Heterologous protein expression in E. coli facilitates high yield and quality, but environmental stress often destabilizes these proteins, affecting their activity. This study investigates a peptide co-expression system to enhance enzyme thermostability, demonstrated here using lipase as a model. Specifically, Late Abundant Embryogenesis (LEA) proteins were employed to protect proteins from damage. Recombinant lipase (MLipA) from Bacillus licheniformis was expressed alongside a LEA-like peptide, inspired by the 11 repetitive amino acid sequences of LEA proteins. Four LEA-like peptide co-expression systems were evaluated for their impact on lipase thermostability using a standard lipase assay. The purified recombinant MLipA was tested at 45 °C, above its optimal temperature, revealing a two-fold increase in activity when co-expressed with the LEA-II-like peptide. Sequence analysis indicated that LEA-II contains more polar and aliphatic residues, enhancing the stability of the MLipA-LEA II complex at elevated temperatures. Molecular docking suggested that specific residues of LEA-II interact with lipase to facilitate enzyme–substrate complex formation with pNPL. Molecular dynamics simulations further confirmed that the MLipA-LEA II complex is more stable at 318.15 K compared to MLipA alone. MM-PBSA analysis showed improved binding affinity of pNPL in the MLipA-LEA II complex. Together, these findings establish LEA-like peptide co-expression as a novel, sequence-independent strategy for enhancing enzyme thermostability, bridging experimental and computational approaches with potential applications in both industrial biocatalysis and therapeutic protein stabilization.

The kidney injury molecule-1 (KIM-1) serves as a well-recognized biomarker for kidney injury, making the establishment of accurate and sensitive detection methods highly valuable for clinical applications. This research involved computational design and analysis of protein-binding peptide fusion; MBP-CCT3-BP03 and MBP-CCT3BP04 were engineered to bind to KIM-1. Because peptides are generally small in size, both designed binding peptides were fused with maltose binding protein (MBP) to assist in their expression and biosensor fabrication. The affinity study and the development of biosensors were conducted electrochemically. The molecular docking binding affinity of CCT-BP03 and CCT3-BP04 against KIM-1 was predicted at -8.8 kJ/mol and - 8.1 kJ/mol respectively, while their calculated MM/PBSA were recorded at -45.70 kJ/mol and - 36.1 kJ/mol, respectively. In addition. The MD simulation structural analysis reveals that MBP-CCT3-BP04 exhibits better structural stability and folding during the 100 ns simulation while interacting with KIM-1, hence it was chosen for the development of the KIM-1 biosensor. Under optimal conditions, the developed biosensor showed a linear correlation of MBP-CCT3-BP04 related to the concentration of KIM-1 with a detection limit of 1.02 ng/mL, where it falls within the clinical range of KIM-1 in urine. The designed MBP-CCT3-BP04 also demonstrates high specificity and selectivity in both the spiked buffer and the simulated urine sample. Overall, this research establishes the viability of using computational procedure in designing binding peptides as well as employing protein-binding peptide fusion approach for biosensor development. This approach provides a practical framework for creating cost-effective and reliable KIM-1 detection platforms that could have significant clinical utility.

