Chicken Egg White Lysozyme Research Articles

Elucidation of the Binding Interaction between β-sitosterol and Lysozyme using Molecular Docking, Molecular Dynamics and Surface Plasmon Resonance Analysis.

In this study, the binding behavior of β-sitosterol with lysozyme (LZM) was elucidated by surface plasmon resonance (SPR), computational molecular docking and molecular dynamics simulation studies. Chicken egg white lysozyme (CEWLZM) served as a model protein. Tri-N-acetylchitotriose (NAG3) was used in the redocking experiments to generate precise binding location of the protein. β-sitosterol displayed a slightly better binding energy (-6.68±0.04 kcal/mol) compared to NAG3. Further molecular dynamics simulations and MMPBSA analysis revealed that residues Glu35, Gln57-Asn59, Trp62, Ile98, Ala107 and Trp108 contribute to the binding energy. Then, 2.5 mg/mL CEWLZM, 1X PBS buffer (pH 7.4) as running and coupling buffers, 30 μL/min as flow rate were applied for SPR analysis. Serial β-sitosterol injections (20-150 μM) were performed through SPR sensor surface. According to SPR binding study, KD value for β-sitosterol-CEWLZM binding interaction was calculated as 71.34±9.79 μM. The results could provide essential knowledge for nutrition, pharmaceutical science, and oral biology.

Surface plasmon resonance, molecular docking, and molecular dynamics simulation studies of lysozyme interaction with tannic acid.

Lysozyme (LZM) is an important enzyme in medicine and industry. Tannic acid (TA) is used in brewing, wine industry, and as a food flavor enhancer. In nutritional and food science, LZM interacts with TA, notably in wine and saliva. This study aimed to investigate the binding interaction between LZM and TA using surface plasmon resonance, molecular docking, and molecular dynamics simulation. Chicken egg white lysozyme (CEWLZM) was applied as a model protein. Tri-N-acetylchitotriose (NAG3), the known inhibitor of CEWLZM, was used in the redocking experiments to determine the precise binding location within the complex. The average binding energies obtained from docking NAG3 and tannic acid to the target structure of CEWLZM were found to be -6.46 ± 0.05 kcal/mol and -7.52 ± 0.39 kcal/mol, respectively. The binding free energy of the CEWLZM-TA complex was then calculated as -27.61 kcal/mol by MMPBSA based on MD simulation trajectories. The observed interactions between the ligands and the lysozyme structure were mainly driven by hydrophobic, van der Waals, and H-bond interactions formed by the active site residues. MD simulations showed consistent and satisfactory binding distances between CEWLZM and TA throughout the analysis. SPR analysis was performed using 1X PBS buffer (pH 7.4) as coupling and running buffers, 30 μL/min as flow rate, and 2.5 mg/mL CEWLZM. Serial concentrations of TA (20-150 μM) were injected through immobilized CEWLZM, and the K D value of CEWLZM-TA binding was obtained as 4.17 × 10-5 M. This study could enhance existing literature and pave the way for future research in food science and oral biology.

Enhancement of Ligand Immobilization on Gold Surface by Optimizing Monolayer Concentration and Pre‐Concentration Studies for Surface Plasmon Resonance Analysis of Rutin‐Lysozyme Interaction

AbstractSurface plasmon resonance (SPR) is a versatile tool for real time monitoring of molecular interactions, providing biophysical data such as affinity and kinetics. Rutin is found in various plants and fruits and is utilized in several industries. Because of its therapeutic and industrial importance, the binding of rutin to one of the major proteins found in blood and saliva, lysozyme (LZM), was investigated to determine the affinity constants of the rutin‐LZM interaction. Chicken egg white lysozyme (CEWLZM) was selected as a model ligand. 11‐mercaptoundecanoic acid (MUA) was chosen for the self‐assembled monolayer (SAM) design on the gold chip surface. After optimizing MUA concentration and pre‐concentration studies to enhance ligand immobilization, optimum MUA concentration in SAM formation, ideal running and coupling buffers, flow rate and ligand concentration were determined to perform SPR analysis. Various concentrations of rutin (20–300 μM) were injected into the SPR for kinetic analysis. The association (ka), dissociation (kd) rate constants, and equilibrium constant (KD) were calculated from the kinetics of the binding curves. SPR binding results indicated that the KD value of rutin‐CEWLZM binding was determined as 80.07±1.22 μM. This study demonstrates the binding affinity of rutin to CEWLZM, offering potential insights for medical and pharmaceutical sciences.

