Search Results (1,223)

The gelatinization and pasting behavior of starch play a critical role in governing its suitability for various food and non-food applications. Although Chlamydomonas reinhardtii is the most-studied microalga, its starch gelatinization and pasting properties have remained elusive. In this study, we applied nitrogen limitation to promote the starch accumulation of C. reinhardtii and recovered the starch using high-pressure homogenization. The multiscale structure and properties of C. reinhardtii starch (CRS) were comprehensively analyzed and compared with those of commonly used terrestrial plant starch. Results showed that CRS possesses a unique multiscale structure characterized by an exceptionally high degree of branching (18.6%) and a thinner crystalline lamellae (9.29 nm). While maintaining an A-type crystalline pattern, CRS granules exhibited higher crystallinity compared with other microalgal starches. CRS had an irregular red blood cell-like morphology with a small size (~1 μm diameter). Physicochemical analysis revealed that CRS has an intermediate gelatinization temperature and a pasting profile defined by low viscosity and remarkable shear resistance, suggesting high stability during hydrothermal processing. Significantly, cooked CRS demonstrated a lower hydrolysis rate and higher resistant starch content than several common terrestrial starches. It is attributed to its higher degree of branching and superior thermostability. This study extends the fundamental knowledge of CRS and provides a critical scientific basis for its application as a novel, sustainable ingredient with special gel properties in the future food industry. Full article

The development of thermostable and pH-robust endo-polygalacturonases (endo-PGases) is crucial for industrial applications such as food processing. This study aimed to engineer the thermostability of an acidic, thermophilic endo-PGase (PoxaEnPG28B) by rigidifying its flexible regions. We employed an integrated computational strategy combining molecular dynamics (MD) simulations at elevated temperatures with in silico analyses of unfolding free-energy changes to identify and design stabilizing mutations. This approach successfully yielded the mutant D249K, which exhibited a 5 °C higher optimal temperature (70 °C) and a 68.8% longer half-life at 55 °C, and it retained over 76.8% activity at 75 °C. Notably, D249K maintained the wild-type’s optimal pH (5.0) and broad pH stability (3.0–8.0). Although it is not the absolute top performer in every single metric, D249K achieves the best overall balance between thermostability and pH robustness among all reported thermophilic endo-PGases. MD simulations revealed that its enhanced stability sems from reduced global and local flexibility and a more compact structure. In juice extraction applications, D249K increased yields by up to 98.5%, significantly surpassing the wild-type. This study demonstrates the efficacy of MD-guided flexible region engineering for the GH28 family and presents D249K as a highly promising industrial biocatalyst. Full article

Bacterial poly(A) polymerases (PAPs) play an important role in RNA metabolism but remain poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemically characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identified Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; the active enzyme was purified as a fusion protein with the DsbA protein and used for functional assays. Mli PAP exhibited optimal activity at 30 °C and similar thermostability to E. coli PAP-1. ATP was the preferred substrate, with K_m comparable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg²⁺ (10 mM) was identified as the optimal cofactor. Mli PAP displayed salt-dependent activity, with the most effective polyadenylation in KCl and inhibition by NaCl and ammonium salts, contrasting with the halophilic nature of its host. This study provides the first functional insights into PAPs from Alphaproteobacteria, broadening the understanding of PAP diversity and biochemical properties, as well as the potential applications of PAPs in biotechnology. Full article

Background: Thermostability remains a key bottleneck for equitable access to mRNA-LNPs vaccines, mainly due to cold-chain requirements. Objectives and methods: Here, we optimized freeze-drying formulations by screening excipients (sugars, sugar alcohols, and proteins) and buffers to preserve mRNA-LNPs as solid formulations under ambient and refrigerated conditions. Physicochemical properties (size, polydispersity index [PDI], and encapsulation efficiency [EE]) and functional integrity, assessed by fluorescence-based in vitro transfection assays, were evaluated during long-term storage of up to six months. Results: Preliminary screening identified 20% sucrose and trehalose with Tris or histidine buffers as optimal for preserving physicochemical properties during freeze-drying, including high encapsulation efficiency (>90%), particle size (~200 nm), and low polydispersity (PDI < 0.2). Mannitol, gelatin, and PBS-based buffers showed adverse effects. At 4 °C, formulations F1–F3 maintained physicochemical stability and functional transfection activity for up to four months. In contrast, 20 °C storage caused progressive destabilization, with increased size, PDI, and encapsulation loss (>60% by six months). Among all formulations, 20% sucrose with 5 mM Tris (F1) showed the most robust preservation of physicochemical integrity and in vitro transfection efficiency under refrigerated and ambient conditions. Conclusions: Sugars outperformed sugar alcohols and gelatin as cryoprotectants. All formulations were stable, including functionally active at 4 °C for up to four months, while a sucrose/Tris formulation retained acceptable stability at 20 °C. Overall, the results demonstrate the feasibility of storing mRNA drug products as solid formulations at non-freezing temperatures. Full article

