enzyme diffusion into amorphous starch domains, enabling efficient hydrolysis and pore development without excessive granule degradation. CMFT + EH treatment reduced enzyme requirements and hydrolysis time by 33% compared to single enzymatic hydrolysis while markedly increasing water and oil absorption capacities. Porous starch prepared by CMFT + EH exhibited enhanced ordering of double-helical structures, with RC% increasing from 25.48% (native) and 24.74% (enzymatic hydrolysis alone) to approximately 28%. Furthermore, CMFT + EH significantly improved curcumin encapsulation efficiency from 40% (native) to ~88% and increased curcumin stability under various storage conditions. This study provided an effective strategy to enhance enzymatic hydrolysis efficiency for porous starch preparation with reduced enzyme addition and hydrolysis time. Full article

In recent years, biofuels and bioenergy derived from algae have gained increasing attention, fueled by the growing demand for renewable energy sources and the urgent need to lower CO₂ emissions. This research examines the generation of bioethanol and biomethane using freshly harvested and sedimented algal biomass. Employing a factorial experimental design, various trials were conducted, with ethanol yield as the primary optimization target. The findings indicated that the sodium hydroxide concentration during pretreatment and the amylase dosage in enzymatic hydrolysis were key parameters influencing the ethanol production efficiency. Under optimized conditions—using 0.3 M NaOH, 25 μL/g starch, and 250 μL/g cellulose—fermentation yielded ethanol concentrations as high as 2.75 ± 0.18 g/L (45.13 ± 2.90%), underscoring the significance of both enzyme loading and alkali treatment. Biomethane potential tests on the residues of fermentation revealed reduced methane yields in comparison with the raw algal feedstock, with a peak value of 198.50 ± 25.57 mL/g volatile solids. The integrated process resulted in a total energy recovery of up to 809.58 kWh per tonne of algal biomass, with biomethane accounting for 87.16% of the total energy output. However, the energy recovered from unprocessed biomass alone was nearly double, indicating a trade-off between sequential valorization steps. A comparison between fresh and dried feedstocks also demonstrated marked differences, largely due to variations in moisture content and biomass composition. Overall, this study highlights the promise of integrated algal biomass utilization as a viable and energy-efficient route for sustainable biofuel production. Full article

α-Arbutin is the fourth generation whitening factor in the field of cosmetics, which can block the synthesis of melanin in epidermal cells and has the advantages of good stability and less toxic side effects. Moreover, α-arbutin has potential application value in food, medicine, and other fields. However, the extraction yield from plant tissues is relatively low, which restricts its application value. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Amylosucrase (ASase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. To discover new resources of amylosucrase (ASase), this study synthesized α-arbutin using low-cost sucrose as a glycosyl donor. Probe sequences were used to identify homologous sequences from different microbial strains in protein databases as candidate ASases. Recombinant plasmids were constructed, and the enzymes were successfully expressed in Escherichia coli, followed by the enzymatic synthesis of α-arbutin. One ASase from Calidithermus terrae, named CtAs, was selected for its effective α-arbutin synthesis. The expression conditions for CtAs were optimized, its enzymatic properties were analyzed, and the conditions for the enzymatic synthesis of α-arbutin were further refined to improve its molar yield. The optimal induction conditions for CtA expression were achieved by adding IPTG at a final concentration of 0.5 mmol/L to LB medium when OD₆₀₀ reached 1.0, followed by an incubation at 20 °C and 200 r/min for 18 h. The optimal temperature and pH for CtAs were found to be 42 °C and 9.5, respectively, with good stability across the pH range of 5.0–12.0. CtAs was activated by Na⁺, K⁺, Mg²⁺, EDTA, methanol, and ethanol, but inhibited by Ca²⁺, Zn²⁺, Ba²⁺, and Ni²⁺. The kinetic parameters were V_max = 6.94 μmol/min/mL, K_m = 89.39 mmol/L, K_cat = 5183.97 min⁻¹, and K_cat/K_m = 57.99 L/(mmol·min). At 42 °C and pH 9.5, the hydrolysis/polymerization/isomerization reaction ratios were 23.27:32.96:43.77 with low sucrose concentrations and 38.50:37.12:24.38 with high sucrose concentrations. The optimal conditions for the enzymatic synthesis were determined to be at 25 °C and pH 5.0 using sucrose at a final concentration of 42 mmol/L and hydroquinone at 6 mmol/L (donor-to-acceptor ratio of 7:1), with the addition of 200 μL (0.2 mg/mL) of purified enzyme and 0.10 mmol/L ascorbic acid, under dark conditions for 6 h. The final molar yield of α-arbutin was 62.78%, with a molar conversion rate of hydroquinone of 74.60%, nearly doubling the yield compared to pre-optimization. Full article

Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5′ side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2′-deoxyuridine (DHU), 2′-deoxyuridine (dU), alpha-2′-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron–electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1. Full article

The enzymatic hydrolysis of the non-reducing disaccharide trehalose in yeasts is carried out by trehalase, a highly specific $\alpha$–glucosidase. Two types of such trehalase activity are present in yeasts, and are referred to as neutral and acid enzymes. They are encoded by distinct genes (NTH1 and ATH1, respectively) and exhibit strong differences in their biochemical and physiological properties as well as different subcellular location and regulatory mechanisms. Whereas a single gene ATH1 codes for acid trehalase, the genome of some yeasts appears to predict the existence of a second redundant neutral trehalase, encoded by the NTH2 gene, a paralog of NTH1. In S. cerevisiae the corresponding two proteins share 77% amino acid identity, leading to the suggestion that NTH2 codes for a functional trehalase activity. However, Nth2p lacks any measurable neutral trehalase activity and disruption of NTH2 gene has no effect on this activity compared to a parental strain. Likewise, single nth1$\Delta$ and double nth1$\Delta$/nth2$\Delta$ null mutants display no detectable neutral activity. Furthermore, disruption of NTH2 does not cause any apparent phenotype apart from a slight involvement in thermotolerance. To date, no evidence of a duplicated NTH gene has been recorded in other archetypical yeasts, like C. albicans or C. parapsilosis, and a possible regulatory mechanism of Nth2p remains unknown. Therefore, although genomic analysis points to the existence, in some yeasts, of two distinct genes encoding trehalase activities, the large body of biochemical and physiological evidence gathered from NTH2 gene does not support this proposal. Indeed, much more experimental evidence would be necessary to firmly validate this hypothesis. Full article

Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects. Full article

The study of amino acid interactions in the active site of scorpion venom phospholipases A₂ could help to gain insights into the structure–function relationship and the biological activities of the enzyme. In the secreted phospholipase A₂ of Scorpio maurus venom glands, Glutamate 63 and Tyrosine 122 amino acids play critical roles in the catalytic mechanism through interactions with residues around the calcium-binding loop. We constructed mutants at these positions by overexpression in Escherichia coli cells. After refolding and purification of recombinant enzymes, we studied their kinetic properties using pH-stat and monolayer techniques. The mutant Glutamate 63–Aspartate (E63D) exhibited a reduced activity, while the second mutant Tyrosine 122–Arginine (Y122R) retained some activity with a 14-fold reduction in catalytic efficiency. However, both mutants remained stable in pH values ranging from 2 to 12 whereas the double mutant D63–R122 was catalytically inactive. Comparative analysis of wild-type and mutant 3-D models showed various modifications of the hydrogen-binding network linking residues Glutamate 63 and Tyrosine 122. These modifications of interactions could explain the reduction in enzymatic activity. The kinetic behavior on phosphatidylcholine and phosphatidylethanolamine monolayers of three mutants was evaluated using a baro-stat system to assess the potential association between the hydrolysis of erythrocyte membrane phospholipids and the enzyme’s capability to penetrate phospholipid monolayers at high surface pressure. Mutants’ kinetic behaviors were similar to the wild-type form with slightly modified specific activities at high surface pressure. All mutants were more active on phosphatidylethanolamine than phosphatidylcholine films at high surface pressure. This study provided new information to further elucidate structure–function relationships of scorpion venom-secreted phospholipases A₂ and the design of novel potent drug molecules. Full article

