Combined high voltage atmospheric cold plasma and ultraviolet-cold plasma inhibited Aspergillus flavus growth and improved physicochemical properties of protein in peanuts.

To improve the application value of peanuts, the fungicidal effect and physicochemical properties of the protein in peanuts were investigated by combining high voltage atmospheric cold plasma (HVCP) and ultraviolet-cold plasma (UVCP) in this study. Compared to the single HVCP or UVCP treatment, the combined treatments exhibited a higher fungicidal efficiency of A. flavus spores in peanuts, decreasing by 0.79-2.97 log10 cfu/g after 8-min treatment. The A. flavus growth and aflatoxin production in peanuts during storage were also lower than the single plasma groups. Moreover, cold plasma treatments could modify the molecular structures of protein in peanuts by changing secondary and tertiary structures, decreasing particle size and increasing zeta potential, which contributed to improve the solubility and emulsification of protein. Overall, this research provides a unique strategy for the combined application of cold plasma in grain decontamination and protein modification.

Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity.

Pulsed electric field (PEF) is an innovative physical method for food processing characterized by low energy consumption and short processing time. This technology represents a sustainable procedure to extend food shelf-life, enhance mass transfer, or modify food structure. The main mechanism of action of PEF for food processing is the increment of the permeability of the cell membranes by electroporation. However, it has also been shown that PEF may modify the technological and functional properties of proteins. Generating a high-intensity electric field necessitates the flow of an electric current that may have side effects such as electrochemical reactions and temperature increments due to the Joule effect that may affect food components such as proteins. This article presents a critical review of the knowledge on the extraction of proteins assisted by PEF and the impact of these treatments on protein composition, structure, and functionality. The required research for understanding what happens to a protein when it is under the action of a high-intensity electric field and to know if the mechanism of action of PEF on proteins is different from thermal or electrochemical effects is underlying.

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins.

KEY MESSAGE: Analysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins. Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins.

Perfect Partners: Biocatalytic Halogenation and Metal Catalysis for Protein Bioconjugation.

Flavin-dependent halogenases (FDHs) are the most extensively researched halogenases and show great potential for biotransformation applications. These enzymes use chloride, bromide, or iodide ions as halogen donors to catalyze the oxygen-dependent halogenation of electron-rich aryl moieties, requiring stochiometric amounts of FADH2 in the process. This makes FDH-catalyzed aryl halogenation a highly selective and environmentally friendly tool for the synthesis of aryl halides. The latter in turn serve as valuable intermediates for transition metal catalyzed cross coupling reactions for C-C bond formation. Previous research made extensive use of this approach to halogenate small molecules as building blocks for late-stage functionalization by transition-metal catalyzed cross-coupling reactions. Based on these results, several groups have managed to expand this research to protein targets over the past two years. Their work indicates an emerging methodology for bioconjugation using late-stage biocatalytic halogenation in conjunction with biorthogonal Suzuki-Miyaura cross-coupling. This strategy could present an attractive alternative to existing approaches due to the stability of the C-C bond bridging the generated biaryl moiety and the ease of late-stage enzymatic modification while maintaining excellent selectivity under mild conditions.

Aqueous ozone effects on wheat gluten: Yield, structure, and rheology.