Inhibitory Impacts of Fulvic Acid-Coated Iron Oxide Nanoparticles on the Amyloid Fibril Aggregations.

Alzheimer's disease is considered as multi-factor diseases, the main hallmarks of which are extracellular amyloid-beta and intracellular tau protein aggregations, leading to neural death. With this in mind, most of the studies have been focused on eliminating these aggregations. Fulvic acid is one of the polyphenolic compounds which exhibits strong anti-inflammation and anti-amyloidogenic effects. On the other hand, iron oxide nanoparticles are able to reduce/eliminate the amyloid aggregations. Here in, the effect of fulvic acid-coated iron-oxide nanoparticles on the commonly used in-vitro model for amyloid aggregation studies, i. e., lysozyme from chicken egg white was investigated. The chicken egg white lysozyme forms the amyloid aggregation under acidic pH and high heat. The average size of nanoparticles was 10.7±2.7 nm. FESEM, XRD, and FTIR confirmed that fulvic acid was coated onto the surface of the nanoparticles. The inhibitory effects of the nanoparticles were verified by Thioflavin T assay, CD, and FESEM analysis. Furthermore, the toxicity of the nanoparticles on the neuroblastoma SH-SY5Y was assessed through MTT assay. Our results indicate that these nanoparticles efficiently inhibit amyloid aggregation formation, while exhibiting no in-vitro toxicity. This data shed light on the anti-amyloid activity of the nanodrug; paving the way for future drug development for treating Alzheimer's disease.

How does dilution affect the conductivity, the propensity to aggregate and the biological activity of enzymes?

The current theories do not provide a convincing explanation for many of the protein' manifestations in solution. Therefore, to shed some light on this critical question, the present work analyzed the effect of dilution on the ability of proteins to catalyze chemical reactions, the so-called biological activity. As models enzymes, laccase from Trametes versicolor and chicken egg white lysozyme were used.The results show that the enzymes' specific biological activity increases on dilution. Three explanations for the intriguing observation were advanced and submitted to experimental scrutiny. Amongst the three hypotheses, only one was corroborated by experiments. According to this explanation, when dissolved in water, proteins reveal two populations: one biologically active whose relative occurrence increases on dilution and another which is not active and whose molecular proportion varies in the opposite direction.Therefore, the reported experimental facts strongly support the chemical behaviour of the proteins in the solution. According to the herein-advocated concepts, they could undergo a dissociation process similar to that found in electrolyte chemistry.

Lactobacillus johnsonii CPN23 vis-à-vis Lactobacillus acidophilus NCDC15 Improves Gut Health, Intestinal Morphometry, and Histology in Weaned Wistar Rats.

The present investigation was carried out with the aim to establish the comparative efficacy of a canine-sourced probiotic meant for canine feeding and a conventional dairy-sourced probiotic. For this purpose, canine-origin Lactobacillus johnsonii CPN23 and dairy-origin Lactobacillus acidophilus NCDC15 were evaluated for potential probiotics health benefits in the rat model. Forty-eight weaned Wistar rats enrolled in this experiment of 8weeks were fed a basal diet and divided into three dietary treatments. Rats of group I enrolled as control (CON) were given MRS placebo at 1mL/head/day, while rats of group II (LAJ) and III (LAC) were administered with overnight MRS broth grown-culture of L. johnsonii CPN23 and L. acidophilus NCDC15, respectively, at 1mL/head/day (108cfu/mL). The average daily gain and net gain in body weight were significantly higher (p < 0.05) in LAJ and LAC than in CON. Fecal and digesta biochemical attributes altered (p < 0.05) positively in response to both probiotics. Total fecal and pooled digesta SCFAs were higher (p < 0.05) in both LAJ and LAC than in CON. The microbial population in cecal and colonic digesta responded (p < 0.05) positively to both probiotics. The diameter of intestinal segments was higher (p < 005) in LAJ as compared to CON. The number and height of villi in jejunum tended to be higher in LAJ as compared to CON. The humoral immune response to sheep erythrocytes as well as chicken egg-white lysozyme was higher in LAJ as compared to CON. Overall, the results of the study have demonstrated the effectiveness of the canine-sourced L. johnsonii CPN23 as a potential probiotic, with a comparatively better response than the dairy-sourced L. acidophilus NCDC15. It could thus be recommended for use in feeding dogs to help augment their health.

