Search Results (141)

Snakebite envenoming remains a critical public health issue, and the molecular variability of venoms limits the cross-species efficacy of conventional antivenoms. Here, we conducted a comparative proteomic analysis of Bitis arietans venom to identify conserved peptide regions derived from enzymatic toxins and evaluate their potential relevance for complementary immunotherapeutic applications. Enzyme-enriched venom fractions were isolated through sequential affinity and ion-exchange chromatography and were subsequently characterized using fluorogenic FRET substrates and inhibitor assays. LC–MS/MS analysis identified 1099 proteins and revealed 36 conserved peptides within snake venom metalloproteinases (SVMPs), serine proteases (SVSPs), and phospholipase A₂ (PLA₂), particularly located near catalytic residues and structurally essential motifs such as the HExxHxxGxxH zinc-binding site in SVMPs, the His-Asp-Ser catalytic triad in SVSPs, and the Ca²⁺-binding loop in PLA₂, across Viperidae venoms. These conserved regions were also observed in homologous toxin isoforms from additional Viperidae genera, supporting the evolutionary conservation of key functional domains. While sequence conservation alone does not guarantee neutralization capacity, the identified regions represent strong candidates for structural epitope mapping and targeted antibody development. This study provides a peptide-level framework for advancing complementary antibody-based therapies designed to broaden cross-species toxin recognition, reduce antivenom dosage requirements, and improve clinical outcomes in snakebite envenoming. Full article

Methyl farnesoate (MF) is a sesquiterpenoid hormone that controls a variety of physiological processes in crustaceans, including morphogenesis, development, reproduction, and molting. MF action is mediated by a transcriptional signaling cascade consisting of Methoprene-tolerant (Met), Steroid receptor coactivator (Src), Krüppel homolog 1 (Kr-h1), and Ecdysone response gene 93 (E93) transcription factors (TFs), and transcriptional co-regulators CREB-binding protein (CBP) and C-terminal-binding protein (CtBP). Phylogenetic and sequence analyses revealed that these genes were highly conserved across pancrustacean species. Met and Src were characterized as basic helix-loop-helix, Period (Per)-Aryl Hydrocarbon Nuclear Translocator (ARNT)-Single-minded (Sim) protein (bHLH-PAS) TFs; Kr-h1 was characterized as a C₂H₂ zinc finger TF with seven zinc finger motifs; E93 was characterized as a helix-turn-helix, pipsqueak (HTH_Psq) TF. CBP was identified by several zinc finger-binding regions with Transcription Adaptor Zinc Finger 1 and 2, Really Interesting New Gene, Plant homeodomain, and Z-type zinc finger domains; the Kinase-inducible Domain Interacting-transcription factor docking site; the Bromodomain-acetylated lysine recognition and binding site; the histone acetyltransferase domain; and a C-terminal CREB-binding region containing a nuclear receptor co-activator-binding domain. CtBP had a dehydrogenase domain with arginine-glutamate-histidine catalytic triad. 81 Met contigs, 45 Src contigs, 136 Kr-h1 contigs, 66 E93 contigs, 60 CBP contigs, and 172 CtBP contigs were identified across pancrustacean taxa, including decapod crustaceans. Bioinformatic identification and annotation of these TFs and co-regulators in brachyuran Y-organ (YO) transcriptomes suggests that MF signaling influences YO ecdysteroidogenesis; functional tests in the YO are needed to establish causality. Full article

