SDS-PAGE Analysis Research Articles

Characterization of an aminotransferase TlBCAT from Trichoderma longibrachiatum UN32 involved in dendrobine-type total alkaloids biosynthesis.

Trichoderma longibrachiatum UN32 is an industrially important fungus capable of producing Dendrobine-Type Total alkaloids (DTTAs). Several reports have pointed out that branched-chain amino acid aminotransferases (BCATs) participate in backbone modification or promote the production of secondary metabolites. We previously proposed that cobalt chloride increased DTTAs production in T. longibrachiatum UN32, which was associated with enhanced expression of the gene BCAT, TlBCAT (Genbank accession No. PP465542). Following cloning and characterization, the cDNA of TlBCAT was found to consists of 1191bp, encoding a protein of 397 amino acid residues. The molecular mass of TlBCAT was about 42kDa through SDS-PAGE analysis. The predicted pI value was 5.54. Recombinant TlBCAT can catalyze L-leu with a catalytic efficiency of 15.91 mM- 1S- 1. In the binding pocket, residues interacting with PLP, including Tyr68, Arg93, Tyr136, and Lys194, are highly conserved. TlBCAT exhibits a broad spectrum of substrate specificity, typical for catalyzing the transamination reaction of various branched-chain and hydrophobic amino acids, with α-ketoglutarate as the amino acceptor. Exogenous branched-chain amino acids (BCAAs) positively affect Trichoderma growth and DTTAs production. These findings offer insights into the physiological significance of BCAAs and present a novel approach for enhancing DTTAs production in Trichoderma mycelium cultures.

Identification and Molecular Analysis of Proteins Associated with Non-spinning Disease in the Silkworm Bombyx mori L Induced by BmNPV and BmDNV

Aims: The present study combines morphological and molecular biology investigations to uncover the underlying causes of the non-spinning syndrome in silkworms. To understand the molecular mechanism and immune mechanism after infection of viral pathogen in silkworm Bombyx mori L. Study Design: Infected silkworm samples were collected from the field and isolated the genomic DNA and total proteins. Analyzed the samples by using genomic tools and proteomic tools. Place and Duration of Study: Central Sericultural Research and Training Institute, Mysuru, Karnataka. 01.03.2023 to 31.07.2023 total 04 months. Methodology: Our findings indicate that infections of BmDNV and BmNPV are associated with abnormal growth of the silk gland, leading to the non-spinning phenotype. To confirm pathogen infection in non-spinning silkworms, we collected genomic DNA and conducted PCR analysis using specific primers for DNV and NPV. Results: Our results showed amplification of both viral infections in the isolated samples. SDS-PAGE analysis revealed differential regulation of several proteins involved in physiological and immunological processes in the hemolymph of non-spinning silkworms. MALDI-TOF/MS was used to identify an up-regulated protein of interest, which was found to be a Lepidopteran low molecular weight lipoprotein. This protein is known to play an important role in insect immune mechanisms. Conclusion: Major disease-causing agents such as BmDNV and BmNPV infections and investigate their impact on protein expression patterns in the hemolymph, shedding light on the factors contributing to non-spinning behavior in silkworms.

Study on the Structural Characteristics and Foaming Properties of Ovalbumin—Citrus Pectin Conjugates Prepared by the Maillard Reaction

This study explored the structural features and foaming properties of ovalbumin (OVA) and its glycosylated conjugates with citrus pectin (CP) formed through the Maillard reaction. The results demonstrated that OVA and CP were successfully conjugated, with the degree of grafting increasing to 43.83% by day 5 of the reaction. SDS-PAGE analysis confirmed the formation of high-molecular-weight conjugates. Fourier-transform infrared (FT-IR) and fluorescence spectroscopy further revealed alterations in the secondary and tertiary structures of OVA, including an enhanced β-sheet content, a reduced β-turn content, and the depletion of tryptophan residues. Moreover, the surface hydrophobicity of the OVA–CP conjugates significantly increased, enhancing foaming properties. Furthermore, the analysis of foaming properties exhibited that the Maillard reaction improved the foaming capacity of OVA to 66.22% and foaming stability to 81.49%. These findings highlight the potential of glycosylation via the Maillard reaction to significantly improve the foaming properties of OVA, positioning it as a promising novel foaming agent.

