Lysozyme Solutions Research Articles

Mechanism of polymer molecular weight-dependent suppression and promotion of liquid-liquid phase separation of a protein solution by the addition of polymer.

Polyethylene glycol (PEG) is a widely used precipitant to concentrate proteins. The effect of PEG is generally understood to be an entropic attraction between proteins due to the depletion effect of PEG around proteins. However, measurements by Bloustine et al. [Phys. Rev. Lett. 96, 087803 (2006)] of the liquid-liquid phase separation (LLPS) temperature have shown that a lysozyme solution is stabilized and destabilized by the addition of low and high molecular-weight PEG, respectively. They also presented a theoretical model of the LLPS temperature as a virial expansion of the free energy and concluded that, in addition to the depletion effect, the attractive interaction between protein and PEG is necessary to explain the experiments. In the present study, theoretical calculations based on liquid-state density functional theory utilizing coarse-grained models are conducted to demonstrate that the protein-PEG effective attraction is responsible for the suppression and promotion of LLPS upon the addition of low- and high-weight PEG, respectively. In contrast, if the interactions between the protein and the PEG are solely due to the excluded volume effect, PEG of any molecular weight destabilizes the solution. These results suggest the necessity to reconsider the conventional understanding of the effects of polymer addition, which have been historically attributed to solely the depletion force.

Versatile Capillary Cells for Handling Concentrated Samples in Analytical Ultracentrifugation.

In concentrated macromolecular dispersions, far-from-ideal intermolecular interactions determine the dispersion behaviors including phase transition, crystallization, and liquid-liquid phase separation. Here, we present a novel versatile capillary-cell design for analytical ultracentrifugation-sedimentation equilibrium (AUC-SE), ideal for studying samples at high concentrations. Current setups for such studies are difficult and unreliable to handle, leading to a low experimental success rate. The design presented here is easy to use, robust, and reusable for samples in both aqueous and organic solvents while requiring no special tools or chemical modification of AUC cells. The key and unique feature is the fabrication of liquid reservoirs directly on the bottom window of AUC cells, which can be easily realized by laser ablation or mechanical drilling. The channel length and optical path length are therefore tunable. The success rate for assembling this new cell is close to 100%. We demonstrate the practicality of this cell by studying: (1) the equation of state and second virial coefficients of concentrated gold nanoparticle dispersions in water and bovine serum albumin (BSA) as well as lysozyme solution in aqueous buffers, (2) the gelation phase transition of DNA and BSA solutions, and (3) liquid-liquid phase separation of concentrated BSA/polyethylene glycol (PEG) droplets.

Preparation and characterization of a jelly fig (Ficus awkeotsang Makino) polysaccharide-based bioactive 3D scaffold for improved vascularization and skin tissue engineering applications

Jelly fig polysaccharides (JFP) were extracted from Ficus awkeotsang Makino achenes. The yield of JFP was approximately 10–15 %. FT-IR spectrum of the extracted JFP confirmed that it was made of low methoxyl pectin (LMP). 3D scaffolds of JFP (JFP scaffold) were fabricated using ionic crosslinking of 2 % (w/v) JFP solution with Ca2+ ions and freeze-drying. The JFP scaffold showed 73.46 ± 1.97 % porosity and a 12-fold swelling capacity. The porous morphology was also observed in SEM micrographs. JFP scaffolds were completely degraded in 14 days when incubated in 1 mg/mL lysozyme solution, compared to the 50 % degradation observed in PBS alone. The antioxidant activity of the JFP and JFP scaffold was approximately 40 %. The hemolytic assay of the JFP scaffold showed <5 % (3.0 ± 0.4) RBC lysis. The cytocompatibility of the JFP scaffold was evaluated using L929 mouse fibroblasts and human dermal fibroblasts (HDF). The in vitro studies using L929 cells showed that the JFP scaffold is cytocompatible. HDF cells cultured in the presence of JFP scaffolds show a higher fold cell viability, proliferation, and migration. Collagen expression and deposition were also studied, and no significant changes occurred with JFP scaffold treatment. In vivo CAM assay showed an increase in the number and thickness of blood vessels by 1.185-fold and 1.19-fold, respectively. These results confirm the angiogenic property of the JFP scaffold. These biocompatible and bioactive properties of the JFP scaffold could be beneficial for tissue engineering and regenerative medicine applications.