Green technologies are actively being used to produce nanosized zinc oxide, which is in demand for water purification processes to remove pollutants. Despite the success of the green synthesis of ZnO nanoparticles, no scientific approach exists for selecting plant extracts to produce nanoparticles with the desired properties. This study shows that the antioxidant activity of the plant extracts used is a key parameter influencing the properties of the resulting ZnO nanoparticles. This conclusion is based on the results of nanoparticle synthesis with the use of various plant extracts. The antioxidant activity of the extracts increases in the following order: plum–gooseberry–black currant–strawberry–sea buckthorn. The synthesized ZnO nanoparticles were characterized by UV–visible spectroscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The catalytic properties of ZnO nanoparticles were tested under the degradation of a synthetic methylene blue dye after exposure to UV light. We found that with an increase in the AOA of plant extracts, the size of the nanoparticles decreases, while their photocatalytic activity increases. The smallest (d = 13 nm), most uniform in size (polydispersity index 0.1), and most catalytically active ZnO nanoparticles with a small band gap (2.85 eV) were obtained using the sea buckthorn extract with the highest AOA, pH 10 of the reaction mixture and 0.1 M Zn(CH₃COO)₂∙2H₂O as a precursor salt. ZnO nanoparticles synthesized in the sea buckthorn extract demonstrated the highest dye photodegradation efficiency (96.4%) compared with other nanoparticles. The established patterns demonstrate the “antioxidant activity–size–catalytic activity” triad can be considered as a practical guide for obtaining ZnO nanoparticles of a given size and with given properties for environmental remediation applications. Full article

Cold-active lipolytic enzymes enable low-temperature biocatalysis, but remain underexplored in Antarctic actinomycetes. Here, we report the discovery and first-step characterization of a CALB-like cold-active lipolytic enzyme (PanLip) from Pseudonocardia antarctica. Sequence and structure analyses revealed a canonical α/β-hydrolase fold with a conserved Ser–Asp–His triad and short helical elements around the pocket reminiscent of CALB’s α5/α10 lid. Mature PanLip was expressed primarily as inclusion bodies in E. coli; an N-terminally truncation (PanLipΔN) improved solubility and PanLipΔN was purified by Ni–NTA. Far-UV CD confirmed a folded α/β architecture. PanLipΔN favored short-chain substrates (p-NPA, k_cat/K_M = 2.4 × 10⁵ M⁻¹·s⁻¹) but also showed measurable hydrolytic activity toward natural triglycerides, consistently with a lipase-family esterase. The enzyme showed an activity optimum near 25 °C and pH 8.0. The enzyme tolerated low salt (maximal at 0.1 M NaCl), mild glycerol, and selected organic solvents (notably n-hexane), but was inhibited by high salt, Triton X-100, and SDS. AlphaFold predicted high local confidence for the catalytic core; DALI placed PanLip closest to fungal lipases (AFLB/CALB). Temperature-series MD and CABS-flex indicated enhanced surface breathing and flexible segments adjacent to the active site—including a region topologically matching CALB α10—supporting a flexibility-assisted access mechanism at low temperature. Structure-based MSAs did not support a cold adaptation role for the reported VDLPGRS motif. Taken together, these findings position PanLip as a promising cold-active catalyst with CALB-like access control and potential for low-temperature biocatalysis. Full article

Ethyl butyrate is a typical flavor ester with pineapple-banana scents, but the poor yield from natural fruits limits its feasibility in food and fragrance industries. In this study, dendritic fibrous nano-silica (DFNS) was hydrophobically modified with octyl groups (DFNS-C₈) to immobilize Candida antarctica lipase B (CALB) for solvent-free esterification of ethyl butyrate. The immobilized lipase CALB@DFNS-C₈, with the enzyme loading of 354.6 mg/g and the enzyme activity of 0.064 U/mg protein, achieved 96.0% ethyl butyrate conversion under the optimum reaction conditions where the molar ratio of butyric acid to ethanol was 1:3, with a reaction temperature and time of 40 °C and 4 h. Under the solvent-free catalytic reactions, CALB@DFNS-C₈ presented the maximum catalytic efficiency of 35.1 mmol/g/h and retained 89% initial activity after ten reuse cycles. In addition, the immobilized lipase can efficiently catalyze the synthesis of various flavor esters (such as butyl acetate, hexyl acetate, butyl butyrate, etc.) and exhibits excellent thermostability and solvent tolerance. A molecular docking simulation reveals that the hydrophobic cavity around the catalytic triad stabilizes the acyl intermediate and ensures the precise orientation of both acid and alcohol substrates. This work provides new insights into the sustainable production of flavor esters using highly active and recyclable immobilized lipases through rational carrier hydrophobization and structural confinement design. Full article

