Mycotoxins, especially aflatoxins produced by fungi of the genus Aspergillus, threaten global food security and public health due to their carcinogenic and immunosuppressive effects. To exploit Key enzymatic steps in the aflatoxin biosynthetic pathway for therapeutic applications, the aflQ gene (also Known ordA) -wich encodes acytochrome P450 monooxygenase capable of generating reactive oxidative species -was targeted as a candidate antimicrobial agent. This study aims to extract and characterize the aflQ gene (known as ordA) from the Aspergillus niger fungus isolated from grain silos in Anbar Governorate, ands proteomically evaluate it as an innovative antimicrobial. The methodology involved isolating the fungus and molecularly characterizing it by amplifying and sequencing a segment ofthe18S rRNA gene. The species identity was confirmed utilizing the BLAST tool in the NCBI database. The sequence was deposited in GenBank and received an Accession number (PV455323). Subsequently, amplification of the aflQ gene was performed using polymerase chain reaction (PCR) and cloned into the pET-30b( +) expression vector. The hybrid plasmid was transformed into E. coli BL21 bacteria, and gene expression was induced using IPTG. The protein production was confirmed by SDS-PAGE analysis at the predicted molecular weight of 63–64 kDa. The results showed that the recombinant AflQ protein exhibited significantly enhanced inhibitory potency, with inhibition diameters reaching 24 mm against Gram-positive bacteria (Staphylococcus and Enterococcus) and 12.5 mm against Gram-negative bacteria (E. coli and K. pneumoniae). Conversely, the control bacterial culture showed no inhibitory activity, verifying that the observed efficacy specific to the AflQ protein. The study concludes that the AflQ protein represents a new class of oxidoreductase/Cytochrome P450antimicrobial agents. It highlights the significance of utilizing cellular debris to reduce industrial production costs by eliminating expensive purification steps.
Staphylococcus aureus is a major human pathogen causing diseases ranging from mild skin infections to life-threatening conditions due to its diverse virulence factors. The emergence of antibiotic-resistant strains underscores the urgent need for an effective vaccine, though none has yet achieved clinical success. The nasal mucosa serves as a natural reservoir in about 30
Drug-induced cardiotoxicity remains a significant challenge in pharmaceutical development. While patch-clamp remains the gold standard for evaluating ion channel effects, its low throughput limits early-phase screening. Multi-electrode array (MEA) systems offer higher throughput but often fail to mechanistically distinguish between various sodium channel modulators, such as agonists versus antagonists. The purpose of this study was to develop a cardiac safety assessment method capable of mechanistically distinguishing sodium channel modulators. In this study, we developed a cardiac safety assessment method using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with fluorescence-based intracellular Na+ flux imaging. We characterized hiPSC-CM batches by verifying the expression of key ion channel genes, including SCN5A, ATP1A1, and SLC8A1. Functional responses to sodium channel modulators were evaluated using MEA analysis. Intracellular Na+ intensity changes were quantified using the sodium sensitive dye CoroNa Green. MEA analysis confirmed functional responses to various Nav1.5 inhibitors and activators, though distinguishing specific modes of action was limited. By utilizing the sodium-sensitive dye CoroNa Green, we successfully quantified real-time Na+ intensity changes. Our results demonstrate that Nav1.5 activators (e.g., Aconitine) and Na+/K+-ATPase inhibitors (e.g., Ouabain, Digoxin) significantly increase intracellular Na+ levels at both 1-h and 24-h intervals. This imaging-based approach provides a mechanistic tool for distinguishing sodium channel modulators, offering an alternative for next-generation cardiovascular safety pharmacology.
Extracts from two native species, Cinnamomum tamala (C. tamala) and Ocimum sanctum (O. sanctum), were analyzed for their antioxidant and antibacterial abilities. The radical scavenging activities of the C. tamala and O. sanctum extracts were noted to be more pronounced in a 50–50
As a member of the DnaE intein family, Ter DnaE-3 (TE3) is limited by a relatively low splicing rate, significant undesired N-terminal cleavage reactions, and dependence on native exteins. Building on eight previously identified mutation sites from directed evolution, we employed site-directed mutagenesis to systematically investigate how these residues regulate wild-type mini-TE3 splicing and cleavage activities across various expression systems. In the pKH kanamycin resistance system, the E23G mutation reduces N-cleavage propensity, which may be attributed to reduced steric hindrance and enhanced structural flexibility. Conversely, in both pKH and pMST systems, the R50Q mutation increases the accumulation of N-terminal cleavage products, suggesting that intein catalysis may be influenced by a distributed regulatory network rather than by isolated residues. These results suggest that the catalytic behavior of TE3 is governed not only by conserved catalytic cores and adjacent extein sequences but also by distal residues that modulate the intein structure. The discovery that R50Q enhances N-terminal cleavage provides a valuable tool for future protein engineering, particularly for optimizing intein-based protein purification and expressed protein ligation (EPL) technologies.