V. parahaemolyticus has several virulence factors, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and two separate type III secretion systems (T3SSs), T3SS1 and T3SS2. T3SS1 is responsible for cytotoxicity, primarily through the activity of its effector VP1680. To gain a detailed understanding of the relationship between the amount of effector, its expression timing, and cytotoxicity, a system is required to regulate protein expression levels and timing. In the present study, we developed an effector protein expression system controlled by an arabinose-dependent transcription factor and found that cytotoxicity toward mammalian cells increased in a VP1680-dependent manner. To ensure specific protein degradation, we also established a targeted protein degradation system, including VP0917 (ClpP) and VP0918 (ClpX)-, or VP0917 and VP1014 (ClpA)-mediated degradation of ssrA-tagged proteins (proteins bearing the C-terminal degradation tag encoded by tmRNA). By combining these systems, more than 50% of the targeted protein could be degraded within 20 min. As a byproduct of creating the systems, we obtained an enhanced green fluorescent protein variant that emits strong fluorescence in V. parahaemolyticus. The protein degradation system developed in this study has demonstrated the potential to control intracellular protein levels to a certain extent. Moreover, experimentally controlling intracellular protein levels will allow for a more detailed examination of the relationship between protein quantity and cellular phenotype, potentially overcoming the limitations of the “all-or-nothing” model. Full article

Cellulases catalyze the hydrolysis of cellulose and can be produced through fermentation processes, such as sequential fermentation (SeqF), which combines submerged and solid-state fermentation. The objective of this study was to evaluate the production of cellulases (endoglucanase and β-glycosidase) by fungi of the genus Aspergillus using coffee husks as substrate. Three Aspergillus strains were evaluated, with A. japonicus URM5620 showing the highest endoglucanase (0.368 U mL⁻¹) and β-glucosidase (0.652 U mL⁻¹) activities by SeqF. Based on the complete factorial design 2², a 9-fold and 3-fold increase in the production of endoglucanase (3.44 U mL⁻¹) and β-glucosidase (2.12 U mL⁻¹), respectively, was observed. Both enzymes showed maximum activity at 60 °C and pH 5.0. The kinetic/thermodynamic parameters indicated a high affinity of the enzymes for their respective substrates and a high catalytic potential. In addition, the half-life and decimal reduction values demonstrate the good thermal stability of endoglucanase (t_1/2 = 8.82 ± 0.34 and D = 29.32 ± 1.13 h) and β-glucosidase (t_1/2 = 26.61 ± 0.74 and D = 88.38 ± 2.47 h) at 60 °C. The thermostability results indicate potential for use in the pretreatment of raw materials. Full article

Concentrated solutions of polycarbosilane (PCS) are critically important for the development of continuous SiC precursor fibers, where solvent–polymer interactions govern rheology, viscoelastic stability, and spinnability. In this work, PCS solutions in two nonpolar hydrocarbon solvents with different molecular architectures as linear n-heptadecane and bicyclic decalin were systematically investigated over a wide concentration range, with emphasis on the semi-dilute entangled and concentrated regimes relevant to solution-based fiber spinning. A combined experimental approach involving steady and oscillatory rheometry and Fourier transform infrared (FTIR) spectroscopy was used to elucidate the influence of solvent structure on solvation, viscoelastic response, microstructural organization, and local intermolecular interactions. Despite similar dilute-solution interaction parameters, the concentrated regimes exhibit pronounced solvent-dependent differences in elasticity and flow behavior. For the first time, linear heptadecane is identified as a viable and technologically promising solvent for PCS, enabling the formation of thermostable homogeneous concentrated solutions with enhanced deformability. This behavior opens a realistic pathway toward a new solution-based fiber-spinning route based on elasticity-controlled processing. The results demonstrate that solvent molecular geometry governs the structure–rheology–processability relationship of concentrated PCS systems rather than solubility parameters alone, providing a new framework for solvent selection in SiC precursor fiber technologies. Full article