Valorizing sardine scales: a circular approach to sustainable collagen for cosmetics and nutrition applications

Background and ObjectiveIn recent years, the consumption of fish products has led to a worrying trend where approximately two-thirds of the total amount of fish is discarded as waste. At the same time, scientific interest in exploring natural collagen sources for cosmetics and dietary supplements has increased. This study explores the potential of valorizing sardine scales (Sardina pilchardus), a by-product of the canning industry, through the extraction of collagen for potential use in dermocosmetic formulations and food supplements.MethodsCollagen from sardine scales was obtained though acid and enzymatic extraction. The collagen extracts were characterized by UV-Vis, FTIR spectroscopy, SDS-PAGE, powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). The collagen was hydrolysed with papain to small peptides. Subsequently, the biological activities of acid-soluble collagen as well as the collagen peptides in terms of antioxidant and antimicrobial activity were evaluated. Furthermore, the capacity of collagen peptides to permeate the intestinal barrier, simulated with caco-2 cells, was evaluated.ResultsPurified collagen extracts were obtained from sardine scales, with enzymatic extraction method having a yield three times higher than the acid method. The SDS-PAGE analysis confirmed the extraction of type I collagen as well as its hydrolysis into small fragments (25–12 kDa). In terms of biological activities, collagen and collagen peptides have not demonstrated antimicrobial activity. However, regarding antioxidant activity, collagen peptides showed three times more capacity compared to non-hydrolyzed collagen. Meanwhile, in 6 h, about 6.37% of collagen peptides could permeate the intestinal barrier.ConclusionThis work represents a continuous effort to advance our understanding and utilization of Portuguese marine waste resources, with focus on the valorization of sardine co-products for the development of food supplement or cosmetic formulations, contributing to the sustainable evolution of the circular blue economy.

Enhanced Oligopeptide and Free Tryptophan Release from Chickpea and Lentil Proteins: A Comparative Study of Enzymatic Modification with Bromelain, Ficin, and Papain.

Plant-based foods offer a sustainable alternative to meet the growing protein demand. Legumes are the most promising of these, as they contain relatively high concentrations of protein, low digestible starch, and dietary fiber, as well as them possibly featuring low levels of fat. Enzymatically modified legume proteins provide us with tempting perspectives in terms of enhancing foods' biological values. However, their bioavailability and digestibility are generally less sufficient than that of proteins of animal origin, which may be improved by well-tailored enzyme modification. In this study, the efficacy of three plant-based proteases (bromelain, ficin, and papain) were evaluated at two distinct concentrations (2.5% and 10%) and three hydrolysis durations (1, 2, and 12 h) when transforming chickpea and lentil proteins. The degree of hydrolysis (DH), peptide profiles, and free amino acid content were analyzed to determine the efficiency of each enzyme. Results showed significant variations in DH, which was influenced by enzyme type, concentration, and hydrolysis duration. Papain exhibited the highest DH, particularly at a 10% concentration, reaching 27.8% efficiency in chickpea and 34.8% in lentils after 12 h. Bromelain and ficin were proven to be less effective, with ficin showing the least hydrolytic activity. SDS-PAGE analysis revealed substantial protein degradation, especially subsequent to papain treatment, pointing out that most proteins were cleaved into smaller peptides. SEC-HPLC indicated a predominant release of peptides within the 200-1000 Da range, suggesting enhanced bioavailability. Papain and bromelain treatments resulted in a significant release of oligopeptides and dipeptides. UHPLC analysis highlighted a marked post-hydrolysis increase in total free amino acids, with arginine, leucine, and lysine being the most abundant ones. Notably, tryptophan, being undetectable in untreated samples, was released in measurable amounts post-hydrolysis. These findings demonstrate papain's superior performance in protein hydrolysis and its potential in producing bioactive peptides, highlighting its applicability in food processing and the development of both nutraceuticals and functional foods.