This study investigates the impact of aqueous ozone (AO) on the yield, molecular structure, and rheological properties of wheat gluten separated using the batter procedure. Employing strong gluten flour (SGF) and weak gluten flour (WGF), we demonstrate that AO pretreatment significantly enhances the yield and purity of separated starch and gluten. Surface hydrophobicity, free sulfhydryl groups, Fourier transform infrared spectroscopy (FTIR), Raman, and size exclusion-high-performance liquid chromatography (SE-HPLC) analyses were used to evaluate the effects of AO on the molecular structure of gluten. Our analysis reveals that low concentrations of AO induce specific modifications in gluten proteins. AO treatment increases cross-linking in glutenin macropolymer (GMP), reduces surface hydrophobicity, and stabilizes secondary and tertiary structures. These changes include an increase in ß-sheet content by approximately 9% and a corresponding decrease in ß-turn structures, leading to enhanced viscoelastic properties of the gluten. The research highlights AO's potential as a sustainable and efficient agent in wheat flour processing, offering advancements in both product quality and eco-friendly processing techniques. Future research should optimize AO treatment parameters and explore its effects on different cereal types further to enhance its applicability and benefits in food processing. PRACTICAL APPLICATION: Our work substantially advances the existing knowledge on wheat flour processing by demonstrating the multifaceted benefits of AO pretreatment. We unveil significant improvements in the yield and purity of starch and gluten when compared to conventional separation methods. Moreover, our in-depth analysis of molecular changes induced by AO, including increased cross-linking, alterations in surface hydrophobicity, and modifications in glutenin macropolymer content, provides new insights into how AO affects the viscoelastic properties of gluten. This contribution is pivotal for the development of more efficient, sustainable, and eco-friendly wheat flour processing technologies.

Novel high internal phase emulsion gels stabilized solely by hemp protein isolate: Enhancement of cannabidiol chemical stability and bioaccessibility.

This study aims to fabricate high internal phase emulsion gels (HIPEGs) using modified hemp protein isolates for microencapsulating cannabidiol (CBD) to enhance their chemical stability and bioaccessibility. Importantly, the combined effect of CBD concentrations (0.1 vs 0.5 wt%) and post gel storage conditions (before-refrigeration (BR) vs after-refrigeration (AR)) on the properties of HIPEGs were investigated. The results showed that the CBD concentration above 0.4 % is necessary to fabricate a stable HIPEG. The rheological properties of HIPEGs were influenced by CBD concentration and refrigeration. The AR gels with 0.5 % CBD showed the highest gel strength (up to 91.7 Pa) and solid-like structures. These properties allowed to HIPEGs maintain good physical stability during storage at 4, 25, and 37 °C for 14 days due to the interconnected polyhedral protein matrices and thick interfacial protein layers. These unique protein architectures offered superior protection against CBD degradation (<2 % of initial added amount) for 100 days during exposure to light and temperature (25 or 37 °C). The INFOGEST digestion results showed the BR gels effectively protected CBD during digestion and consequently improved their stability and bioaccessibility up to 95 % and 74 %, respectively. Overall, the fabricated HIPEGs could be valuable for nutraceutical delivery.

Non-natural MUC1 Glycopeptide Homogeneous Cancer Vaccine with Enhanced Immunogenicity and Therapeutic Activity.

Glycopeptides derived from the glycoprotein mucin-1 (MUC1) have shown potential as tumor-associated antigens for cancer vaccine development. However, their low immunogenicity and non-selective conjugation to carriers present significant challenges for the clinical efficacy of MUC1-based vaccines. Here, we introduce a novel vaccine candidate based on a structure-guided design of an artificial antigen derived from MUC1 glycopeptide. This engineered antigen contains two non-natural amino acids and has an α-S-glycosidic bond, where sulfur replaces the conventional oxygen atom linking the peptide backbone to the sugar N-acetylgalactosamine. The glycopeptide is then specifically conjugated to the immunogenic protein carrier CRM197 (Cross-Reactive Material 197), a protein approved for human use. Conjugation involves selective reduction and re-bridging of a disulfide in CRM197, allowing the attachment of a single copy of MUC1. This strategy results in a chemically defined vaccine while maintaining both the structural integrity and immunogenicity of the protein carrier. The vaccine elicits a robust Th1-like immune response in mice and generates antibodies capable of recognizing human cancer cells expressing tumor-associated MUC1. When tested in mouse models of colon adenocarcinoma and pancreatic cancer, the vaccine is effective both as a prophylactic and therapeutic use, significantly delaying tumor growth. In therapeutic applications, improved outcomes were….

Soy proteins modified using cavitation jet technology.