Polyethylene glycol–mediated genetic transformation and fluorescent labelling of Colletotrichum sublineola for studying sorghum anthracnose pathogenicity

AbstractAnthracnose, a disease primarily caused by the fungus Colletotrichum sublineola, affects sorghum production in China. However, the infection mechanisms and complex host interactions of C. sublineola remain to be elucidated. Thus, this study aimed to establish and optimize a protoplast transformation system of C. sublineola so as to lay a foundation for further research on the infection mechanisms and complex host interactions of C. sublineola. A genetic transformation system was established and optimized for C. sublineola strain HX‐5‐1 and used to introduce fluorescent reporter genes, namely, green fluorescent protein (GFP) and mCherry. Results showed that the growth of HX‐5‐1 was completely inhibited on a regenerating medium containing 300 μg mL−1 hygromycin B, indicating that hygromycin B acted as a transformant‐selective marker. HX‐5‐1 produced the highest yield of protoplasts when the mycelia were cultured for 60 h and dissolved in a 0.1% lysozyme solution (0.7 M NaCl) for 3 h. Subsequently, a 106 mL−1 protoplast solution transformed using 5 μg of plasmid DNA exhibited the highest transformation efficiency. Laser confocal microscopy showed that GFP and mCherry fluorescent signals were successfully expressed in the mycelia and spores of HX‐5‐1, indicating that they were stably inherited in the transformants and did not affect colony morphology, growth rate, or pathogenicity. In conclusion, we successfully established a genetic transformation system for C. sublineola and employed it to obtain stable fluorescent protein expression. This study may serve as a foundation for understanding the infection process of C. sublineola.

Enhancing protein stability under stress: osmolyte-based deep eutectic solvents as a biocompatible and robust stabilizing medium for lysozyme under heat and cold shock.

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml-1. Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.

What determines sub-diffusive behavior in crowded protein solutions?

The aqueous environment inside cells is densely packed. A typical cell has a macromolecular concentration in the range 90–450 g/L, with 5%–40% of its volume being occupied by macromolecules, resulting in what is known as macromolecular crowding. The space available for the free diffusion of metabolites and other macromolecules is thus greatly reduced, leading to so-called excluded volume effects. The slow diffusion of macromolecules under crowded conditions has been explained using transient complex formation. However, sub-diffusion noted in earlier works is not well characterized, particularly the role played by transient complex formation and excluded volume effects. We have used Brownian dynamics simulations to characterize the diffusion of chymotrypsin inhibitor 2 in protein solutions of bovine serum albumin and lysozyme at concentrations ranging from 50 to 300 g/L. The predicted changes in diffusion coefficient as a function of crowder concentration are consistent with NMR experiments. The sub-diffusive behavior observed in the sub-microsecond timescale can be explained in terms of a so-called cage effect, arising from rattling motion in a local molecular cage as a consequence of excluded volume effects. By selectively manipulating the nature of interactions between protein molecules, we determined that excluded volume effects induce sub-diffusive dynamics at sub-microsecond timescales. These findings may help to explain the diffusion-mediated effects of protein crowding on cellular processes.

Study of the Precrystallization Solution of Lysozyme by Accelerated Molecular Dynamics Simulation

Study of the Precrystallization Solution of Lysozyme by Accelerated Molecular Dynamics Simulation

Study of the Precrystallization Solution of Lysozyme by Accelerated Molecular Dynamics Simulation

The behavior of a dimer isolated from the crystal structure of tetragonal lysozyme has been simulated using the accelerated molecular dynamics method. The simulation time was 240 ns. The simulation data are compared with the data obtained previously using classical molecular dynamics. It is shown that the dimer studied is stable in both experiments, but the accelerated molecular dynamics method made it possible to reveal additional conformational changes in lysozyme molecules.