The codon optimised gene produces an active human basic fibroblastic growth factor in rice cell suspension culture

The coding sequence of human basic fibroblast growth factor (hbFGF) was optimised for expression in rice. An expression cassette was constructed by fusing the PCR-amplified RAmy3D promoter, along with its 5’UTR, 3’UTR, and terminator sequences, to the codon-optimised hbFGF sequence. This cassette was inserted into the pCAMBIA1304 shuttle vector, which also contained the RAmy3D signal peptide. Agrobacterium tumefaciens strain LBA 4404 was used to transform rice callus. Among the transformed lines, the callus expressing the highest level of bFGF (38.1 mg/kg fresh weight) was identified via ELISA and selected for establishing a cell suspension culture. Expression and secretion of the recombinant bFGF into the culture medium were observed three days after incubating the transgenic rice cells in sucrose-free medium. The presence of recombinant bFGF was confirmed through Western blot and SDS-PAGE analyses. Furthermore, the rice-derived bFGF effectively stimulated the proliferation of NIH/3T3 cells, demonstrating a comparable biological activity to that of commercial bFGF.

Adverse Effects of Glyphosate-Based Herbicide on Hatching Rate, Morphological Alterations, and Acetylcholinesterase (AChE) Expression in Golden Apple Snail Eggs

This research investigated the effects of glyphosate herbicide on golden apple snail (Pomacea canaliculata) eggs, focusing on hatching rates, morphological changes, and acetylcholinesterase (AChE) expression to explore its potential as a biomarker of exposure. Additionally, key bioactive components in golden apple snail eggs were characterized. The study demonstrated that glyphosate toxicity increased with both exposure time and concentration. Optical coherence tomography (OCT) analysis revealed adverse morphological effects on eggs and embryos, including changes in shell structure and organ development. SDS-PAGE analysis identified four main protein bands, including Perivitellin 2 in three isoforms (98, 67, and 31 kDa) and Ovorubin (28 kDa). Lipids, phosphorus, and carbohydrates were identified as key components through Sudan Black B, Methyl Green, and Alcian Blue staining. AChE, with a molecular weight of 71 kDa, was further analyzed by Western blot, showing decreased expression with prolonged and higher concentrations of glyphosate exposure. GC-MS analysis identified major bioactive compounds in untreated eggs, including 3-Fluoro-β, 5-dihydroxy-N-methylbenzeneethanamine, 2-Aziridinylethylamine, and dextroamphetamine, which have pharmaceutical properties such as anti-hypertensive, diuretic, anti-diabetic, and anti-inflammatory effects, as well as potential applications in treating attention deficit hyperactivity disorder and narcolepsy. These compounds were present at lower levels in glyphosate-exposed groups, suggesting glyphosate's impact on the eggs' biochemical defense mechanisms. This study highlights the potential effects of glyphosate on golden apple snail eggs, which may have implications for future snail populations and aquatic ecosystems.

Effects of mono- and divalent- ions and their strength on foaming properties of infant formula protein model system

Metal ions may affect the foaming properties of infant formula products that including milk protein during reconstitution. In this study, an infant formula protein model system (IFPMS) composed of both whey protein and casein was constructed, and the effect of mono (Na+, K+) and divalent (Mg2+, Ca2+) ions and their strength (0–100 mM) on foaming properties was investigated. The mono ions had no significantly impact on foaming capacity (FC), MgCl2 slightly increase FC and reaching a maximum of 116.09 ± 7.46% at 25 mM, whereas CaCl2 significantly decreased FC from 105.78 ± 7.42% to 83.14 ± 6.75% as 0-25 mM (p < 0.05). Foam stability (FS) was significantly improved as all salts above 10 mM (p < 0.05). The dramatically decreased net charge and larger protein aggregates (> 25 mM) of IFPMS with divalent ions indicating their stronger charge-shielding effect, which caused slow adsorption of protein and increased surface tension. Multiple spectral results confirmed that salts enhanced protein interactions via inter-molecular and form a viscoelastic layer to stabilize foam. Absorbed protein and SDS-PAGE analysis revealed that MgCl2 promoted protein adsorption, while CaCl2 reduced casein in the foam phase. Correlation analysis further implied that the FC showed highly significant positive with absorbed protein, solubility, and β-turn (p ≤ 0.01), and FS was significantly correlated with secondary structure and viscosity (p ≤ 0.05). This study may provide useful information for an in-depth understanding of the foaming properties of infant formula.