Lignocellulose biomass is the most abundant renewable feedstock on Earth, composed of polymers (both sugar and aromatic) and thus a highly sustainable resource for developing value-added products. The naturally present biomass is highly complex and tough due to the formation of a lignin-carbohydrate matrix, making it more recalcitrant. Therefore, a pretreatment is necessary to remove lignin and enhance the enzyme accessibility. Depending on the type of biomass, a specific pretreatment approach was selected. It is well known that combining two or more different pretreatment strategies effectively reduces complexity. In the present study, an integrated approach was used in which 1
This study aims to provide an integrated assessment of recombinant protein bioprocessing by combining technological maturity frameworks (TRL/BRL) with environmental sustainability indicators. A structured literature-based analysis was conducted, covering recent advances in bioprocess engineering, including precision fermentation, artificial intelligence-assisted control, and sustainability assessment tools such as life cycle analysis (LCA) and process mass intensity (PMI). Recombinant protein production using microbial systems, such as Escherichia coli, demonstrates high scalability and cost-efficiency, with reported emissions significantly lower than those of conventional pharmaceutical synthesis (≈ 20 vs 351 kg CO2 eq/kg product). However, critical gaps persist between laboratory-scale innovation and industrial implementation, particularly in emerging economies. The integration of digital tools, including AI and digital twins, shows strong potential to improve process optimization and scale-up success rates. Bridging the gap between technological development and industrial deployment requires integrating process design, sustainability metrics, and readiness frameworks. Targeted innovation strategies are essential to accelerate biopharmaceutical development and enhance global health outcomes.
Bacterial interactions within the human gut microbiota are dynamic and closely linked to host health. These interactions are time-dependent and can be modulated by probiotic bacteria through metabolite production, environmental modification such as pH changes, and niche occupation. However, commonly used human-targeted drugs may alter these microbial dynamics. This study evaluated the effect of acetaminophen, a widely used over-the-counter drug, on the growth dynamics of a probiotic lactic acid bacterium within synthetic gut microbiota communities. The growth of Lactococcus lactis subsp. lactis was analyzed in monoculture and in co-culture with the commensal species Clostridium butyricum using 2 mL batch microbioreactors. Biomass accumulation was monitored until stationary phase, while acetaminophen and lactic acid concentrations were quantified by LC–MS. Growth kinetics were characterized using four parameters: carrying capacity, biomass accumulation (AUC), maximum growth rate ( μ_max ), and lag phase duration, which were estimated via regression analysis that coupled the 4Z Gompertz model and the exponential growth model. Co-cultures exhibited greater biomass accumulation and higher carrying capacity than monocultures, indicating mutualism between the species. A minimum effective concentration assay identified 66.1 mM acetaminophen as the concentration for subsequent experiments. Acetaminophen negatively affected all biomass-related kinetic parameters of L. lactis in mono- and co-culture, whereas C. butyricum growth remained unaffected in monoculture. LC–MS analysis suggested drug internalization without metabolic degradation. Lactic acid production reached a saturation concentration of 17.8 mM, indicating that metabolite-related probiotic activity may remain stable despite reduced biomass.