Preparation and characterization of lysozyme loaded liposomal dry powder inhalation using non-ionic surfactants

Delivering protein drugs through dry powder inhalation (DPI) remains a significant challenge. Liposomes offer a promising solution, providing protection for proteins from external environment and controlled release capabilities. Furthermore, the use of non-ionic surfactants plays a crucial role in protecting the activity of proteins because of how the surfactants positioning themselves at the liquid–gas interface during the spray-drying process. In this study, lysozyme-loaded liposomal DPI formulations were prepared using various non-ionic surfactants, including polysorbate 80, poloxamer 188, poloxamer 407, and sucrose stearate. Lysozyme solution and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine liposomes were subjected through high-pressure homogenization to form lysozyme-loaded liposomes. Formulations of homogenized lysozyme liposomes were spray-dried and further characterized. The particle size of reconstituted liposomal lysozyme DPI was from 129.5 to 816.9 nm. The formulations showed encapsulation efficiency up to 32.5% with zeta potential value of around − 30 mV, and spherical structures were observed. The aerosol dispersion performance of the dry powder inhalers was evaluated with emitted doses reaching up to 103% and fine particle fractions up to 28.4%. Significantly higher lysozyme activity was confirmed in formulation with drug to PS 80 ratio of 1: 0.5 w/w (92.1%) compared to that of formulation containing no surfactant (59.8%). The formulation stood out as the only formulation that maintained protein activity while demonstrating good aerosol performance.

The changes in molecular interaction and conformation of lysozyme mediated by ionic liquids

Using Ionic liquids（ILs）to regulate protein crystallization has attracted great attention, but the mechanism of ILs mediating crystallization is still unclear. Herein, we chose 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and 1,3-dimethylimidazolium iodide ([C1mim]I) as additives to mediate lysozyme crystallization, and use situ Raman to track the conformational changes of lysozyme in solution. Meanwhile, molecular simulation was used to explore the binding between ILs and lysozyme molecules. The results demonstrated that the addition of ILs produced new characteristic peaks in Raman spectra, suggesting some changes to occur in solution structure. The changes of peaks at 1340 cm−1 and 1359 cm−1, and the enhancement of peak at 1450 cm−1 indicated the unfolding of lysozyme molecules. The quantitative analysis of amide Ⅰ band showed that the addition of ILs increased the β-sheet content in secondary structures. Molecular docking showed that [C4mim]Cl and [C1mim]I could both bind to the active site of lysozyme molecules, and the binding energy were −3.1 kcal/mol and −4.5 kcal/mol, respectively. Molecular dynamics results showed that [C4mim]Cl could stably bind to lysozyme molecules, while [C1mim]I left the active site after several ns, which means that [C4mim]Cl brought about stronger impact on interaction of lysozyme molecules than [C1mim]I in solution.

Spectroscopic and Molecular Docking Studies of the Interaction of Non-steroidal Anti-inflammatory Drugs with a Carrier Protein: an Interesting Case of Inner Filter Effect and Intensity Enhancement in Protein Fluorescence.

Interaction of diclofenac and indomethacin with lysozyme was studied using several spectroscopic and molecular docking methods. Difference UV-visible spectra showed that the absorption profile of lysozyme changed when both diclofenac and indomethacin were mixed with the former. The sequential addition of both drugs to the lysozyme solution caused the decrease of the intrinsic fluorescence of the latter, however, when the data were corrected for inner filter effect, an enhancement in the fluorescence of lysozyme was detected. Accordingly, the fluorescence enhancement data were analyzed using Benesi-Hildebrand equation. Both, diclofenac and indomethacin showed good interaction with lysozyme, although, the association constants of indomethacin were nearly two-fold higher as compared to that of diclofenac. The binding was slightly more spontaneous in case of indomethacin and the major forces involved in the binding of both drugs with lysozyme were hydrogen bonding and hydrophobic interactions. Secondary structural analysis revealed that both drugs partially unfolded lysozyme. Results obtained through molecular docking were also in good agreement with the experimental outcomes. Both, diclofenac and indomethacin, are bounded at the same site inside lysozyme which is located in the big hydrophobic cavity of the protein.

Antibacterial activity of lysozyme-chitosan oligosaccharide conjugates on two periodontal bacteria.