The increasing demand for high-performance proteases in industrial applications necessitates strategies to enhance enzyme yield, stability, and functional robustness under extreme conditions. This study investigates a serine protease from Bacillus toyonensis, isolated from fermented kitchen waste, and its co-expression with short peptides derived from late embryogenesis abundant (LEA) proteins, LEA-I, and LEA-K. The protease gene was cloned into a pRSFDuet-1 vector and expressed in Escherichia coli BL21(DE3), followed by purification using immobilized metal affinity chromatography. Co-expression with LEA peptides significantly enhanced protease production and stability, with ProLEA-K achieving the highest purification fold (1,638.00) and specific activity (16.38 U/mg) compared to 0.01 U/mg in the crude extract. Functionally, ProLEA-K exhibited a 2.5-fold increase in activity at 60°C and retained substantial activity up to 70°C, whereas the protease expressed alone (ProWL) declined sharply at elevated temperatures and maintained high activity across a broad pH range spanning acidic to alkaline conditions, a rare feature for acidic serine proteases. Evaluation under detergent-containing conditions revealed a differential response, whereby detergents enhanced the activity of ProWL but reduced the activity of LEA-co-expressed variants, indicating a context-dependent effect of LEA peptides on enzyme performance. In silico analysis supported these experimental findings. Multiple sequence alignment revealed strong conservation of the catalytic triad (Asp171, His203, and Ser225) and an amino acid profile consistent with secreted bacterial enzymes. Structural modeling confirmed stable folding, and molecular docking with azocasein indicated stronger substrate binding in LEA-containing variants (-6.9 kcal/mol for ProLEA-K vs -5.8 kcal/mol for ProWL). These results identify LEA peptides, particularly LEA-K, as effective molecular stabilizers, improving both the yield and functional robustness of industrial proteases. IMPORTANCE:This work highlights a simple and effective approach to improve industrial proteases using short late embryogenesis abundant peptides as natural stabilizers. The method enhanced yield, thermal stability, and pH adaptability across acidic and alkaline conditions without genetic redesign of the protease itself, offering a broadly applicable and economical strategy for enzyme improvement.

The prokaryotic expression system has been employed extensively for the production of recombinant proteins. Nevertheless, some proteins are particularly challenging to express at the large-scale production level. One strategy for enhancing the expression of target proteins in host cells is the use of fusion tags. The two short-disordered peptides (LEA II and LEA K) were derived from the group 3 late embryogenesis abundant protein (G3LEA) of Polypedilum vanderplanki, comprising 13 amino acid residues. The objective of this study was to investigate the potential of LEA II and LEA K (LEA-tags) as a means of increasing protein expression at low temperatures, with a particular focus on proteins that are challenging to express. To validate the efficacy of LEA tags as an expression enhancer for improving protein production, green fluorescent protein (GFP), lipase (lip) derived from Bacillus licheniformis, and silicatein-α (sil) derived from Suberites domuncula were employed as model proteins to assess the extent of their enhanced expression following fusion with LEA tags. LEA II improved the expression of lip and sil by 18- and 21-fold, respectively; LEA K improved it by 11- and 18-fold, respectively. Notably, LEA II increased the soluble expression of lipase and silicatein. The protein yield increased without impairing protein function. Using LEA tags represents a promising approach for enhancing the production of challenging proteins in Escherichia coli.

A voltammetric immunosensor for the detection of Newcastle disease virus (NDV) has been developed by employing polyclonal antibody targeting NDV (anti-NDV) as a bioreceptor. Anti-NDV was immobilized on polyethylene glycol (PEG)-containing self-assembled monolayer (SAM) which was activated with N-(3-dimethylaminopropyl)-N '-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) coupling on screen-printed gold electrode (SPGE). The introduction of PEG-containing SAM on the SPGE allowed the bioreceptor to covalently bound to the electrode surface whilst still providing a hydrophilic layer on the electrode which is important to greatly reduce non-specific bindings. The bioreceptor functionalized electrode was then allowed to be incubated with NDV-spiked samples. The electrode surface modification with PEG-containing SAM, immobilization of anti-NDV as bioreceptor, up to the detection of NDV were characterized electrochemically through differential pulse voltammetry (DPV) analysis in [Fe(CN)6]3- as the redox probe. Decrement of anodic current peak (Ipa) of [Fe(CN)6]3- was seen as the concentration of NDV increased from 0.156 to 20 HA mu L- 1 with the limit of detection (LoD) of 1.50 HA mu L- 1 at 3 sigma m- 1. The detection of NDV in HA mu L- 1 unit in this study would ease interlaboratory interpretation as it was the same unit used in hemagglutination (HA) assay of conventional NDV diagnosis. The specificity of anti-NDV used as bioreceptor towards NDV was confirmed through western blot analysis, whilst the selectivity of the bioreceptor-functionalized electrode has been tested with allantoic fluid as the negative control in which no apparent changes of anodic peak (Ipa) has been seen. This simple, fast, and less laborious electrochemical detection method could become an alternative to the conventional method for NDV detection.