This article aims to analyze the effects of enzyme treatment concentration, temperature, and time on peanut protein so as to obtain an optimal enzymatic hydrolysis condition for flavorzyme (Fla) and alkaline protease (Alk). The results were as follows: enzymatic hydrolysis temperature 60 °C and 55 °C, enzyme concentration 10% and 4%, enzymatic hydrolysis time 80 min and 60 min, and double enzyme hydrolysis ratio 2% Fla + 5% Alk, respectively. The BALB/c mice were sensitized with gavage of peanut protein before and after enzyme treatment to evaluate the effects of different enzyme treatments on peanut allergenicity. Compared with the mice sensitized with raw peanuts, the weight growth rate of the mice sensitized with enzyme treatment peanut increased but not as much as the control, the degranulation degree of mast cell and basophils decreased, the inflammatory infiltration and congestion in jejunum and lung tissue decreased, the expression of proinflammatory factors and thymic stromal lymphopoietin (TSLP) gene decreased, and the secretion of specific antibodies (IgE, and IgG) decreased, and the binding ability of peanut protein with peanut-specific IgE antibodies decreased as well. The results above indicate that the allergenicity of peanut protein decreases after enzyme treatment and the dual enzyme (Fla + Alk) treatment can be much more efficient. Full article

The microstructure of legumes plays a crucial role in regulating starch digestion and postprandial glycemic responses. Starch granules are double encapsulated within the outer cell wall and the inner protein matrix of legume cotyledon cells. Despite progress in understanding the role of cell walls in delaying starch digestion, the role of the protein matrix has received little research attention. The aim of this study was to evaluate if the protein matrix and cell wall may present combined physical barriers retarding enzyme hydrolysis of intracellular starch. Intact cotyledon cells were isolated from navy beans and used to assess the barrier effect of the protein matrix on the digestion of starch under conditions simulating the upper gastrointestinal tract. The cells were pretreated with pepsin at 37 °C and pH 2.0 for 1, 4, or 24 h and without pepsin for 24 h (control) to facilitate removal of the intracellular protein matrix prior to cooking and simulated in vitro digestion. A longer pretreatment time resulted in a lower protein content of the cells and a higher initial rate and extent of starch hydrolysis. We suggest that in addition to the primary cell wall barrier, the protein matrix provides a secondary barrier restricting the accessibility of α-amylase to starch. This study provides a new fundamental understanding of the relationship between the structural organization of legume cotyledon cells and starch digestion that could inform the design of novel low glycemic index foods. Full article

In the present study, we prepared pea peptides with high angiotensin-converting enzyme (ACE) inhibitory activity in vitro using an enzymatic hydrolysis of pea protein and compounded them with clam peptides to obtain a pea-clam double peptide. The effects of the two-peptide composite and pea peptides on hypertension and the damage-repair of corresponding organs were studied in spontaneously hypertensive rats (SHRs). We found that both pea peptides and the two-peptide composite significantly reduced the blood pressure upon a single or long-term intragastric administration, with the two-peptide composite being more effective. Mechanistically, we found that the two-peptide composite could regulate the renal renin-angiotensin system (RAS), rebalance gut microbial dysbiosis, decrease renal and myocardial fibrosis, and improve renal and cardiac function and vascular remodeling. Additionally, hippocampal lesions caused by hypertension were also eliminated after two-peptide composite administration. Our research provides a scientific basis for the use of this two-peptide composite as a safe antihypertension ingredient in functional foods. Full article

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH₃ generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis. Full article