This study aimed to evaluate the in vitro antibacterial effects of lysozyme-chitosan oligosaccharide conjugates (LYZOX) against Streptococcus gordonii and Porphyromonas gingivalis. Planktonic S. gordonii and P. gingivalis were treated with various concentrations of LYZOX for 10 min. The treated bacteria were incubated on trypticase soy agar plates, and colony-forming unit (CFU) was calculated. The antibacterial effect of LYZOX was compared with that of lysozyme, chitosan, physiological saline, and benzalkonium chloride solution. Cell morphology before and after LYZOX treatment was observed using a scanning electron microscope (SEM). The antibacterial effect of LYZOX with decanoic acid against the biofilm-like bacteria was also examined via crystal violet staining. The Kruskal-Wallis test and post hoc Dunn tests were performed to compare the difference in antibacterial activity of each treatment. Bacterial CFU numbers were reduced after LYZOX treatment in a concentration-dependent manner. The reduction in CFUs was smaller for corresponding concentrations of chitosan or lysozyme alone. SEM analyses revealed bacterial cells shrank following LYZOX treatment. The combined use of LYZOX and decanoic acid yielded an even higher antibacterial effect against bacterial biofilms. LYZOX exhibits antibacterial activity against two periodontal bacteria and may be a promising plaque control agent.

3D Printed Microfluidic Cell for SAXS Time-Resolved Measurements of the Structure of Protein Crystallization Solutions

A multichannel microfluidic cell (MFC) obtained using 3D printing for studying the structure of complex solutions by small-angle X-ray scattering (SAXS) is described. MFC was tested at the BioMUR beamline of the Kurchatov synchrotron. A comparative analysis of SAXS signal from the standard capillary and from the developed MFC was carried out, with MFC showing significant advantages. The dynamics of SAXS scattering curves for lysozyme solutions with NaCl precipitant were studied when the protein and precipitant concentrations changed. The obtained time series of data are well consistent with the known data for the lysozyme solution.

Reusable and Biodegradable Separation Membranes Prepared from Common Mushrooms for the Removal of Oily and Particulate Contaminants from Water.

Mushroom chitin membranes with controllable pore structures were fabricated through a simple process with naturally abundant Agaricus bisporus mushrooms. A freeze-thaw method was applied to alter the pore structures of the membranes, which consist of chitin fibril clusters within the glucan matrix. With tunable pore size and distribution, mushroom chitin membranes could effectively separate stable oil/water emulsions (dodecane, toluene, isooctane, and chili oil) with various chemical properties and concentrations and particle contaminants (carbon black and microfibers) from water. Chitin fibrils tightly pack with each other to form a dense membrane, leading to no permeation of contaminants or water. An increasing number of applied freeze-thaw cycles confers more tortuous pore structures throughout the mushroom chitin membranes, leading to higher flux while maintaining rejection performance. The 3D simulation constructed by the X-ray computed tomography and GeoDict software also demonstrated capturing a considerable amount of contaminants within the membranes' pores, which can be easily removed by water rinsing for further successive filtration. Furthermore, mushroom chitin membranes were almost completely biodegraded after approximately a month of being buried in the soil or kept in a lysozyme solution while possessing mechanical durability demonstrated by consistent filtration performance for repeated usage up to 15 cycles under ambient and external pressure. This research is a proof of concept that mushroom-derived chitin develops functional and biodegradable materials for environmental applications with scalability.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein–water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay.

Polyphosphazenes represent a class of intrinsically flexible polyelectrolytes with potent immunoadjuvant activity, which is enabled through non-covalent self-assembly with antigenic proteins by charge complexation. The formation of supramolecular complexes between polyphosphazene adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), and a model vaccine antigen, hen egg lysozyme, was studied under physiological conditions using automated dynamic light scattering titration, asymmetric flow field flow fractionation (AF4), enzyme-linked immunosorbent assay (ELISA), and fluorescent quenching methods. Three regimes of self-assembly were observed covering complexation of PCPP with lysozyme in the nano-scale range, multi-chain complexes, and larger aggregates with complexes characterized by a maximum loading of over six hundred protein molecules per PCPP chain and dissociation constant in the micromolar range (Kd = 7 × 10-6 mol/L). The antigenicity of PCPP bound lysozyme, when compared to equivalent lysozyme solutions, was largely retained for all complexes, but observed a dramatic reduction for heavily aggregated systems. Routes to control the complexation regimes with elevated NaCl or KCl salt concentrations indicate ion-specific effects, such that more smaller-size complexes are present at higher NaCl, counterintuitive with respect to PCPP solubility arguments. While the order of mixing shows a prominent effect at lower stoichiometries of mixing, higher NaCl salt reduces the effect all together.