Impact of chromate and dichromate on lysozyme stability: A spectroscopic and molecular docking investigation.

A comparative study of interaction between chicken egg white lysozyme (Lyz) with two hexavalent chromate ions; chromate and dichromate; which are prevalently known for their toxicity, was investigated using different spectroscopic techniques along with a molecular docking study. Both steady-state and time-resolved studies revealed that the addition of chromate/dichromate is responsible for strong quenching of intrinsic fluorescence in Lyz and the quenching is caused by both static and dynamic quenching mechanisms. Different binding and thermodynamic parameters were also calculated at different temperatures from the intrinsic fluorescence of Lyz. The conformational change in Lyz and thermodynamic parameters obtained during the course of interaction with chromate/dichromate were well-supported by the molecular docking results.

Weakly Non-Covalent Docking of Amino-Acid Schiff Base Zn(II) Complex to Lysozyme

Artificial metal enzymes that combine proteins with synthesized unnatural metal complexes as cofactors are attracting attention. The preparation of artificial metal enzymes not only clarifies the behavior of metal ions in biology, but also leads to the development of synthetic chemistry fields such as the discovery of new catalytic reactivity and substrate selectivity that are not observed in nature. In addition, a certain Schiff base zinc (II) complex is known to exhibit antioxidant and anticancer activity, too. Therefore, in this study, we investigated a rapid synthesis method of two known amino acid Schiff base zinc (II) complexes using microwave method and the complexation of zinc (II) complex with chicken egg white lysozyme, which is a relatively low molecular weight protein. Furthermore, investigation of weakly non-covalent intermolecular interaction features between the zinc (II) complexes and lysozyme was also carried out using some spectroscopic measurements.

Unconventional effects of long-term storage of microwave-modified chicken egg white lysozyme preparations

Thermal modification is an effective method that induces significant expansion of the antimicrobial properties and other valuable properties of chicken egg white lysozyme. In our latest research, a new innovative method of enzyme modification was developed, in which microwave radiation was used as an energy source to process liquid lysozyme concentrate (LLC). After modification, high-quality preparations were obtained. However, long-term storage in a concentrated form initiated various processes that caused darkening over time and could also lead to other significant changes to their structure and, consequently, to their functional properties. This necessitated multidirectional research to explain this phenomenon. This paper presents the results of research aimed at assessing the physicochemical changes in the properties of microwave-modified lysozyme in the form of a liquid concentrate after long-term storage under refrigeration conditions. The assessment also considered the conditions under the acidity of the modifying medium and the duration of the microwave modification. The analysis showed that the values of the basic parameters determining the quality and usefulness of the modified enzyme significantly improved during long-term storage of the preparations. The greatest changes were observed in the preparations modified for the longest time and in the most acidic environment (process time 260 s, pH 2.0), the number of oligomers under these conditions increased by 18% after 12 months of holding, and the surface hydrophobicity increased by as much as 31%. In addition, microbiological tests showed that the preparations of microwave-modified lysozyme had an effect on gram-positive bacteria as well as on gram-negative, and this effect was significantly enhanced after 12 months. The results confirm that LLC modification with microwave radiation is a highly efficient method to prepare high-quality and high utility potential lysozyme. Notably, an interesting and important phenomenon was the observation of the unconventional behaviour of the preparations during their long-term storage, which increased their utility potential significantly.