The conventional chemical bleaching of pulp generates substantial environmental burdens due to toxic effluents and high chemical consumption. This study explores a sustainable alternative through the enzymatic biobleaching of pineapple leaf fiber (PALF) pulp using crude laccase extracted from Paramarasmius palmivorus, a white-rot fungus. The effects of enzyme dosage (1–10 U/g pulp) and reaction time (2–10 h) on pulp quality were systematically optimized using Response Surface Methodology (RSM). Key pulp properties, including Kappa number, brightness, and intrinsic viscosity, were evaluated and compared to chemical bleaching using hydrogen peroxide (H2O2). Results showed that laccase treatment reduced the Kappa number by up to 57
Due to their distinct characteristics compared to other proteases, Keratinases present a promising alternative for various industrial applications. This study focused on isolating and screening keratinolytic bacteria from poultry waste-contaminated soil, optimizing keratinase production and thoroughly analyzing the biochemical, physicochemical, and thermodynamic properties of the keratinase produced by the isolated strain, Bacillus sp. MSGU2024. Optimized culture conditions resulted in a 2.9-fold increase in keratinase production compared to the initial unoptimized basal medium. The enzyme exhibited optimal activity at 55 ^∘ C and pH 8. The keratinase displayed stability in the presence of reducing agents, surfactants, and organic solvents, with stability enhanced by 1.5–2.5 times in the presence of non-ionic detergents such as Tween 20 and Tween 80. Its phenylmethylsulfonyl fluoride (PMSF) inhibition confirmed its classification as a serine protease. The enzyme’s K_m and V_max values, 0.102 mM and 0.09 M·min−1 respectively, indicated high substrate affinity and catalytic efficiency. Furthermore, the enzyme’s half-life under varying temperature and pH conditions underscored its robustness. The calculated Z-value revealed that the D-value decreased tenfold with a 5.62 ^∘ C rise in temperature. The thermodynamic analysis provided key insights, with the activation energy for denaturation ( E_d ) measured at 254.6 kJ · mol ^-1 . Gibbs free energy ( Δ G^* ), entropy ( Δ S^* ), and enthalpy ( Δ H^* ) values ranged from 101.59 to 108.57 kJ · mol ^-1 , 436.02 to 443.65 J · mol ^-1 · K ^-1 , and 249.32 to 249.41 kJ · mol ^-1 , respectively. These findings highlight the enzyme’s stability and efficiency, positioning it as a strong candidate for diverse biotechnological applications.
Bio-based fermented products have recently become increasingly important for both human health and sustainability due to the growing interest in healthy sustainable diets. In this study, spirulina was used as a substrate for the cultivation of Lacticaseibacillus rhamnosus 23.2 in a 3 L bioreactor and emphasized the evaluation of the potential health effects of the obtained bioactive products. The antioxidant potential of bioactive products has been extensively studied using assays like DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radicals, demonstrating its capacity to scavenge free radicals and mitigate oxidative stress. MTT and cell migration (scratch) assays reduced viability of Caco-2 cells and hindered cell migration cancer cells FS compared to unfermented spirulina (unFS) revealed that FS significantly reduced cell viability and migration in a dose-dependent manner, with the strongest effects observed at 5X dilution. In contrast, unFS showed weaker bioactivity. These findings highlight fermentation as a promising biotechnological approach to enhance the functional properties of natural compounds for cancer prevention and treatment. To summarize, the current study revealed that the potential of spirulina biomass to be a suitable media substrate for L. rhamnosus 23.2 strain. These findings will provide awareness toward milestone the health and food sector industrialization of FS products.
The genetic instability of genes transposed into the baculovirus genome by Bacmid technology, often caused by mini-F replicon, raises a crucial challenge for large scale production of recombinant proteins via serial passages in insect cells. To address this problem, we reconstructed Bacmid vector by decoupling the mini-F replicon from Tn7 transposition site. Two Bacmid variants, BacDC1 and BacDC2, were generated through homologous recombination. In BacDC1, the KanR-lacZα-attTn7 cassette remains in its original position while the mini-F replicon is relocated to the chitinase/v-cathepsin loci. Conversely, BacDC2 undergoes the opposite rearrangement. Using the classical Bacmid technology, two genes encoding enhanced green fluorescent protein (eGFP) and nano-luciferase (Nluc) C-terminally tagged spike 1 (S1) protein of porcine epidemic diarrhea virus (PEDV) were cloned as reporters to assess stability. Fluorescence observation analysis indicated that the genetic stability of cloned genes was markedly improved by the Bacmid variant-derived baculoviruses with sustained expression for up to 20 passages, appropriately 2–3 times longer than the wild-type control. Similarly, western blot and luciferase activity analysis demonstrated the S1-Nluc secretion from Sf9 cells infected with the dual expression variants remained stable over 10 passages, whereas the control showed significant expression decline by passage 5. These data suggest that decoupling the mini-F replicon from Tn7 transposition site significantly stabilizes cloned gene expression. The Bacmid variants thus represent promising vectors for industrial recombinant protein production and gene therapy applications.