Optimizing aquasoya protein: Insights of various pre-treatments for enhancing the upcycling potential from small black bean by-product

To minimize resource waste and promote recycling, it is essential to recover nutritional components from by-products. This study aimed to optimize of Aquasoya protein (AS-P) recovery from small black bean by-products, focusing on the effects of pH adjustments, sonication, and fractionation on protein yield, solubility, and structural integrity. Alkaline treatment at pH 8.5 significantly enhanced AS-P recovery yield (77.60 %) and protein concentration (421.16 μg/mL), attributed to the increased solubility and effective removal of non-protein components. Sonication for 10 min exhibited the highest protein solubility (88.69 %) post-fractionation, indicating reduced protein aggregation. Fractionation was crucial in minimizing browning reactions and enhancing protein purity by eliminating oligosaccharides and impurities. SDS-PAGE analysis revealed distinct protein bands from alkaline treatment, while sonication resulted in lipoxygenase (LOX) protein degradation. Principal Component Analysis (PCA) emphasized that fractionation notably enhances the stability and quality of AS-P, making it as a viable ingredient for food applications.

Enhanced lipase production and characterization from Aeromonas media VBC8: Applications in biodegradation of lubricating oil waste

This study aimed to explore the potential of a novel indigenous strain for the improved production of lipase from castor oil-contaminated soil. Among the various isolates, Aeromonas media VBC8 was found to be the most effective for lipase production. The effect of different inducer oils (olive, peanut, soybean, rice bran, sunflower, coconut, sesame, and fish liver oil) on the biomass of A. media VBC8 and its lipase activity was determined. Among the various oils assessed, fish liver oil exhibited highest lipase activity, with 89 U/mL with 9.1 g/L of biomass. Furthermore, Box-Behnken Design was used to optimize the cultural conditions resulting in an enhanced lipase activity of 1156 U/mL. The lipase was purified through ammonium salt (60 w/v %) precipitation, desalting and ion exchange column, achieving a yield of 16 % and specific activity of 98.4 U/mL. The purified lipase remained active over a wide range of pH 4.0–11.0 and temperature of 10–80 °C with maximum activity at pH 8.0 and 40 °C. SDS-PAGE analysis revealed the lipase's molecular weight to be 94 kDa. The study also evaluated the role of crude and purified lipase in the biodegradability of lubricating oil waste, achieving a maximum fatty acid conversion of 39 and 76 %, respectively, after 7 h incubation at room temperature.

Serodiagnosis of urogenital schistosomiasis and profiling of immunoreactive protein(s) in Schistosoma haematobium soluble egg and adult worm antigens.

To achieve schistosomiasis eradication plan by 2030, the development of efficient diagnosis is crucial. This study focuses on assessing the immunodiagnostic potential of S. haematobium (Sh) soluble egg antigen (SEA) and worm antigen (SWA) for urogenital schistosomiasis. Urine microscopy identified 50 S. haematobium-positive and 50 negative samples from a total of 500 examined. An additional 50 samples from a non-endemic area were included, bringing the total number of samples used for the assay to 150. Indirect ELISA immunoassays using SEA and SWA as the probing antigens evaluated 50 sera samples each from Sh positive, negative endemic (NE), and non-endemic (NNE) individuals. SDS-PAGE analysis of crude protein extracts was conducted, followed by Western blot analysis using primary antibodies from pooled Sh-infected sera samples. Diagnostic performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The AUC values for Sh SEA and SWA were 0.75 and 0.76 in NE samples, and 0.91 and 0.89 in NNE samples, respectively. Sensitivities 90 (95% CI: 78.64 - 95.65)/ 64.71 (95% CI: 52.17 - 75.92), and specificities 50 (95% CI: 36.64 - 63.36)/ 81.25 (95% CI: 63.56 - 92.79) were recorded for SEA and SWA, respectively in NE samples. In addition, sensitivities 90 (78.64 - 95.65)/ 92 (95% CI: 80.77 - 97.78), and specificities 72 (95% CI: 58.33 - 82.53)/ 72.00 (95% CI: 57.51 - 83.77) were recorded for SEA and SWA, respectively in NNE samples. The mean antibody titer against Sh SEA in infected samples was significantly higher than in non-infected samples (P <0.0001). Eight (8) immunoreactive protein bands; 4 each of SEA and SWA were identified, indicating potential for diagnostic tool development. Sh SEA and SWA demonstrate promise for diagnosing urogenital schistosomiasis in both endemic and non-endemic regions.