Thermal modification of hen egg white lysozyme using microwave treatment

Thermal modification has commonly been used in the processing of chicken egg white lysozyme and has shown good antibacterial results in the field of laboratory study. However, the use of conventional thermal energy sources still presents disadvantages. In this work, microwave radiation was used as a new alternative to conventional sources of thermal energy for the modification of lysozyme. The material for modification was 5% concentration monomer lysozyme solutions with different pH levels. The enzyme was modified by microwave radiation under different microwave powers and different modification times separately. Important lysozyme protein parameters, such as: electrophoresis, hydrolytic activity, hydrophobicity, and solubility, were evaluated. Our research showed that the number of oligomers, especially dimers, and the surface hydrophobicity increased significantly in the preparations obtained after the processes under the appropriate conditions of modification. The highest production efficiency yield was 54.4% oligomers and 30.9% dimers. An additional beneficial effect of using this new method was the fact that the hydrolytic activity did not decrease as much as in the case of other thermal methods. And the solubility of the obtained preparations was high, ranging from 87% to 100%. The research showed that lysozyme dimer formation and other important protein properties changed effectively. Such a method not only changed the protein folding structure but also offered the advantages of a short processing time, high efficiency and easy enzyme control. It will have a greater application potential after further development.

Microcalorimetric Analysis of the Adsorption of Lysozyme and Cytochrome c onto Cation-Exchange Chromatography Resins: Influence of Temperature on Retention.

We studied the adsorption mechanism of two basic proteins, equine cytochrome c (Cyt) and chicken egg-white lysozyme (Lys), adsorbing onto negatively charged chromatography surfaces. In liquid chromatography, the retention volume of Lys was larger than that of Cyt on negatively charged ion-exchange resins. When the temperature increased, the retention volume of Cyt increased, whereas that of Lys clearly decreased. Both Lys and Cyt share similar physical characteristics, so the opposite behavior with increasing temperatures was surprising, indicating a more complex mechanism of adsorption may be involved. We analyzed the adsorption of these proteins by using isothermal titration calorimetry (ITC). The change in adsorption enthalpy determined by ITC allowed the understanding of the reason for and underlying driving forces of protein adsorption that resulted in this opposite behavior. Large exothermic enthalpies of adsorption were observed for Lys (-43.95 kJ/mol), and Lys adsorption was found to be enthalpically driven. On the other hand, endothermic enthalpies were dominant for Cyt adsorption (32.41 kJ/mol), which was entropically driven. These results indicate that dehydration and release of counterions play a more important role in Cyt adsorption and ionic interaction and hydrogen bridges are more significant in Lys adsorption. Understanding of the adsorption mechanism of proteins onto chromatography resins is essential for modeling and developing new, efficient chromatographic processes.

Is the Protein Dynamical Transition useful?

Terahertz spectroscopy potentially provides a fast and easy method to measure the protein dynamical transition [1,2].Typically the THz measurements are performed using solutions, avoiding the difficulty in hydration control conditions typically used in for neutron scattering, however there is some question as to whether the freezing of the solution effects protein structure or dynamics. More significantly, does the rapid onset of biomolecular dynamics occurring ∼200 K provide any new insight into specific protein structural dynamics, or is it strictly a reflection of the hydration water? To address these questions we performed terahertz dynamical transition measurements in the 100-270 K range and between 0.15 - 2.00 THz on buffered solutions, dry glycerol solutions and minimally hydrated glycerol solutions. Buffer solution measurements using chicken egg white lysozyme, myoglobin and orange carotenoid protein with concentrations between 2 - 30 mM follow Beer's law concentration dependence below 15 mM from which terahertz molar absorptivity of the hydrated protein can be extracted. For dry glycerol-protein solutions, the dynamical transition is absent. The transition is present for 0.5 g water/g protein. The transition is identical to the buffer solution results, indicating that the freezing of the bulk water does not effect the measurements and it is not necessary to use the hydrated glycerol method. Finally we examine how light induced structural changes of OCP can induce changes in the dynamical transition, and correlate these changes with other structural flexibility measurements to identify the mechanism responsible for the DT's sensitivity to protein structural change. This work is supported by NSF grants DBI 1556359 and MCB 1616529, DOE grant DE-SC0016317 and NIH STTR R41 GM125486.