Short peptides derived from Late Embryogenesis Abundant (LEA) proteins are critically important because they appear to exert their protective functions through liquid-liquid phase separation (LLPS) in vivo. Excitingly, confocal fluorescence microscopy revealed that green fluorescent protein (GFP) exhibited a heterogeneous intracellular distribution in Escherichia coli cells co-expressing LEA peptides. Guided by this observation, we conducted in vitro LLPS assays and found that the LEA-II and LEA-K peptides attenuated segregative LLPS while enhancing solution stability in the associative LLPS mode. These data support our hypothesis that LEA peptides function as molecular shields that suppress protein aggregation, thereby improving heterologous protein expression.

An integration between co-delivery and the targeting ability of nanoparticles confers an important clinical implication in breast cancer treatment, as the progression, recurrence, and development of resistance towards standard therapy are associated with a crosstalk of aberrant signaling pathways. In this study, lipid-based nanoparticles structurally defined by the presence of an inverse hexagonal phase with aqueous channels within, i.e., hexosomes, were investigated for gemcitabine and thymoquinone (Gem-TQ) co-delivery and surface functionalization with vitamin D-3-methoxypoly(ethylene glycol)(2000) (VD-PEG). These hexosomes were prepared using soy phosphatidylcholine (SPC) and an emulsifier, citric acid esters of monoglycerides (citrem). VD-PEG/SPC/citrem/Gem-TQ hexosomes demonstrated mean hydrodynamic particle size of 336.5 +/- 5.4 nm and entrapment efficiency of 97.7 +/- 0.1 % (Gem) and 99.0 +/- 0.1 % (TQ) at the compositional ratio of 2.5:2.5:0.1 wt % (SPC:citrem:VD-PEG) and co-loading at 2:9 mu M Gem-TQ. Moreover, the half-maximal lethal dose (LD50) of VD-PEG/SPC/citrem/Gem-TQ determined in zebrafish (Danio rerio) embryos at Gem:TQ concentration of 4.6:20.8 mu M was reduced by 1.3-fold compared to LD50 of Gem-TQ (non-encapsulated) solution. In addition, the inhibitory concentrations (IC50) following 24 h treatment using VD-PEG/SPC/citrem/Gem-TQ hexosomes were 33.4 +/- 8.0 mu M and 23.8 +/- 2.9 mu M against MCF7 and T-47D hormone receptor-positive luminal breast cancer cells, respectively. VD-PEG/SPC/citrem/Gem-TQ hexosomes containing methylene blue (MB) as the molecular fluorescent probe demonstrated higher cellular uptake in T-47D compared to MCF7 cells, therefore, the use of VD-PEG for the fabrication of lyotropic liquid crystalline nanoassemblies can be further investigated for its therapeutic potency and targeting mechanisms.

Conventional diagnostic methods often involve long incubation times due to limited fluid mixing in confined spaces, despite offering high sensitivity. Therefore, acoustic streaming was employed to enhance microscale advection, thereby improving biomolecular interactions and reducing assay duration. The micromixing capability was demonstrated by dispersing methylene blue (MB) in deionized water and glycerol solutions, where homogenization time decreased by approximately 80 % in water and 84-88 % in glycerol under acoustic actuation. Biomolecule adsorption was modeled using MB adsorbed onto cellulose acetate-graphene oxide (CA-GO) beads, showing improved adsorption and a reduced time to saturation from 16 to 8 min. Maximum adsorption occurred at 2 MHz frequency and 20 V amplitude. By using these optimized parameters, voltammetric immunosensing of Newcastle disease virus (NDV) was performed on PEG-alkanethiol-modified screen-printed gold electrodes (SPGE). The system incorporating acoustic streaming was compared against one without it. Results demonstrated a comparable limit of detection (1.46 HA μL-1 at 3σ m-1) achieved at shorter assay duration (8 min). These findings underscore the potential of acoustic streaming in electrochemical immunosensors to accelerate diagnostic assays without compromising sensitivity or specificity, particularly for applications utilizing screen-printed electrodes.

