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International Journal of Biological... Jun 2024Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as...
Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as fouling during heat treatments. However, at the state of the art, this effect remains unclear, contrary to that of temperature. This article presents a novel strategy to study the impact of shear rate and concentration on the accumulation of whey protein surficial deposits. It consists in applying a range of shear rates (0-200 s) at controlled temperature (65 °C) on whey protein solutions (5-10 wt%) by a parallel plate rheometer equipped with a glass disc, thus allowing the off-line characterization of the deposits by microscopy. Our results highlight an unequivocal effect of increasing shear stress. At 5 wt%, it fosters the formation of primary deposits (≈ 10 μm), whereas at 10 wt% it results in the development of complex branched structures (≈ 50 μm) especially for shear rates ranging from 140 s to 200 s. Based on the classification by size of the observed populations, we discuss possible hypotheses for the deposit growth kinetics, involving the interplay of different physico-chemical protein-surface interactions and paving the way to future further investigations.
PubMed: 38908625
DOI: 10.1016/j.ijbiomac.2024.133291 -
Frontiers in Nutrition 2024Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable...
BACKGROUND
Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable alternatives to chemical catalysts. However, their catalytic activities are susceptible to denaturation. In this study, we present the discovery of novel protein-based biocatalysts derived from processed foods, including skimmed milk, soy milk, cheese, and dried tofu. These food catalysts exhibit high availability, low cost, safety, and thermo-stability.
RESULTS
Focusing on the physiologically intriguing coenzyme pyrroloquinoline quinone (PQQ), we observed that the reaction with glycine to form imidazolopyrroquinoline (IPQ) did not proceed efficiently when PQQ was present at very low concentrations. Surprisingly, in the presence of protein-based foods, this reaction was significantly accelerated. Notably, skimmed milk enhanced the PQQ detection limit (600 times lower) during high-performance liquid chromatography (HPLC) following IPQ derivatization. Milk appears to facilitate the reaction between PQQ and various amino acids, primary amines, and secondary amines. Further investigations revealed that food catalysis operates through a non-enzymatic mechanism. Additionally, nuclear magnetic resonance spectroscopy demonstrated that milk components interacted with amino substrates due to the ability of amines to react with quinones on colloidal surfaces.
CONCLUSION
These practical food catalysts not only contribute to environmental safety but also hold significance across diverse scientific domains. Non-enzymatic protein catalysts find applications in biocatalysis, organic synthesis, food technology, analytical chemistry, and fundamental nutritional and evolutionary studies.
PubMed: 38903631
DOI: 10.3389/fnut.2024.1391681 -
BioRxiv : the Preprint Server For... Jun 2024How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and...
UNLABELLED
How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from . Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.
SIGNIFICANCE STATEMENT
One can conceptualize KaiB as an on-off switch to regulate circadian rhythms in bacteria, where the "On state" is the Fold-switched state that binds KaiC and other proteins, and the "Off state" is the Ground state. Our work exemplifies how evolution tuned the kinetics of interconversion to align with the hour-long timescale of its biological function. The Ground state is dramatically destabilized at cold temperatures, and the system contains an alternate "off" conformation that exchanges with the primary "off" conformation at faster timescales than the rate-limiting step. Our findings demonstrate a simple principle for evolving a protein switch: one part of a protein domain remains stably folded to serve as a scaffold for the rest of the protein to re-fold.
PubMed: 38895306
DOI: 10.1101/2024.06.03.597139 -
International Journal of Molecular... May 2024Dermatology and cosmetology currently prioritize healthy, youthful-looking skin. As a result, research is being conducted worldwide to uncover natural substances and...