Efficient recovery of rare earth elements (REEs) is essential for sustainable technologies. We evaluated a synthetic cyclizable peptide (peptide 2) for bacterial biosorption. Fluorescence resonance energy transfer analysis, utilizing non-linear regression models, revealed that peptide 2 possesses high affinity for middle-range lanthanides, such as Tb3+, Gd3+, and Dy3+. Disulfide-mediated cyclization significantly enhanced binding affinity, yielding a dissociation constant of 2.6 nM for Tb3+. To develop a practical biosorbent, we engineered Escherichia coli to display peptide 2 on its outer membrane via the OmpC protein. These recombinant strains exhibited a standardized adsorption capacity of up to 1.11 mg Tb/g dry biomass, representing a statistically significant improvement of up to 33
Industrial-scale production of 6′-sialyllactose (6′-SL), a sialylated human milk oligosaccharide, has recently attracted increasing attention. As a crucial enzyme for both microbial synthesis approaches and enzymatic catalysis, several α2,6-sialyltransferases (α2,6-SiaTs) have been reported and characterized. However, the low expression levels and poor stability of α2,6-SiaTs remain major bottlenecks in the construction of efficient 6′-SL production systems. In this study, we applied a deep-learning-based protein generative model, ProteinMPNN, to redesign an α2,6-SiaT from Photobacterium sp. JT-ISH-224 (pst6-224), resulting in a marked improvement in its heterologous expression in Escherichia coli BL21(DE3). Furthermore, machine learning–guided combinatorial mutagenesis was employed to restore catalytic activity in the redesigned enzyme. The expression level of the final variant was increased by approximately tenfold compared with that of the wild-type pst6-224, and a higher 6′-SL titer was achieved in a 1 L cascade catalytic system starting from CMP. This work demonstrates the potential of machine learning–assisted protein engineering and provides an α2,6-SiaT variant with high expression levels for the biological synthesis of 6′-SL.
The immune system combats viral infections through innate and adaptive responses, with macrophages and dendritic cells playing key roles in pathogen clearance. Neutralizing antibodies aid viral elimination but have variable efficacy against SARS-CoV-2, which uses ACE2 for entry. Human recombinant soluble ACE2 (hrsACE2) acts as a decoy to block viral entry but has limitations in affinity and immune uptake. We produced mannosylated hrsACE2 (Mann-hrsACE2) via a baculovirus expression system to enhance uptake by innate immune cells, aiming to improve viral neutralization and stimulate cell-mediated immunity against SARS-CoV-2. Mann-hrsACE2 protein was produced in Sf9 insect cells using recombinant baculovirus, purified by Ni–NTA chromatography, and verified by SDS-PAGE and Western blot. Its interaction with macrophages was assessed by FITC-labeling and analyzed through fluorescence microscopy and flow cytometry. Expressed Mann-hrsACE2 displayed an upward shift SDS-PAGE and Western blot analysis. Flow cytometry revealed macrophage uptake of Mann-hrsACE2 at 3, 28, and 98
Biodiesel offers a sustainable alternative to fossil fuels with reduced greenhouse gas emissions; however, its commercial viability is constrained by high production costs. This study aimed to develop a cost-effective strategy for enhanced lipid and biodiesel production using Fusarium oxysporum NRC 2017, cultivated on sugarcane bagasse hydrolysate enzymatically saccharified by Bacillus cereus 3SME, as an inexpensive carbon source. To improve lipid accumulation, the fungal strain was subjected to chemical and physical mutagenesis using gamma radiation (Ɣ-rays), ethidium bromide (EtBr), ethyl methane sulfonate (EMS), and sodium azide (NaN₃). Genetic variation between the wild type and induced mutants was confirmed by inter-simple sequence repeat (ISSR) analysis. The highest lipid-producing mutant, F. oxysporum NRC 2017-1, derived from gamma radiation, was selected for further optimization. Critical process parameters were optimized using response surface methodology (RSM) to maximize lipid yield. Under optimized conditions, the mutant strain achieved a maximum lipid yield of 3.81 g/L, representing a substantial increase over the wild type. Gas chromatography analysis revealed a favorable fatty acid methyl ester (FAME) profile, dominated by C16–C18 fatty acids, which meets the key compositional requirements for high-quality biodiesel. Furthermore, the physicochemical properties of the produced biodiesel, including cetane number, viscosity, and density, were within the recommended limits of international standards (ASTM D6751 and EN 14214). This study presents an integrated and sustainable approach combining strain improvement via mutagenesis, valorization of lignocellulosic agricultural waste, and statistical process optimization. The findings highlight the potential of F. oxysporum NRC 2017-1 as a promising candidate for scalable, cost-effective biodiesel production.
Basic Fibroblast Growth Factor (bFGF), also known as FGF2, is a multifunctional growth factor involved in several physiological and pathological processes, including angiogenesis, wound healing, and embryonic development. This study aimed to investigate the feasibility and efficacy of producing recombinant bFGF in Nicotiana benthamiana using a geminiviral transient expression vector. The primary objective was to assess the bioactivity of N. benthamiana-produced bFGF in comparison with commercially available recombinant bFGF preparations produced in different heterologous expression systems. NIH-3T3 cells were exposed to varying concentrations (0.5–1000 ng/mL) of plant-produced bFGF for 24 and 48 h. The assessment parameters included cell viability, cellular responses, and wound-healing capacity. The plant-produced bFGF exhibited slightly greater cell viability compared to the non-treated control and a modest increase in cell proliferation. The analysis of the dose–response curve yielded an EC50 value of 0.171 ng/mL highlighting the bioactivity and potency of plant-produced bFGF. These results demonstrate the efficacy of plant-derived bFGF in promoting cellular activities and facilitating wound closure, underscoring its potential for wound healing applications. However, further validation through preclinical and clinical studies is warranted.