LEA peptides, which are designed based on late embryogenic abundant (LEA) protein sequences, have demonstrated chaperone-like functions, such as improving drought stress tolerance of Escherichia coli (E. coli). Previous studies have focused on the biological functions of linear LEA peptides. However, the function of cyclic LEA peptide still unknown. This study aimed to explore the cyclic LEA peptides' bio function like enhance the drought stress tolerance of E. coli by cyclizing the LEA peptide using SICLOPPS (Split Intein Circular Ligation of Peptides and Proteins). The results indicated that cyclization significantly improved the function and extended the potential applications. At the same time, we found that peptides containing numerous lysine residues exhibited reduced performance, which may be due to the exteins' residues affecting the SICLOPPS efficiency.

Late embryogenesis abundant (LEA) proteins are responsible for facilitating tolerance to various environmental stresses across diverse organisms. Group 3 LEA proteins are characterised by the presence of 11-mer amino acid motifs, which inspired the design of short peptides with similar protective functions. Here, we designed a LEA peptide variant (LEA-K) and evaluated its acid tolerance capacity in Escherichia coli BL21 (DE3) at pH4. Expression of LEA-K peptide improved the bacterial viability under acidic stress, suggesting its protective functions. To explore the molecular mechanism of such tolerance, we combined the RNA-sequencing (RNA-Seq) technique and molecular docking simulations. Transcriptome analysis identified 283 differentially expressed genes (DEGs), and revealed metabolic reprogramming and activation of stress-related pathways, including proton pumping, biofilm formation, and stress responsive systems. Functional enrichment analysis suggested a key role of two-component regulatory systems (TCSs) such as reactive chlorine species (RCS), sensor histidine kinase BtsS/transcriptional regulatory protein BtsR, and DNA-binding dual transcriptional regulator OmpR/sensor histidine kinase EnvZ. Protein-peptide docking simulations indicated potential interactions between LEA-K and these TCSs, suggesting a mechanistic basis of the observed transcriptional modulation. These findings propose previously unknown functional roles for LEA peptides, not only acting as molecular shields but also as signal-transducing modulators. This work expands our understanding of stress tolerance mechanisms and presents a new avenue for engineering stress-resilient bacterial systems.

In the industrial production of recombinant proteins, the culture of recombinant cells and the purification of recombinant proteins must be highly efficient, and various approaches and technologies have been developed. The intracellular environment in which recombinant proteins are produced in an environment of molecular crowding, where many biomolecules are in contact with each other. It is stressful for cells to express recombinant proteins in this environment, and it is important to regulate the intracellular environment for highly efficient expression of recombinant proteins. The LEA peptide, designed from the repeat sequence of the LEA protein, has a protective function for biomolecules. Co-expression of LEA peptides in cells can boost expression of recombinant proteins by serving as a mediator between biomolecules implicated in protein expression through molecular shielding function of the peptide. The application of LEA peptides to proteins that are difficult to express is expected and the technological applications will be presented.

The displacement of an electroactive monitoring agent, i.e., polyamidoamine dendrimers encapsulated gold nanoparticles (PAMAM-Au) upon the presence of a target antibody via acoustic streaming has been studied. Acoustic streaming has been used to improve the mass transfer and reduce the sample incubation rate, thus this study investigated its ability in enhancing the PAMAM-Au displacement efficiency of our immunosensor. For this purpose, the bio-nanogate components of maltose-binding protein carrying the antigenic determinant (MBP-aD) of hepatitis B surface antigen (HBsAg) as a bioreceptor was functionalized, followed by the monitoring agent i.e. PAMAM-Au on the electrode prior to the incubation with targeted anti-hepatitis B surface antigen (anti-HBsAg) antibody. The modified electrode was then coupled with a piezotransducer and connected to the signal transducer to induce acoustic streaming upon sample incubation. Under optimal acoustic actuation, the sample incubation time has been reduced from 20 min to 8 min via the enhancement of PAMAM-Au displacement induced by acoustic streaming. The result also demonstrated that the specificity and selectivity of the sensing platform under acoustic actuation are comparable to the static incubation in detecting the targeted antibody.