Propionylated derivatives of gastrodin are valuable due to their enhanced lipophilicity and bioavailability. This study investigated the molecular basis for the differential catalytic efficiency of Aspergillus oryzae whole cells in gastrodin propionylation. A high conversion rate of 96.84% was achieved with soybean oil induction, compared to only 8.23% under glucose induction. Comparative transcriptomic analysis identified 20,342 differentially expressed genes (DEGs), which were significantly enriched in lipid metabolism and signal transduction pathways. From 26 upregulated lipase-related DEGs, a candidate triacylglycerol lipase gene (CL24.Contig40_All) was prioritized. Homology modeling and molecular docking supported its potential role by demonstrating that the encoded enzyme possesses a typical α/β hydrolase fold with a catalytic triad and favorable binding with both gastrodin and vinyl propionate. These findings indicate that soybean oil may enhance lipase expression by activating lipid metabolic and phosphatidylinositol signaling pathways, providing crucial transcriptional-level insights and genetic targets for the rational design of efficient whole-cell biocatalysts. Full article

Sperm proteases are involved in several gamete interaction events leading to fertilization. This report presents a detailed analysis of the expression and localization of serine protease PRSS38 in human and in mouse spermatozoa and its involvement in fertilization-related events, using bioinformatics, cellular, biochemical, molecular, and functional approaches. Bioinformatics analyses included genomics and data analysis, prediction of protein subcellular localization and post-translational modifications, Self-Organizing Maps (SOMs) unsupervised training with other serine proteases, protein modeling (AlphaFold), and genetic variant analysis. For cellular, biochemical, and functional studies, human semen samples were obtained from healthy normozoospermic volunteers, and cauda epididymal sperm were collected from adult Balb-c/C57 mice. PRSS38 presence was detected in human and mouse sperm protein extracts by Western immunoblotting. Sperm PRSS38 subcellular localization was determined by fluorescence immunocytochemistry. Human sperm–oocyte interaction events were assessed by means of the mouse Cumulus Penetration Assay (CPA) using mouse COCs, the Human Hemizona Assay (HZA), and the ZP-free hamster egg Sperm Penetration Assay (SPA). Mouse sperm–oocyte interactions were evaluated by means of in vitro fertilization (IVF) with COCs and denuded oocytes. PRSS38 is proposed to be a GPI-anchored serine protease (active site: His-100, Asp-150, and Ser-245) based on bioinformatics analyses. Using commercial antibodies, protein forms of the expected Mr (human: 31 kDa; mouse: 32 and 24 kDa) were specifically immunodetected in protein sperm extracts. Immunocytochemical analysis revealed a specific PRSS38 signal in the human sperm acrosomal region, equatorial segment, and flagellum. Mouse sperm PRSS38 was immunolocalized in the equatorial segment and hook. Human sperm preincubation with specific antibodies resulted in inhibition (p < 0.05) of CPA, HZA, and SPA. Mouse sperm preincubation with PRSS38 antibodies impaired (p < 0.05) homologous IVF using COCs and denuded oocytes. Genetic variants affecting residues involved in the GPI anchor and the catalytic triad were found in individuals from the general population whose PRSS38 protease function could be altered. This study provides, for the first time, an integrated analysis of PRSS38 in human and mouse sperm, contributing to our understanding of mammalian fertilization and male infertility. Full article