Development of Cashew and Pistachio Ladders through a Food-Processing Approach.

Following successful oral immunotherapy (OIT) for peanut allergy using boiled peanuts (BOPI trial), this study investigated the potential of wet-thermal-processing-induced allergen modification, specifically soaking and boiling (1-4 h) to reduce the allergenicity of cashew and pistachio allergens. In addition, this study provides a foundation of understanding for developing safer forms of cashew/pistachio administration for application in OIT by gradual exposure to increasing doses of modified allergens with reduced potency as an "allergen ladder". An SDS-PAGE analysis and an intrinsic-fluorescence spectroscopy revealed altered tertiary structures of the allergens, leading to their denaturation and even degradation. Notably, the reduction in both allergen-specific polyclonal IgG and human-specific IgE (sIgE) binding correlated with the treatment time, with the most significant decrease observed after 4 h of boiling. In contrast, shorter soaking treatments showed negligible effects on the IgE-binding capacity of these nuts, therefore indicating a further need for optimization. These findings indicate that extended boiling effectively reduced the amounts of the highly potent allergenic component Ana o 3 in cashew and Pis v 1 in pistachio, as confirmed by ELISA using polyclonal anti-Ana o 3 antibodies, and an immunoblot showed decreased IgE epitope binding in cashew and pistachio allergens, which further modified their allergenic profiles. This approach shows promise as a viable method for offering a safer therapeutic option for cashew/pistachio allergy.

Optimization, Purification and Characterization of Lipase from Streptomyces sp. A3301, with Application of Crude Lipase for Cooking Oily Wastewater Treatment

Streptomyces sp. A3301, which produces lipase isolated by Panyachanakul et al. [1]. This optimization was done for the subsequent purification and characterization of the biological lipase produced by the isolate. The results showed that the strain produced lipase with a maximum activity of 321 U/mL using the optimal medium and conditions (1.5 % (w/v) xylose and 2 % (w/v) yeast extract, pH 7.0 at 30 °C, 150 rpm for 3 days). The specific activity of the purified lipase was 27,000 U/mg, which was 544 times the pre-purification level, based on hydrophobic chromatography. After ion-exchange chromatography, the specific activity was 5,600 U/mg, which was 113 times the pre-purification level, with a single-peak purification profile. The purified lipase had a single band based on SDS-PAGE analysis and the molecular mass was 45 kDa. The optimum temperature and thermo-stability of A3301 lipase were 60 and 30 - 55 °C, respectively. The optimum pH of the purified enzyme was pH 9.0, and the enzyme was stable in the pH range of 8.0 - 9.0. The purified lipase was stable in acetone, chloroform and toluene, with a high relative activity of 63 - 76 %. The immobilized strain was then applied to oily-wastewater treatment. The strain can remove oil and grease in synthetic wastewater containing oil at 1 - 3 % (v/v), with removal rates of 100, 99.82 and 99.68 %, respectively, after incubation for 6 days. It was then applied to oily-wastewater treatment from a restaurant, achieving the highest degradation rates of 98.53 % after treatment for 6 days. In addition, it also affected the Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) values decreasing. HIGHLIGHTS Optimization of lipase production by Streptomyces A3301. Purification and characterization of the lipase produced by the Streptomyces A3301. Application of the immobilized Streptomyces A3301 strain in the treatment of oily cooking wastewater. The immobilized Streptomyces A3301 strain has the capability to remove oil and grease in both synthetic mediums and restaurant wastewater. GRAPHICAL ABSTRACT