Synthesis of core-shell material rich in carboxyl groups and its application in the selective adsorption of cationic peptides.

The ability to extract peptides and proteins from biological samples with excellent reusability, high adsorption capacity, and great selectivity is essential in scientific research and medical applications. Inspired by the advantages of core-shell materials, we fabricated a core-shell material using amino-functionalized silica as the core. Benzene-1,3,5-tricarbaldehyde and 3,5-diaminobenzoic acid were used as model organic ligands to construct a shell coating by alternately reacting the two monomers on the surface of silica microspheres. The resultant material featured an outstanding capability for the adsorption of cationic peptides, most likely owing to its porous structure, a large number of carboxylic functional groups, and low mass-transfer resistance. The maximum saturated adsorption capacity reached 833.3mg/g and the adsorption process took only 20min. Under optimized adsorption conditions, the core-shell material was used to selectively adsorb cationic peptides from the tryptic digestive solution of lysozyme and bovine serum albumin, Specifically, the analysis results showed seven cationic peptides in the eluate and twenty anionic peptides in the supernatant, which indicates the efficient trap of most cationic peptides in the digestive solution.

Dynamic Electric Field Alignment Determines the Water Rotational Motion around Protein.

Water rotational dynamics in biomolecular solution is crucial to evaluating and controlling biomolecule stability. In this molecular dynamics simulation (MD) study on lysozyme solutions, we present how the exerted internal electric field determines water rotational dynamics. We find that the relaxation time of water rotation is equivalent to that of the reorientation of the exerted overall electric field for every single water molecule, regardless of its translation mode. Namely, water molecular rotation synchronizes with the exerted field reorientation. We also map the reorientation process of the electric field at fixed points relative to protein in the solution, which displays the local hydration dynamics commensurate with the reported time-dependent fluorescence Stokes shift (TDFSS) measurements. Comparing the spatial distribution of local field reorientation relaxation time with that of rotational relaxation time, we further suggest that water rotation dynamics are subject to the reorientation of the local overall field within the hydration layer. While outside the hydration layer, the relaxation time of the local electric field reorientation is short enough (subpicosecond) to assume the δ function, showing the electric force with randomly changing orientation is applied to each water molecule.

The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions.

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules "connect" through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

Liquid-Liquid Phase Separation Prediction of Proteins in Salt Solution by Deep Neural Network.

Liquid-liquid phase separation (LLPS) underlies the formation of membrane-free organelles in eukaryotic cells and plays an important role in the development of some diseases. The phase boundary of metastable liquid-liquid phase separation as well as the cloud point temperature of some globular proteins characterize the phase behavior of proteins and have been widely studied theoretically and experimentally. In the present study, we used a regression and classification neural network to deal with the phase behavior of lysozyme and bovine serum albumin (BSA). We predicted the cloud point temperature and solubility of a lysozyme solution containing sodium chloride by regression and the reentrant phase behavior of BSA in YCl3 solution containing a surfactant dodecyl dimethyl amine oxide (DDAO) by classification. Specifically, our network model is capable of predicting (a) the solubility of lysozyme in the range: pH 4.0-5.4, temperature 0-25 °C, and NaCl concentration 2-7% (w/v); (b) the cloud point temperature of lysozyme in the range: pH 4.0-4.8, NaCl concentration 2-7%, and lysozyme concentration 0-400 mg/mL; and (c) the phase behavior of BSA in the range: DDAO 1-60 mM, BSA 30-100 mg/mL, and YCl3 1-20 mM. We experimentally tested the model at some prediction points with a high accuracy, which means that deep neural networks can be applicable in qualitative and quantitive analysis of liquid-liquid phase separation.