Effect of the initial concentration on the equilibrium liquid phase concentration at salting out of proteins

AbstractBACKGROUNDProtein precipitation is a unit operation commonly employed in bioprocesses. The most important thermodynamic property for designing an industrial precipitation operation is the solubility. The solubility is defined as the protein concentration in a certain liquid phase in equilibrium with its solid precipitate. In some cases, the protein concentration in the liquid phase at equilibrium depends on the initial concentration of the protein, therefore being an apparent solubility. Here an assessment of the effect of the molecular mass of proteins on the dependence of the apparent solubility on the initial protein concentration is reported.RESULTSProteins with different molecular masses (bovine insulin, 5.7 kDa; chicken egg white lysozyme, 14.7 kDa; porcine trypsin, 23.4 kDa; bovine serum albumin (BSA), 66.0 kDa) at different initial concentrations were precipitated with NaCl at constant pH and temperature. Lysozyme was the only protein that showed no change in its solubility as a function of the initial protein concentration at the studied conditions. For insulin, the effect of the initial concentration on the apparent solubility was verified only at low salt concentrations. The apparent solubility of proteins with higher molecular mass, i.e. trypsin and albumin, showed dependence on the initial protein concentration: the higher the initial protein concentration, the higher the apparent solubility.CONCLUSIONBased on the data of this work and the literature, the molecular mass appears to affect the dependence of the apparent solubility of proteins on their initial concentration in precipitation processes. © 2019 Society of Chemical Industry

Radiation-damage investigation of a DNA 16-mer.

In macromolecular crystallography, a great deal of effort has been invested in understanding radiation-damage progression. While the sensitivity of protein crystals has been well characterized, crystals of DNA and of DNA-protein complexes have not thus far been studied as thoroughly. Here, a systematic investigation of radiation damage to a crystal of a DNA 16-mer diffracting to 1.8 Å resolution and held at 100 K, up to an absorbed dose of 45 MGy, is reported. The RIDL (Radiation-Induced Density Loss) automated computational tool was used for electron-density analysis. Both the global and specific damage to the DNA crystal as a function of dose were monitored, following careful calibration of the X-ray flux and beam profile. The DNA crystal was found to be fairly radiation insensitive to both global and specific damage, with half of the initial diffraction intensity being lost at an absorbed average diffraction-weighted dose, D1/2, of 19 MGy, compared with 9 MGy for chicken egg-white lysozyme crystals under the same beam conditions but at the higher resolution of 1.4 Å. The coefficient of sensitivity of the DNA crystal was 0.014 Å2 MGy-1, which is similar to that observed for proteins. These results imply that the significantly greater radiation hardness of DNA and RNA compared with protein observed in a DNA-protein complex and an RNA-protein complex could be due to scavenging action by the protein, thereby protecting the DNA and RNA in these studies. In terms of specific damage, the regions of DNA that were found to be sensitive were those associated with some of the bound calcium ions sequestered from the crystallization buffer. In contrast, moieties farther from these sites showed only small changes even at higher doses.

Protein adsorption characteristics of nanoparticle-assembled hollow microspheres of hydroxyapatite and their composites with PLLA microporous membranes

Nanoparticle-assembled hydroxyapatite (HA) hollow microspheres have a high surface area and are convenient to handle, owing to their characteristic structure. In this study we characterized the protein adsorption of HA hollow microspheres prepared from CaCl2 and K2HPO4 by a water-in-oil-in-water (W/O/W) emulsion method assisted by two surfactants: Span 80 and Tween 20. The HA hollow microspheres adsorbed bovine serum albumin, bovine γ-globulin, equine skeletal muscle myoglobin, and chicken egg white lysozyme in 10 mM sodium phosphate buffer (pH 6.8) in a Langmuir-type adsorption and desorbed the proteins in 800 mM sodium phosphate buffer (pH 6.8). The maximum adsorbed amounts of the HA hollow microspheres were 7.5–9.0 times higher than those of the microrods with a similar size range. The composite membranes of the HA microspheres and the poly(l-lactic acid) (PLLA) microporous membranes exhibited a high adsorption capacity for γ-globulin.