Characterization of newly isolated Bacillus cereus D3 Co-producing α-amylase and protease and its application in food raw materials

Bacillus cereus, which effectively secretes various extracellular enzymes, has been widely used in the food, feed, and fermentation industries. One strain co-producing α-amylase and protease was newly isolated from Daqu and identified as B. cereus D3. It was shown that the production of α-amylase and protease co-produced by B. cereus D3 reached 13.89 ± 0.07 and 2468.45 ± 38.96 U/mL through optimization of fermentation conditions (nutrients (g/L): 20 of glucose, 20 of urea, 0.7 of MnCl2; incubation conditions: 24 h of incubation, initial medium pH of 7.0, temperature of 37 °C, 8% inoculum size, and 30% working volume). Moreover, the enzymatic genetic co-expression exhibited a similar pattern to the changes in enzymatic activities (growth-phase-dependent pattern). The two enzymes performed excellent thermal stability and were stable over wide pH ranges of 6.0–11.0 and 2.0–8.0, respectively. Finally, the hydrolysis degrees of raw materials including rice, sorghum, corn, and soybeans by α-amylase from B. cereus D3 reached 66.59 ± 4.21, 57.99 ± 4.39, 57.44 ± 4.67, and 38.01% ± 1.73%, respectively, producing glucose and maltose as the main products. The hydrolysis degrees of proteinous materials including mackerel heads and soybeans by protease from B. cereus D3 were 39.93 ± 1.99 and 30.44% ± 1.39%. SDS-PAGE analysis displayed extensive breakdown of macromolecular proteins, forming peptides of varied sizes. This is a systematic study on the co-production of α-amylase and protease by B. cereus, which is crucial to widen the understanding of this species co-producing multi-enzymes and explore its potential application in the food fermentation industry.

Sonication-Assisted Decellularization of Waste Tilapia (Oreochromis niloticus) Heads for Extracellular Matrix Extraction

Tilapia (Oreochromis niloticus), which is extensively farmed globally and ranks as the second most cultivated fish in the Philippines, generates significant amounts of waste that are often underutilized. One specific type of waste material consists of fish heads, which contain a valuable source of extracellular matrix (ECM). This study aims to evaluate the effects of sonication as a viable decellularization method for the extraction of ECM from tilapia fish heads. Particularly, two treatments were tested on the head samples: sonication-assisted decellularization (dWS) using a water bath sonicator, and decellularization without sonication (dNS), each with different contact times (5 min and 10 min). Histological analysis with H and E staining and DNA quantification revealed that sonication-assisted samples (dWS) showed a greater reduction in basophilic components and DNA content, achieving a 93.7% removal rate. These dWS samples also had the highest protein loss, retaining only 33.86% of the original protein. SDS–PAGE analysis indicated that both dWS and dNS samples maintained similar collagen structures, as evidenced by identical subunit bands. ATR–FTIR spectra confirmed the presence of collagen type I in all samples, detecting characteristic amides A, B, I, II, and III. The results revealed that varying treatments and contact times had significant effects on the physical and mechanical properties of the decellularized extracellular matrix (ECM). These findings highlight the effectiveness of sonication in the decellularization process, particularly for utilizing waste tilapia heads.

Caspase-3 interactins with calpain and cathepsin L: implications for protein stability and quality in fish fillets during postmortem storage

The degradation of structural proteins during fish postmortem storage is a critical factor affecting meat quality, leading to economic losses in the seafood industry. The complex interplay between caspase-3 and structural protein stability during fish postmortem storage remains largely unexplored. By treating grass carp fillets with a caspase-3 inhibitor, we establish a model for inhibiting apoptosis, delineating the role of caspase-3 in protein degradation. The effect of caspase-3 on grass carp proteins following postmortem storage and its cascade interaction with calpain and cathepsin L was further investigated. The result demonstrated that the destabilisation of the mitochondrial membrane during apoptosis led to modifications in the B-cell lymphoma (Bcl) family proteins, subsequently triggered the activation of caspase-9/caspase-3. Additionally, caspase-3 reduced the expression of the calpastatin and lysosomal membrane protein 1 (LAMP-1) gene, resulting in increased calpain and cathepsin L activity. SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that caspase-3 inhibitor treatment reduced actin fractures and preserved critical structural proteins. This investigation unveils the molecular mechanisms through which caspase-3 affects protein degradation in fish fillets during storage, providing valuable insights into the postmortem degradation processes.