Dermatology and cosmetology currently prioritize healthy, youthful-looking skin. As a result, research is being conducted worldwide to uncover natural substances and carriers that allow for controlled release, which could aid in the battle against a variety of skin illnesses and slow the aging process. This study examined the biological and physicochemical features of novel hydrogels containing cannabidiol (CBD) and α-terpineol (TER). The hydrogels were obtained from ε-caprolactone (CL) and poly(ethylene glycol) (PEG) copolymers, diethylene glycol (DEG), poly(tetrahydrofuran) (PTHF), 1,6-diisocyanatohexane (HDI), and chitosan (CHT) components, whereas the biodegradable oligomers were synthesized using the enzyme ring-opening polymerization (e-ROP) method. The in vitro release rate of the active compounds from the hydrogels was characterized by mainly first-order kinetics, without a "burst release". The antimicrobial, anti-inflammatory, cytotoxic, antioxidant, and anti-aging qualities of the designed drug delivery systems (DDSs) were evaluated. The findings indicate that the hydrogel carriers that were developed have the ability to scavenge free radicals and impact the activity of antioxidant enzymes while avoiding any negative effects on keratinocytes and fibroblasts. Furthermore, they have anti-inflammatory qualities by impeding protein denaturation as well as the activity of proteinase and lipoxygenase. Additionally, their ability to reduce the multiplication of pathogenic bacteria and inhibit the activity of collagenase and elastase has been demonstrated. Thus, the developed hydrogel carriers may be effective systems for the controlled delivery of CBD, which may become a valuable tool for cosmetologists and dermatologists.
Topics: Hydrogels; Cannabidiol; Skin; Humans; Cyclohexane Monoterpenes; Antioxidants; Regeneration; Polymers; Biocompatible Materials; Keratinocytes; HaCaT Cells; Drug Carriers; Drug Delivery Systems; Anti-Infective Agents
PubMed: 38892121
DOI: 10.3390/ijms25115934 -
Foods (Basel, Switzerland) Jun 2024Soy remains the legume protein of excellence for plant-based meat alternatives due to its fiber-forming potential. In this study, protein-rich powders from soy protein...
Soy remains the legume protein of excellence for plant-based meat alternatives due to its fiber-forming potential. In this study, protein-rich powders from soy protein isolate (SPI), concentrate (SPC), and their mixture (SPM) were thoroughly characterized for their proximate composition, nutritional quality, and physicochemical properties to understand their structuring behavior during high-moisture extrusion. SPI presented higher degrees of protein denaturation and aggregation, least gelation concentration and lower essential amino acid contents. Thus, an SPI:SPC combination (1:9 ratio, 70% protein) was extruded at three different screw speeds (300, 350, and 400 rpm) and two temperature profiles (120 and 140 °C maximum temperature). The effects of the processing parameters on the extrudates were evaluated for their appearance (fibrousness), texture (TPA, cutting force, and anisotropy), color, protein structure (FTIR), and trypsin inhibitors. Higher temperatures resulted in softer and darker extrudates, with increased visual and instrumental anisotropy. Increasing screw speeds led to softer and lighter extrudates, without a clear fibrousness effect. β-sheet structures decreased and intermolecular aggregates (A1) increased after extrusion, especially at 140 °C, together with the formation of intramolecular aggregates (A2). Extrusion also significantly decreased the amount of trypsin inhibitors (>90%). This study demonstrates that extrusion parameters need to be carefully selected to achieve meat analogs with optimal textural and nutritional characteristics.
PubMed: 38890977
DOI: 10.3390/foods13111748 -
Frontiers in Nutrition 2024Composite natural emulsifiers such as whey protein isolate (WPI) and chitosan (CS) are commonly used in Pickering emulsions to address the effect of thermal deformation...
Composite natural emulsifiers such as whey protein isolate (WPI) and chitosan (CS) are commonly used in Pickering emulsions to address the effect of thermal deformation of proteins before complexation with CS and heating after complexation. In this study, the properties of WPI and CS composites were investigated by complexing CS with either unmodified WPI or thermally denatured WPI (DWPI). Three types of composite particles were prepared, WPI-CS, DWPI-CS, and D(WPI-CS). Atomic force microscopy revealed that the composite particles formed larger aggregates with increased contour size and surface roughness compared to CS and WPI, whereas the interfacial tension decreased, indicating improved emulsifying abilities. Fourier-transform infrared analysis revealed differences in the hydrogen bonds between CS and WPI/DWPI. All three composite particles formed stable emulsions with droplet sizes of 20.00 ± 0.15, 27.80 ± 0.35, and 16.77 ± 0.51 μm, respectively. Thermal stability experiments revealed that the curcumin emulsion stabilized with WPI-CS and DWPI-CS exhibited relatively better thermal stability than that stabilized with D(WPI-CS). experiments results indicated that the bioaccessibility of the curcumin emulsion stabilized with WPI-CS was 61.18 ± 0.16%, significantly higher than that of the emulsions prepared with the other two composite particles ( < 0.05). This study will enable the customized design of WPI composite-based Pickering emulsions for application in the food and nutrition industries.