Development of a Microfluidic Chip for Protein Crystallization by the Microbatch Method

A composite microfluidic chip (MFC), which comprises a technological system for protein crystal growth followed by X-ray crystallography, was developed. Crystallization tests were performed using chicken egg-white lysozyme, aminotransferase from Methanococcus vannielii, and esterase PMGL2 from the permafrost metagenomic library. Materials (borosilicate glass and poly(methyl methacrylate)) for MFC fabrication were compared. The use of poly(methyl methacrylate) was shown to allow comparative in situ X-ray crystallography of protein crystals directly in the chip. Investigations and experiments were carried out at the BELOK beamline at the Kurchatov synchrotron radiation source (National Research Centre “Kurchatov Institute”).

The Effect of Crystal Contact Forces on the Protein Global Motions

Protein crystals are widely used for structural determination using X-ray crystallography and solid state NMR. X-ray free electron lasers have enabled direct dynamical structural movies of photo or chemically initiated biochemical reactions. However the perturbation of protein dynamics by the crystal contact forces has been persistently under debate. If crystal contact forces are sufficiently strong, the time resolved X-ray diffraction measurements will not accurately reflect the in vivo biological dynamics. Here we probe the effect of crystal contact forces on protein structural dynamics by directly measuring the collective intramolecular vibrations as a function of crystal symmetry for chicken egg white lysozyme (CEWL). These vibrations extend throughout the protein, involving coherent motion of entire domains. While the collective vibrational density of states is a broad continuous spectrum peaked near 100 cm−1, (3 THz), polarization dependent terahertz absorption isolates vibrations with displacements primarily along the polarization direction [1]. Triclinic, monoclinic, tetragonal and orthorhombic CEWL crystals are grown according to previous published protocols. X-ray measurements verify their crystal structure and quality before and after terahertz measurements. The anisotropic terahertz absorbance is measured using our new technique: ideal polarization varying anisotropic THz microscopy (IPV-ATM). This near field THz microscopy method allows rapid determination of the anisotropic THz absorption. By starting from measurements of the lowest symmetry, triclinic crystals, which have a single protein per unit cell and absolute alignment of the protein molecule array, we can predict the spectra for higher symmetry groups, neglecting crystal contact perturbations. We compare our predicted spectra to the measurements of the monoclinic, orthorhombic and tetragonal crystals, attaining a measure of the impact of the intermolecular crystal forces on the intramolecular vibrations. [1]G. Acbas, K. A. Niessen, E. H. Snell, and A. G. Markelz, Nat. Comm.(2014) https://doi.org/10.1038/ncomms4076.

Does poly(ionic liquid) modulate the non-covalent interactions of chicken egg white lysozyme? Elucidation of biomolecular interactions between biomolecules and macromolecular solvents

Stabilizing and destabilizing effects of different poly(ionic liquid) (PIL) concentrations on chicken egg white lysozyme as a reason for bimolecular interactions.

Poli(Hidroksietilmetakrilat-Genişletilmiş Perlit) Kompoziti ile Yumurta Akından Lizozimin Tek Basamakta Saflaştırılması

In this study, we investigated adsorption behaviour of lysozyme on poly(hydroxyethylmethacrylate-expanded perlite) [P(Hema-Ep)]. For this purpose, we synthesized P(Hema-Ep) composite by bulk polymerization and it was characterized by SEM, FTIR and swelling ratio. The fresh chicken egg white lysozyme was used as a model protein, had an isoelectric point (pI) 10.7 and molecular weight (MW) 14 kDa. Adsorption capacity was found 51 mg g-1 at room temperature and in 0.1 M pH=8.0 phosphate buffer. Experiments on desorption and reusability were also performed. It appears that P(Hema-Ep) composites can be used ten times without lost of efficiency. Lysozyme purification, without causing any denaturation, can be performed from chicken egg white by using this new synthesized composite in a single step.

Application of Empirical Phase Diagrams for Multidimensional Data Visualization of High-Throughput Microbatch Crystallization Experiments

Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphologic features, such as crystal size, as well as kinetic features, such as crystal growth time. Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols. These techniques have limitations in terms of handling large data sets, comprehensiveness or completeness. To eliminate these limitations, morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram method. Morphologic features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength, and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the empirical phase diagram method can support high-throughput crystallization experiments in its data amount as well as its data complexity.