Soy proteins are seen as a promising alternative food source for meat with environmentally friendly properties. The problem is that the functional properties of soy proteins do not meet the needs of the food industry, and some existing modification technologies have adverse effects. Recently, cavitation jet technology (CJT) has been studied because it generates high heat, high pressure, strong shear and strong shock waves. This review summarizes the history and mechanism of cavitation jets. The energy generated during the cavitation jet process can open molecular structures, and the shock waves and microjets generated can pulverize the materials by erosion. The impact of the CJT on the morphology, structure, and functionality of soy proteins is discussed. The impact of combining CJT with other techniques on the production of soy proteins was also reviewed. The modification of proteins using two or more methods with complementary strengths, avoiding the disadvantages of certain techniques, makes the modification of proteins more effective. One of the most prominent effects is the combined treatment of cavitation jets with physical techniques. Finally, the review provides a comprehensive analysis of the application of modified soy proteins in the food industry and highlights promising avenues for future research.

Proteomic insights into adventitious root formation in Larix kaempferi.

The adventitious root formaton (ARF) in excised plant parts is essential for the survival of isolated plant fragments. In this study, we explored the complex mechanisms of ARF in Larix kaempferi by conducting a comprehensive proteomic analysis across three distinct stages: the induction of adventitious root primordia (C1, 0-25 d), the formation of adventitious root primordia (C2, 25-35 d), and the elongation of adventitious roots (C3, 35-45 d). We identified 1976 proteins, with 263 and 156 proteins exhibiting increased abundance in the C2/C1 and C3/C2 transitions, respectively. In contrast, a decrease in the abundance of 106 and 132 proteins suggests a significant demand for metabolic processes during the C2/C1 phase. The abundance of IAA-amino acid hydrolase and S-adenosylmethionine synthase were increased in the C2/C1 phase, underscoring the role of auxin in adventitious root induction. The decrease in abundance of photosynthesis-related proteins during the C2/C1 phase highlights the significance of initial light conditions in adventitious root induction. Moreover, variation in cell wall synthesis and metabolic proteins in the C2/C1 and C3/C2 stages suggests that cell wall metabolism is integral to adventitious root regeneration. Gene Ontology enrichment analysis revealed pathways related to protein modification enzymes, including deubiquitinases and kinases, which are crucial for modulating protein modifications to promote ARF. Furthermore, the increased abundance of antioxidant enzymes, such as peroxidases and glutathione peroxidases, indicates a potential approach for enhancing ARF by supplementing the culture medium with antioxidants. Our study provides insights into metabolic changes during ARF in L. kaempferi, offering strategies to enhance adventitious root regeneration. These findings have the potential to refine plant propagation techniques and expedite breeding processes. SIGNFICANCE: The main challenge in the asexual reproduction technology of Larix kaempferi lies in adventitious root formation (ARF). While numerous studies have concentrated on the efficiency of ARF, proteomic data are currently scarce. In this study, we collected samples from three stages of ARF in L. kaempferi and subsequently performed proteomic analysis. The data generated not only reveal changes in protein abundance but also elucidate key metabolic processes involved in ARF. These insights offer a novel perspective on addressing the challenge of adventurous root regeneration.

Yeast Proteins: Proteomics, Extraction, Modification, Functional Characterization, and Structure: A Review.

Proteins are essential for human tissues and organs, and they require adequate intake for normal physiological functions. With a growing global population, protein demand rises annually. Traditional animal and plant protein sources rely heavily on land and water, making it difficult to meet the increasing demand. The high protein content of yeast and the complete range of amino acids in yeast proteins make it a high-quality source of supplemental protein. Screening of high-protein yeast strains using proteomics is essential to increase the value of yeast protein resources and to promote the yeast protein industry. However, current yeast extraction methods are mainly alkaline solubilization and acid precipitation; therefore, it is necessary to develop more efficient and environmentally friendly techniques. In addition, the functional properties of yeast proteins limit their application in the food industry. To improve these properties, methods must be selected to modify the secondary and tertiary structures of yeast proteins. This paper explores how proteomic analysis can be used to identify nutrient-rich yeast strains, compares the process of preparing yeast proteins, and investigates how modification methods affect the function and structure of yeast proteins. It provides a theoretical basis for solving the problem of inadequate protein intake in China and explores future prospects.