Background: bacterial pathogens and their toxins present analytical challenges for rapid and specific detection, contributing to over 600 million cases of illness annually and worsening antimicrobial resistance (AMR). Conventional detection methods are useful but limited. Single-domain antibodies (sdAbs) offer alternative recognition elements with unique biochemical and engineering benefits, enabling the development of nanobody-based immunoassays and biosensing platforms that provide fast, highly selective, and reliable detection of bacterial pathogens and toxins in both food and clinical environments. Objectives: this systematic review assesses the analytical and functional performance of nanobody-based immunoassays and sensing formats for detecting bacteria and toxins across food and clinical samples. Methods: following PRISMA guidelines, major scientific databases were used to gather research, resulting in 32 eligible studies published between 2011 and 2025. Results: data collected included assay platforms, target bacteria and toxins, limit of detection, sensitivity, specificity, matrix recovery, and practicality. Risk of bias was evaluated using an adapted QUADAS-2 framework. The review shows that nanobody-based immunoassays have achieved high performance, thermostability, compatibility with genetic engineering, and versatile assay design. When combined with advanced transduction and signal amplification strategies, these systems contribute to the development of highly sensitive and user-friendly bioanalytical platforms for detecting bacteria and toxins. Conclusions: however, most studies relied on spiked samples and lacked large-scale validation, emphasizing the need for standardized benchmarking and real-world testing. Full article

In laying hens, the undigested starch in conventional corn can reduce the efficiency of their diet. Therefore, the current study evaluated the effects of replacing conventional corn with a corn hybrid expressing thermostable α-amylase (Enogen corn) in standard and energy-deficient diets in laying hens. A total of 320 White Leghorn hens were assigned to four dietary treatments in a 2 × 2 factorial design of corn type (conventional and Enogen) and energy density (standard and 200 kcal/kg reduction) from 18–45 weeks of age. Enogen corn diet improved overall feed efficiency by 8% (p < 0.001) and increased egg production by 6–10% (p < 0.05) during 35–45 weeks compared to conventional corn diet. Yolk weight was also improved by Enogen corn at 35 and 45 weeks (p < 0.05), with no significant effect on body weight and body composition (p > 0.05). Reducing dietary energy led to higher feed intake during 36–45 weeks (p = 0.017), worsened overall feed efficiency (p = 0.030), and decreased cortical bone mineral density (p = 0.035). In conclusion, replacing conventional corn with Enogen corn improved feed efficiency and egg production, whereas a 200 kcal/kg energy reduction triggered compensatory intake and impaired cortical bone quality. Full article

Naturally derived cosmetic enzymes from food-grade plant sources are increasingly sought after as sustainable and skin-compatible alternatives to conventional exfoliating agents; however, many existing plant proteases exhibit poor thermal stability, limiting their practical use in cosmetic formulations. In this study, a thermostable keratinolytic protease extracted from Momordica charantia (bitter melon), a widely consumed edible and medicinal plant, was characterized to overcome these limitations and evaluated for its cosmetic applicability. The enzyme demonstrated strong keratin-degrading activity and retained over 80% of its activity at 70 °C, indicating superior thermal stability compared with commonly used cosmetic enzymes. In vitro assays using RAW264.7 murine macrophages confirmed low cytotoxicity and revealed significant inhibition of lipopolysaccharide-induced nitric oxide production, along with moderate elastase inhibitory activity, suggesting additional skin-beneficial properties. To assess practical exfoliating efficacy and skin compatibility, a four-week in-use test was conducted with 11 healthy adult volunteers using a formulation containing the M. charantia-derived enzyme. Significant reductions in desquamation index and improvements in skin smoothness (SEsm), measured using a Visioscan^® VC20 Plus, and hydration, assessed with a Corneometer^® CM825, were observed (p < 0.001), with no adverse effects reported. Collectively, these findings indicate that this naturally sourced, plant-derived keratinase offers a thermally stable and effective enzymatic exfoliation strategy, supporting its potential use as a sustainable cosmetic bioactive ingredient. Full article