PubMed: 38887503
DOI: 10.3389/fnut.2024.1418120 -
Journal of Pharmacy & Bioallied Sciences Apr 2024contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including...
contains andrograpanin, which is both anti-inflammatory and anti-infective. comprises over 150-200 species from the family . exerts various properties, including anti-inflammatory property. Herbal mouthwash was made using and extract. The anti-inflammatory effect was evaluated using an albumin denaturation assay and egg albumin denaturation. The percentage of protein denaturation that is inhibited by the formulation of and indicates that it has strong anti-inflammatory effect. According to the findings, as concentration is raised, the formulation's anti-inflammatory activity rises. The formulation's percentage inhibition values are also equivalent to those of a typical anti-inflammatory medicine, indicating that it may be effective as a natural anti-inflammatory agent.
PubMed: 38882775
DOI: 10.4103/jpbs.jpbs_581_23 -
BioRxiv : the Preprint Server For... May 2024display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of...
display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing -(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.
PubMed: 38853888
DOI: 10.1101/2024.05.29.596443 -
Current Health Sciences Journal 2024Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed...
Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed to the release of free radicals by immune cells, prooxidants that can also cause protein alteration. This study was performed in order to assess the potential anti-inflammatory effect of two bee venom samples harvested from Apis mellifera. Free radical scavenging capacity was investigated using DPPH and ABTS.+ tests and protective effect on proteins through the inhibitory activity on thermal denaturation of albumin.
PubMed: 38846469
DOI: 10.12865/CHSJ.50.01.11 -
RSC Advances May 2024An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of...
An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of 5-amino-7-(substituted)--(4-sulfamoylphenyl)-4,7-dihydro-[1,2,4]-triazolo[1,5-][1,3,5]triazine-2-carboxamide derivatives generated the reaction of 2-hydrazinyl--(4-sulfamoylphenyl)-2-thioxoacetamide, cyanoguanidine and a variety of aldehydes was achieved under green conditions. This experiment was conducted to evaluate the anti-inflammatory effect of the newly synthesized compounds using indomethacin as a reference medication; all compounds were tested for anti-inflammatory activity using the inhibition of albumin denaturation, RBC hemolysis technique and COX inhibition assay. The results showed that all evaluated compounds exhibited significant anti-inflammatory efficacy leading to excellently effective RBC membrane stabilization, inhibition of protein denaturation, and inhibition of COX enzymes when compared to those of indomethacin. At concentrations of 50, 100, 200, and 300 μg ml, these compounds decreased COX-1 and COX-2 activities more than indomethacin and have IC values in the range of 40.04-87.29 μg ml for COX-1 and 27.76-42.3 μg ml for COX-2 while indomethacin showed IC = 91.57 for COX-1 and 42.66 μg ml for COX-2. The anti-inflammatory findings show the need for more investigation to define the properties underlying the evaluated compounds' anti-inflammatory abilities. The enzyme cyclooxygenase-2 (COX 2) (PDB ID: 5IKT) was docked with ten synthetic substances. With docking scores () of -8.82, -7.82, and -7.76 kcal mol, 7-furan triazolo-triazine (4), 7-(2-hydroxy phenyl) triazolo-triazine (11), and 7-(4-dimethylamino phenyl) triazolo-triazine (12) had the greatest binding affinities, respectively. Therefore, these substances have COX-2 (PDB ID: 5IKT) inhibitory capabilities and hence may be investigated for COX 2 targeting development. Furthermore, both the top-ranked compounds (4 and 11) and the standard indomethacin were subjected to DFT analysis. The HOMO - LUMO energy difference (Δ) of the mentioned compounds was found to be less than that of indomethacin.
PubMed: 38832248
DOI: 10.1039/d4ra02970d