Typical 2-Cys peroxiredoxins (2-Cys Prxs, AhpC/Prx1 subfamily) are ubiquitous thiol peroxidases that efficiently reduce H₂O₂ and other hydroperoxides via a reactive peroxidatic Cys (C_P). Under elevated hydroperoxide levels, C_P can be hyperoxidized to sulfinic (C_P-SO₂H) or sulfonic (C_P-SO₃H) acids, leading to enzyme inactivation. Notably, eukaryotic 2-Cys Prxs are orders of magnitude more sensitive to hyperoxidation (sensitive Prxs) by H₂O₂ than their bacterial counterparts (robust Prxs). Sensitivity to hyperoxidation also correlates with the catalytic triad composition: enzymes containing threonine (Thr-Prx) are more prone to hyperoxidation by H₂O₂ than those with serine (Ser-Prx). While hyperoxidation is reversed in eukaryotes by an enzyme (sulfiredoxin), it is generally considered irreversible in bacteria. Here, we compared the hyperoxidation susceptibility of three typical 2-Cys Prxs: human Prx2 (Thr-Prx, sensitive), P. aeruginosa (Thr-Prx, robust) and S. epidermidis (Ser-Prx, robust) to lipid hydroperoxides derived from linoleic acid, containing one or two peroxide moieties per molecule. Employing structural analysis, molecular simulations and kinetic assays, we found that lipid peroxides proved to be potent hyperoxidizing agents for all 2-Cys Prx tested, inactivating the enzymes up to 10,000 times faster than H₂O₂. These results may have implications for understanding bacterial oxidative stress responses and antimicrobial resistance. Full article

Procyanidins are oligomeric flavonoids with several bioactive properties. Their antidiabetic potential is related to their capacity to inhibit enzymes responsible for the absorption of dietary carbohydrates, such as pancreatic α-amylase. Procyanidins possess great structural diversity, including types of monomers and interflavanic bonds (A- or B-), and the degree of polymerization. However, there is a lack of evidence that systematically analyzes the effect of these structural features on their α-amylase inhibitory activity. In this paper, the activity of a mammalian pancreatic α-amylase was assessed using two different substrates, and the inhibitory activity of five commercially available procyanidins and three monomeric flavonoids was compared. The enzyme-binding sites of the eight compounds were predicted by in silico analysis to help explain the different enzyme-inhibitory activities. The inhibitory activity of procyanidins and monomeric flavonoids depended on the substrate used. A-type dimers presented the best activity against a polymeric substrate, while a B-type dimer was the best inhibitor for an oligomeric substrate. The predicted binding site for dimers and monomers was close to the active site. For the B-type trimer, the binding site was on the back side (approximately 180°) of the catalytic triad. In silico predictions suggested that dimeric procyanidins, especially A-type, could better enter the active site cavity, which could explain their superior inhibitory activity. We may conclude that inhibition of pancreatic α-amylase by procyanidins is mainly related to the type of interflavanic bond and the degree of polymerization. Dimers could be the most effective procyanidins to mildly inhibit this enzyme and present antidiabetic potential. Full article

Phthalic acid esters (PAEs) are important plasticizers that have led to the heavy pollution of farmland, which has aroused significant and widespread concern for soil health and food safety. Microbial degradation has been recognized as an efficient pathway for removing PAEs from the environment. In this study, the PAE-degrading strain FR1 was isolated from sewage and determined to belong to Glutamicibacter. This strain degraded PAEs efficiently under a wide range of conditions—10–50 °C, pH of 6.0–11.0, and 0–8% salinity—demonstrating its great potential in PAE bioremediation. Genome sequencing provided complete genomic information, showing that the strain comprises one chromosome (3,404,214 bp) and three plasmids (112,089 bp, 80,486 bp, and 40,002 bp). The chromosome harbors 3238 protein genes, of which the PAE hydrolase genes dphGB1 and mphGB2 have been cloned. The hydrolase DphGB1 from hydrolase family I contained the catalytic triad Ser75-Asp194-His221. After heterogeneous expression and purification, the recombinant protein DphGB1, of about 30 kDa, was obtained. This hydrolase showed strong hydrolytic ability toward DEHP. The protein MphGB2 could also hydrolyze MBP. The molecular docking revealed interaction between DphGB1 and DBP. The main hydrolases of strain FR1-degrading PAEs were functionally identified. These results will promote the elucidation of the catalytic mechanisms of PAE hydrolases and the application of strain FR1 in farmland soil remediation. Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