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses. Full article

Linear epitopes, comprising continuous amino acid sequences, are critically implicated in the immunological cross-reactivity of livestock products owing to their stability during food processing. This paper aims to comprehensively review linear epitope-mediated cross-allergic reactions in major livestock products, including milk, eggs, and meat. Furthermore, this review systematically delineates the pathophysiological mechanisms underlying classical cross-allergic reactions, including α-Gal syndrome and avian egg syndrome, while addressing novel cross-reactive allergens, such as the thermostable poultry meat allergen Gal d 7 and the egg–milk cross-reactive protein α-vitellin, alongside their respective linear B-cell epitope profiles. Finally, this study summarizes the roles of these cross-reactive allergens in precise diagnosis and targeted therapy, providing a theoretical basis for future in-depth research on food cross-allergenic mechanisms. Full article

Background: Killed Whole Cell Genome-Reduced Bacteria (KWC/GRB), a versatile vaccine platform, can produce very low cost, thermostable, easily manufactured vaccines expressing complex immunogens that include potent immunomodulators. This system supports iterative optimization through a Design–Build–Test–Learn (DBTL) workflow aimed at enhancing immunogenicity. We applied this approach to developing HIV-1 gp41 Membrane-Proximal External Region (MPER) vaccines using the scaffolded MPER antigen, 3AGJ, a recombinant heterologous protein engineered to mimic MPER structures recognized by broadly neutralizing monoclonal antibodies (bNAbs). Methods: Five KWC/GRB vaccines expressing versions of 3AGJ were designed, including versions linked to immunomodulators and multimers of the immunogen. Display on the surface of the bacteria was evaluated by flow cytometry using the broadly neutralizing monoclonal antibody 2F5. Outbred HET3 mice were vaccinated intramuscularly, and MPER-specific antibody responses were assessed by ELISA and by the ability of the vaccines to induce neutralizing antibodies. Neutralization was measured against tier 1 and tier 2 HIV-1 pseudoviruses. Results: All five vaccines were strongly expressed on the bacterial surface and induced clear MPER-specific antibody responses in every mouse. About 33% of the animals showed detectable HIV-1 neutralization. Conclusions: These results demonstrate that a KWC/GRB-based scaffold-MPER (3AGJ) vaccine can elicit HIV-1 neutralizing antibodies in a subset of animals. Although further optimization will be required to improve the consistency and magnitude of neutralizing responses, the findings provide an initial validation of the concept. There are many strategies that can be used to enhance and extend immune responses induced by KWC/GRB vaccines that can be employed to yield improved anti-HIV-1 immune responses. Full article

As synthetic biology advances, prokaryotic microorganisms have become critical platforms for heterologous biosynthesis in cell factory applications. However, conventional free enzyme systems encounter substantial challenges, including inefficient intermediate transfer, toxic intermediate accumulation, and vulnerability to temperature and pH fluctuations. Enzyme complex catalytic systems offer promising solutions to these limitations. Bacillus licheniformis, a Generally Recognized as Safe (GRAS) host with exceptional protein secretion capacity, represents an ideal chassis for enzyme complex construction. This study developed a peptide-mediated platform in B. licheniformis to enable enzyme complex self-assembly and evaluated its effects on metabolic pathway performance. Five peptide elements were screened through fusion with enhanced orange/green fluorescent proteins (eOFP/eGFP) and transglutaminase (TGase). Effective peptide pairs were identified by measuring fluorescence intensity, visualizing complex formation via laser confocal microscopy, and assessing TGase activity. Subsequently, recombinant strains expressing peptide-fused key metabolic enzymes (gadTt and KdgA) were constructed for whole-cell biotransformation using gluconate as substrate to investigate the impact of peptide-mediated enzyme complexes on pyruvate synthesis. In the fluorescent protein system, P18/D18—amphipathic peptides that drive enzyme self-assembly via intermolecular hydrophobic interactions—increased extracellular fluorescence intensity of eOFP and eGFP by 31.11% and 25.21%, respectively. The D18 peptide significantly elevated TGase activity by enhancing structural stability to over 1.3-fold that of the control. For pyruvate synthesis, the peptide-mediated enzyme complex exhibited remarkable advantages in substrate conversion rate (up to 53.08%) and thermostability, confirming the platform’s ability to enhance substrate channeling despite no optimization for absolute yield. This study established a novel peptide-mediated multienzyme self-assembly platform in B. licheniformis, providing a valuable strategy for artificial metabolic channel design in synthetic biology. Full article

The thermophilic bacterium Thermus thermophilus represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from T. thermophilus strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both Escherichia coli and T. thermophilus. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from Thermus spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting T. thermophilus strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications. Full article