Abstract Heterozygous protein expression in E. coli facilitates high yield and quality. However, the challenges of protein instability due to environmental stress are still an issue that affects the activity of the protein produced. In this study, the improvement of protein thermostability was done using a peptide co-expression system. The developed system exploited the usefulness of Late Abundant Embryogenesis (LEA) proteins to protect proteins from damage. Recombinant lipase from Bacillus licheniformis was expressed along with the LEA-like peptide, whose design was inspired by the 11 repetitive amino acid sequences of the LEA protein. In total, four LEA-like peptide co-expression systems were assessed. The evaluation of improvements in protein thermostability was conducted using a standard lipase assay. The purified lipase was challenged at 45 °C, a higher temperature than its optimal temperature. Two-fold lipase activity was recorded from the protein co-expressed with the LEA-II-like peptide. Based on amino acid sequence comparison, LEA-II has the advantage of containing more polar residues with several aliphatic amino acids, which may improve LipAB.licheniformis-LEA II complex stability at higher temperatures. Next, molecular docking and molecular dynamic simulation were employed to analyze the stability of the lipase in the presence and absence of LEA II. The findings of the RMSD, MM-GBSA and related analyses showed that the LipAB.licheniformis-LEA II complexes have better stability than the LipAB.licheniformis alone, thus supporting the lipase assay. These findings successfully unravel the potential of the LEA-like peptide co-expression system as a novel approach to improve enzyme thermostability.

Lyotropic liquid crystalline nanoassemblies (LLCNs) are internally self-assembled (ISA)-somes formed by amphiphilic molecules in a mixture comprising a lipid, stabilizer, and/or surfactant and aqueous media/dispersant. LLCNs are unique nanoassemblies with versatile applications in a wide range of biomedical functions. However, they comprise a nanosystem that is yet to be fully explored for targeted systemic treatment of breast cancer. In this study, LLCNs proposed for gemcitabine and thymoquinone (Gem-TQ) co-delivery were prepared from soy phosphatidylcholine (SPC), phytantriol (PHYT), or glycerol monostearate (MYVR) in optimized ratios containing a component of citric and fatty acid ester-based emulsifier (Grinsted citrem) or a triblock copolymer, Pluronic F127 (F127). Hydrodynamic particle sizes determined were below 400 nm (ranged between 96 and 365 nm), and the series of nanoformulations displayed negative surface charge. Nonlamellar phases identified by small-angle X-ray scattering (SAXS) profiles comprise the hexagonal, cubic, and micellar phases. In addition, high entrapment efficiency that accounted for 98.3 ± 0.1% of Gem and 99.5 ± 0.1% of TQ encapsulated was demonstrated by the coloaded nanocarrier system, SPC/citrem/Gem-TQ hexosomes. Low cytotoxicity of SPC-citrem hexosomes was demonstrated in MCF10A cells consistent with hemo- and biocompatibility observed in zebrafish (Danio rerio) embryos for up to 96 h postfertilization (hpf). SPC/citrem/Gem-TQ hexosomes demonstrated IC50 of 24.7 ± 4.2 μM in MCF7 breast cancer cells following a 24 h treatment period with the moderately synergistic interaction between Gem and TQ retained (CI = 0.84). Taken together, biocompatible SPC/citrem/Gem-TQ hexosomes can be further developed as a multifunctional therapeutic nanodelivery approach, plausible for targeting breast cancer cells by incorporation of targeting ligands.

Cry toxins from Bacillus thuringiensis (Bt) have been extensively applied in agriculture to substitute the use of chemical insecticides. We have previously reported the use of a coexpression system in which late embryogenesis abundant (LEA) peptides under the control of the lac promoter increase the expression of insecticidal proteins in Bt. The use of lactose to induce the expression of LEA peptides may be a desirable alternative to isopropyl β‐D‐thiogalactopyranoside, the most frequently used inducer for recombinant protein expression. In this study we investigated the use of lactose as an inducer for optimal protein expression. We observed enhanced insecticidal Cry protein expression by applying a simple technique based on intermittent induction, and then optimized concentration and the point of induction time from the 11th h to the 15th h. Our data suggest that intermittent induction of lactose might be a new technique for the enhancement of bacterial protein expression.