Phthalic acid esters (PAEs), ubiquitously employed as a plasticizer, have been classified as priority environmental pollutants because of their persistence, bioaccumulation, and endocrine-disrupting properties. As a characterized PAE-degrading strain of marine origin, Mycolicibacterium phocaicum RL-HY01 utilizes di-(2-ethylhexyl) phthalate (DEHP) as its sole carbon and energy source. Genome sequencing and RT-qPCR analysis revealed a previously uncharacterized hydrolase gene (dehpH) in strain RL-HY01, which catalyzes ester bond cleavage in PAEs. Subsequently, recombinant expression of the cloned dehpH gene from strain RL-HY01 was established in Escherichia coli BL21(DE3). The purified recombinant DehpH exhibited optimal activity at 30 °C and pH 8.0. Its activity was enhanced by Co²⁺ and tolerant to most metal ions but strongly inhibited by EDTA, SDS, and PMSF. Organic solvents (Tween-80, Triton X-100, methanol, ethanol, isopropanol, acetone, acetonitrile, ethyl acetate, and n-hexane) showed minimal impact. Substrate specificity assay indicated that DehpH could efficiently degrade the short and long side-chain PAEs but failed to hydrolyze the cyclic side-chain PAE (DCHP). The kinetics parameters for the hydrolysis of DEHP were determined under the optimized conditions, and DehpH had a V_max of 0.047 ± 0.002 μmol/L/min, K_m of 462 ± 50 μmol/L, and k_cat of 3.07 s⁻¹. Computational prediction through structural modeling and docking identified the active site, with mutagenesis studies confirming Ser228, Asp324, and His354 as functionally indispensable residues forming the catalytic triad. The identification and characterization of DehpH provided novel insights into the mechanism of DEHP biodegradation and might promote the application of the target enzyme. Full article

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

This study used all-atom molecular dynamics simulations to investigate the structural dynamics of Ves a 1, a phospholipase from Vespa affinis venom, and its interactions within a lipid membrane environment, both alone and in the presence of the inhibitor voxilaprevir. Simulations conducted over 1 $\mathsf{\mu }$s for triplicate runs demonstrated system stability and convergence of structural properties. Our findings reveal that Ves a 1 engages in dynamic interactions with the lipid bilayer, involving key regions such as its lids, catalytic triad, and auxiliary site. The presence of voxilaprevir was observed to subtly alter these membrane interaction patterns and influence the enzyme’s catalytic area, reflecting the inhibitor’s impact within its physiological context. These results emphasize the crucial role of the lipid bilayer in shaping enzyme function and highlight voxilaprevir as a promising candidate for further inhibitor development, offering vital insights for rational drug design targeting membrane-associated proteins. Full article

Lactiplantibacillus plantarum B22 exhibits a high esterase/lipase activity, but the genomic and probiotic potential remains unclear. We employed an integrated approach combining whole-genome sequencing, molecular docking studies, and phenotypic assays to dissect the genomic and functional basis underlying the high lipolytic activity and probiotic traits of L.plantarum B22. This strain exhibited a robust lipase activity (3.45 ± 0.13 U/mL), with whole-genome analysis revealing that the complete genome of this strain spans 2,027,325 bp, encoding 2005 genes with a guanine-cytosine (GC) content of 35.06%. Notably, 13 esterase/lipase genes were identified, 4 of which (gene3060, gene3059, gene2553, gene0798) harbor conserved catalytic triads (Ser-His-Gly/Ala), essential for lipase function. Molecular docking studies confirmed strong binding affinity to tributyrin (ΔG ≤ –5.52 kcal/mol) and elucidated the interaction mechanisms, involving hydrogen bonding and hydrophobic interactions between the esterase/lipase enzymes and tributyrin. Phenotypic and genomic analyses further demonstrated that L. plantarum B22 possesses excellent tolerance to simulated human gastrointestinal tract conditions, along with potent antioxidant and antimicrobial activities, highlighting its strong probiotic potential. Genomic annotation also identified 68 genes associated with lipid metabolism and an intact fatty acid synthesis pathway. Importantly, the analysis of phenotypes and genes involved in virulence factors, and the production of harmful metabolites suggests that L. plantarum B22 is safe. Collectively, this study offers novel insights into the genome-guided functional characterization of L. plantarum B22, providing a robust foundation for its development as a functional probiotic strain. Full article