Bridging the gap: RNAylation conjugates RNAs to proteins.

In nature, the majority of known RNA-protein interactions are transient. Our recent study has depicted a novel mechanism known as RNAylation, which covalently links proteins and RNAs. This novel modification bridges the realms of RNA and protein modifications. This review specifically explores RNAylation catalyzed by bacteriophage T4 ADP-ribosyltransferase ModB, with a focus on its protein targets and RNA substrates in the context of Escherichia coli-bacteriophage T4 interaction. Additionally, we discuss the biological significance of RNAylation and present perspectives on RNAylation as a versatile bioconjugation strategy for RNAs and proteins.

Controlling (E/Z)-Stereoselectivity of -NHC=O Chlorination: Mechanism Principles for Wide Scope Applications.

Organic halogen compounds are cornerstones of applied chemical sciences. Halogen substitution is a smart molecular design strategy adopted to influence reactivity, membrane permeability and receptor interaction. Chiral bioreceptors may restrict the stereochemical requirements in the halo-ligand design. Straightforward (but expensive) catalyzed stereospecific halogenation has been reported. Historically, PCl5 served access to uncatalyzed stereoselective chlorination although the stereochemical outcomes were influenced by steric parameters. Nonetheless, stereochemical investigation of PCl5 reaction mechanism with carbamoyl (RCONHX) compounds has never been addressed. Herein, we provide the first comprehensive stereochemical mechanistic explanation outlining halogenation of carbamoyl compounds with PCl5; the key regioselectivity-limiting nitrilimine intermediate (8-Z.HCl); how substitution pattern influences regioselectivity; why oxadiazole byproduct (P1) is encountered; stereo-electronic factors influencing the hydrazonoyl chloride (P2) production; and discovery of two stereoselectivity-limiting parallel mechanisms (stepwise and concerted) of elimination of HCl and POCl3. DFT calculations, synthetic methodology optimization, X-ray evidence and experimental reaction kinetics study evidence all supported the suggested mechanism proposal (Scheme 2). Finally, we provide mechanism-inspired future recommendations for directing the reaction stereoselectivity toward elusive and stereochemically inaccessible (E)-bis-hydrazonoyl chlorides along with potentially pivotal applications of both (E/Z)-stereoisomers especially in medicinal chemistry and protein modification.

Protein S-Nitrosylation: A Chemical Modification with Ubiquitous Biological Activities.

Nitric oxide (NO) induces protein posttranslational modification (PTM), known as S-nitrosylation, which has started to gain attention as a critical regulator of thousands of substrate proteins. However, our understanding of the biological consequences of this emerging PTM is incomplete because of the limited number of identified S-nitrosylated proteins (S-NO proteins). Recent advances in detection methods have effectively contributed to broadening the spectrum of discovered S-NO proteins. This article briefly reviews the progress in S-NO protein detection methods and discusses how these methods are involved in characterizing the biological consequences of this PTM. Additionally, we provide insight into S-NO protein-related diseases, focusing on the role of these proteins in mitigating the severity of infectious diseases.

Nanoparticles that Distinguish Chemical and Supramolecular Contexts of Lysine for Single-Site Functionalization of Protein.

Lysine is one of the most abundant residues on the surface of proteins and its site-selective functionalization is extremely challenging. The existing methods of functionalization rely on differential reactivities of lysine on a protein, making it impossible to label less reactive lysines selectively. We here report polymeric nanoparticles that mimic enzymes involved in the posttranslational modifications of proteins that distinguish the chemical and supramolecular contexts of a lysine and deliver the labeling reagent precisely to its ε amino group. The nanoparticles are prepared through molecular imprinting of cross-linkable surfactant micelles, plus an in situ, on-micelle derivatization of the peptide template prior to the imprinting. The procedures encode the polymeric nanoparticles with all the supramolecular information needed for sequence identification and precise labeling, allowing single-site functionalization of a predetermined lysine on the target protein in a mixture.

Proteins from Legumes, Cereals, and Pseudo-Cereals: Composition, Modification, Bioactivities, and Applications.