In the present study, an experiment was carried out on the postharvest of cucumber fruit during a 14-day shelf life. The aim was to assess the impact of calcium nanoparticles (CaNPs) blended with different concentrations of salicylic acid (SA) on the shelf life of cucumbers during the seasons of 2018 and 2019. The investigation further monitored the influences of CaNPs-SA on some physical properties of cucumber, including the percentage weight loss, color, and fruit firmness. In addition, chemical properties, such as total soluble solids (SSC%), total acidity (TA%), total soluble sugars, and chlorophyll pigmentation of the fruit skin, were assessed during a 14-day shelf lifeCell wall degradation enzymes (CWEAs) such as polygalacturonase (PG), cel-lulase (CEL), xylanase (XYL), and pectinase (PT) were also researched. In addition, the generation rates of H2O2 and O2•− were calculated, as well as the reduction of DPPH. The lipid peroxidation (malondialdehyde, MDA) and cell membrane permeability (IL%) of cell wall composites were also determined. CaNPs-SA at 2 mM suppressed CWEAs, preserved fruit quality, reduced weight loss throughout the shelf-life period, and reduced the percent leakage value. At this concentration, we also found the lowest levels of MDA and the highest levels of DPPH.

Mercury is one of the priority metals classified as a human carcinogen, according to the U.S. Environmental Protection Agency and the International Agency for Research on Cancer. This metallic element has a high degree of toxicity, is known to induce multiple organs damage, and has severe adverse effects on human health and the environment, even at low levels of exposure. It has many forms in soil, such as inorganic mercury. In this work, two types of tripeptides: proline-cysteine-histidine (PCH) and γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH), were individually immobilized onto gold nanoparticles (AuNPs) surfaces with 20 nm in size via covalent coupling. In a monoligand system, only a particular tripeptide-AuNPs will be used as capturing agents for Hg2+, while in a heteroligand system, two different tripeptide-AuNPs will be used simultaneously in a mixture. Both tripeptides formed stable complexes with soft and hard metal ions. The interaction of heteroligand enhances the sensitivity and selectivity of the plasmonic sensor for Hg2+. This system was incorporated with β-mercaptoethanol (BME), which acts as a spacer between the surface-bound ligand. The heteroligand PCH/GSH-AuNPs was found to be more effective compared to both monoligand systems PCH-AuNPs and GSH-AuNPs with absorbance reading (A730/A524) of 0.808, 0.373 and 0.609, respectively. Upon the addition of metal ions, the red-to-blue color change and the degree of AuNPs aggregation formed by the heteroligand system were doubled when compared to the monoligand system. The finding was supported by absorption spectra, Transmission Electron Microscopy (TEM) analysis, and Dynamic Light Scattering (DLS) spectroscopy. The limit of detection (LOD) for Hg2+ detection was 25 parts per billion (ppb). The heteroligand system was further validated by conjugating tripeptides onto different sizes of AuNPs (20 nm and 100 nm). This new approach can constitute a more effective detecting system targeting small molecules.

In spore-forming bacteria such as Bacillus and Clostridium, the vegetative cells form highly durable hard shells called endospores inside the bacteria to survive as the growth environment deteriorates. Because of these properties, endospores can cause food poisoning and medical accidents if they contaminate food, medicine, or other products, and it is required for technology to detect the spores at the manufacturing site. In this study, we focused on the surface-enhanced Raman scattering (SERS) method for the sensitive detection of dipicolinic acid (DPA), a molecular marker of endospores. We constructed Fe3O4/Ag core–shell functional silver nanoparticles that specifically bind to DPA, and investigated a method for the qualitative detection of DPA by SERS and the quantitative detection of DPA by fluorescence method using a terbium complex formed on the surface. As a result, the concentration of the functional silver nanoparticles constructed could detect spore-derived DPA by fluorescence detection method, and SERS was several tens of nM. The functionalized nanoparticles can detect DPA quantitatively and qualitatively, and are expected to be applied to detection technology in the production of food and pharmaceuticals.