The interaction between medicinal plants and their root-associated microbes plays a key role in shaping rhizosphere chemistry and bioactivity. This study evaluates the antioxidant potential and metabolic profiles of Viola odorata roots and associated Rhizobium sp. BR7 using in vitro DPPH, ABTS, FRAC based antioxidant assays and LC–MS-based untargeted metabolomics. Both plant and microbial extracts exhibited significant free radical-scavenging capacity. The isolate BR7 showed the most potent antioxidant activity (IC50 = 69 µg/mL), followed by AH3, JN9, and SGA9. Isolates BR5 and KB11 showed lower efficacy. The inverse correlation was observed between flavonoid content and ABTS based antioxidant activity, indicating the key role of flavonoids in antioxidant potential. LC–MS based analysis revealed overlapping antioxidant metabolites such as caffeic acid derivatives, kaempferol-3-O46 glucoside, apigenin, and chlorogenic acid, indicating metabolic complementarity between plant and bacteria. Besides these, BR7 also contained unique compounds including indole-3-acetic acid (IAA) fragments, rhamnolipid-like biosurfactants, and alkylresorcinols, suggesting strong rhizosphere adaptability. Overall, Rhizobium sp. BR7 emerges as a promising source of antioxidant co-metabolites with potential applications in biopharmaceutical and functional food development.
RNA extraction from some plants is difficult owing to the presence of pigments and secondary metabolites in vacuoles and cell walls, which hinder molecular biological studies. In this study, we developed a simple method for high-yield RNA extraction from pitchers of the carnivorous plant Nepenthes × ventrata that is recalcitrant to RNA isolation. RNA was extracted from pitchers of Nepenthes × ventrata at different developmental stages using conventional methods. Additionally, a modified cetyltrimethylammonium bromide (CTAB) method, in which an ethanol-precipitation step at high LiCl concentration was introduced, was used to extract RNA. Conventional purification methods yielded low or negligible amounts of RNA from pitcher tissues of Nepenthes, and the extracts contained visible pigment contamination. The conventional CTAB method, which is effective for RNA extraction from sweet potato tissues, also yielded only a small amount of RNA from Nepenthes tissues; most of the RNA remained in the supernatant discarded during the LiCl precipitation step, along with the contaminating pigments. By replacing this step with ethanol precipitation in the presence of high concentration of LiCl, the contaminating pigments were removed, and RNA with high purity and yield was recovered. Purified RNA could be efficiently used for full-length cDNA synthesis. The modified method requires no expensive extraction kits and is relatively safe because it does not use phenol. Therefore, this method may be effectively applied to plants that are recalcitrant to RNA isolation.
The biotechnological potential of microbial assimilation of C6 tricarboxylates relies heavily on the availability of transporters. Hydroxycitrate (HCA), a structural analog of citrate, accumulates abundantly in Garcinia plants and could serve as a valuable biosynthetic precursor; however, its uptake systems are poorly known. This study aimed to identify and functionally characterize Bacillus subtilis citrate transporters and their ability to enable HCA uptake in the heterologous Escherichia coli system. Growth of wild-type and transporter-deficient derivatives of B. subtilis (ΔcitM, ΔcimH, ΔcitH, and ΔyraO) was profiled in presence of HCA and citrate using plate and broth assays. Genes encoding these transporters were independently expressed in E. coli to confirm their functional potential, in correlation with structural and topological predictions derived from PSIPRED and MEMSAT-SVM analysis. Deletion of citM severely diminished the growth of B. subtilis on HCA and citrate while that of cimH distinctly impaired growth on HCA, unlike citH and yraO knockouts. However, only citM overexpression enabled HCA uptake in E. coli. Favorable membrane-insertion topology predicted by the PSIPRED/MEMSAT analysis further supported CitM’s exclusive functionality as an HCA transporter. CitM was identified as primarily necessary and sufficient for HCA uptake in both B. subtilis and E. coli. The potential of CimH untapped under the tested conditions could be determined through host-optimization strategies. Such mechanistic insights into the expanded transporter promiscuity provide an important framework for engineering microbial systems capable of valorizing HCA-rich agrowaste into value-added products.