This review presents a comprehensive analysis of plant-based proteins from soybeans, pulses, cereals, and pseudo-cereals by examining their structural properties, modification techniques, bioactivities, and applicability in food systems. It addresses the critical need for a proper utilization strategy of proteins from various plant sources amidst the rising environmental footprint of animal protein production. The inherent composition diversity among plant proteins, their nutritional profiles, digestibility, environmental impacts, and consumer acceptance are compared. The innovative modification techniques to enhance the functional properties of plant proteins are also discussed. The review also investigates the bioactive properties of plant proteins, including their antioxidant, antimicrobial, and antitumoral activities, and their role in developing meat analogs, dairy alternatives, baked goods, and 3D-printed foods. It underscores the consideration parameters of using plant proteins as sustainable, nutritious, and functional ingredients and advocates for research to overcome sensory and functional challenges for improved consumer acceptance and marketability.

Properties of Myofibrillar Protein in Frozen Pork Improved through pH-Shifting Treatments: The Impact of Magnetic Field.

The present study demonstrates the effects of pH-shifting treatments and magnetic field-assisted pH-shifting treatments on the properties of myofibrillar protein (MP) in frozen meat. The solubility results indicate that the pH-shifting treatments increased the solubility of MP from 16.8% to a maximum of 21.0% (pH 9). The values of surface hydrophobicity and protein particle size distribution indicate that the pH-shifting treatment effectively inhibited protein aggregation through electrostatic interactions. However, under higher pH conditions (pH 10, 11), the treatments assisted by the magnetic field increased the degree of aggregation. The total thiol content and SDS-PAGE results further suggest that the magnetic field-assisted pH-shifting treatment accelerated the formation of covalent bonds among MPs under the alkaline environment. The results of the Differential Scanning Calorimetry (DSC) and protein secondary structure analysis indicate that the magnetic field promoted the unfolding of protein structures in an alkaline environment, markedly reducing the effective pH levels of pH-shifting. Electron paramagnetic resonance (EPR) data indicate that the phenomenon might be associated with the increased concentration of free radicals caused by the magnetic field treatment. In summary, the application of magnetic field-assisted pH-shifting treatments could emerge as a potent and promising strategy to improve the protein properties in frozen meat.

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases.

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.

Tailoring soy protein/corn zein mixture by limited enzymatic hydrolysis to improve digestibility and functionality.

This study aimed to modify plant protein mixture to improve their functionality and digestibility by limited hydrolysis. Soy protein isolate and corn zein were mixed at the ratio of 5:1 (w/w), followed by limited hydrolysis using papain from 15 to 30 min. The structural characteristics, in vitro digestibility, and functional properties were evaluated. Also, DPPH radical scavenging activity was determined. The results indicated that the molecular weight of different modified samples was largely reduced by limited hydrolysis, and the proportion of random coil was significantly increased. Furthermore, the solubility, foaming, emulsifying and water-holding capacity of hydrolyzed protein mixture were significantly improved, which were close to those of whey protein isolate. In vitro digestibility after 30-min limited hydrolysis was remarkably elevated. In addition, the hydrolyzed protein mixture exhibited a higher antioxidant activity than those of untreated proteins. Overall, limited hydrolysis of protein mixture led to improved digestibility, functionality and antioxidant activity.

Highly selective chemical modification of poly-His tagged peptides and proteins.

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.

A single backbone amide modification method to achieve single site-specific chemical protein conjugation.

Efficient and precise chemical protein modification methods are highly sought after in biotechnology. However, chemically distinguishing a single site within a large protein is challenging. This study introduces a Copper Assisted Sequence-specific Conjugation Tag (CAST) method, enabling rapid (second order rate 8.1 M-1s-1) and site-specific chemical modification of the protein backbone with pinpoint accuracy. The versatility of this method is demonstrated through the preparation of antibody-drug conjugates, showcasing high plasma stability and potent efficacy in both in vitro and in vivo settings. Thus, CAST emerges as an efficient and quantitative approach for attaching payloads to large, native proteins.