Structural modification and functional improvement of lactoferrin through non-covalent and covalent binding to coffee polyphenol

Studies have suggested that milk may enhance or neutralize the bioavailability of coffee polyphenols, possibly due to reversible and irreversible interactions between coffee polyphenols and milk proteins. The effects of non-covalent and covalent binding of lactoferrin (BLF) to caffeic acid (CAA) on protein structure and function were investigated. SDS-PAGE analysis confirmed the covalent interaction between BLF and CAA. Multispectral experiments characterized the BLF-CAA complexes and conjugates, revealing alterations in the tertiary structure of the proteins in the BLF-CAA conjugates. Molecular docking and kinetics results demonstrated that hydrogen bonding, electrostatic interaction, and hydrophobic forces were the primary internal forces between CAA and BLF. When combined with CAA, the covalent conjugates exhibited superior functional properties including solubility, oxidation resistance, thermal stability, emulsification and foamability, bioaccessibility, and antimicrobial properties. This study offers a theoretical foundation and technical benchmark for the preparation of protein-based delivery vectors with synergistic effects.

Cold plasma-induced covalent binding with epigallo-catechin 3-gallate: A strategy for antigenicity reduction and antioxidant improvement of β-lactoglobulin

The application of cold plasma (CP) treatment was investigated for its effectiveness in promoting the covalent grafting of β-lactoglobulin (β-LG) and epigallo-catechin 3 -gallate (EGCG), as well as improving the antioxidant capacity of β-LG and eliminating its antigenicity. The results indicated that CP treatment significantly enhanced the covalent grafting of β-LG and EGCG. The maximum EGCG content in β-LG-EGCG complexes reached 47.08 ± 0.58 mg/g (with 45 s treatment), as confirmed by SDS-PAGE analysis and FTIR spectroscopy. Additionally, LC-MS/MS identifies two binding sites (T76 and K77) of EGCG in the β-LG-EGCG conjugates. After 60 s treatments, there was a significant reduction in the IgG binding capacity of β-LG-EGCG conjugates (29.97 ± 0.05%). Further analysis suggested that the change in the conformational epitopes of β-LG, due to the conjugating with EGCG, was likely the main factor responsible for the reduced binding Immunoglobulin G (IgG) capacity. The maximum antioxidant activities of β-LG-EGCG conjugates were found to be 974.14 ± 1.94 μmol TE g−1 for ABTS and 748.48 ± 9.66 μmol TE g−1 for DPPH). These values represent an eightfold increase in DPPH activity and a twofold increase in ABTS compared to the control sample. This study demonstrated that the CP treatment is highly effective technology for facilitating the covalent grafting of polyphenols to proteins, thereby enhancing their antioxidant properties and eliminate their antigenicity.

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built. Only if HA greatly exceed Asp in the support, immobilization did not work. While only using the Asp-agarose support immobilized enzyme molecules were only ionically adsorbed after glutaraldehyde treatment (visualized in SDS-PAGE analysis), the mixed supports gave covalent immobilization. The glutaraldehyde modification of these biocatalysts permitted to establish covalent bonds with the support, and this was more effective when using higher amounts of HA in the support. When around 60 % of the groups in the support were HA, the treatment with glutaraldehyde fully suppressed enzyme release from the support after boiling in SDS. The glutaraldehyde treated biocatalysts were more stable than just the adsorbed enzymes or the enzyme adsorbed only on Asp supports and then treated with glutaraldehyde (the optimal biocatalyst retained 90 % of the initial activity while the just adsorbed ficin retained 50 % of the initial activity). This strategy can be utilized to immobilize other proteins with high isoelectric points following this immobilization strategy.

Production of Tobacco Etch Virus Protease (TEV) Expressed in the Endotoxin-Free Bacillus subtilis and Its Application.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.