Stability Of Lysozyme Research Articles

Determinants for Sugar-Induced modulation of thermodynamic stability of lysozyme

The current study focuses on the experimental evidence to describe the molecular mechanism by which the sugars modulate the thermodynamic stability of hen egg-white lysozyme (Lyz) at pH 2.3. Analysis of thermal and chemical denaturation curves of Lyz under variable concentrations of sugar (trehalose, sucrose, maltose, glucose, ribose, glycerol) at pH 2.3 depicted important results: (i) sugar increases the thermodynamic stability of Lyz, and it typically tracks the order as: trehalose > sucrose > maltose > glucose > ribose > glycerol, and (ii) sugar increases the thermodynamic stability of Lyz via preferential exclusion of sugar at the protein surface, but the repulsive enthalpic interactions of sugar with protein also add to the sugar-mediated increase in thermodynamic stability of the protein. Analysis of the sugar effect on denaturant concentration dependent denaturation free energy of Lyz at pH 2.3 revealed that the sugar counteracts the denaturant (urea, guanidinium chloride (GdnCl)) efficacy to decrease the thermodynamic stability of Lyz. Furthermore, the counteraction efficiency of sugar on denaturant impact to decrease thermodynamic stability of Lyz typically tracks the order as: trehalose > sucrose > maltose > glucose > ribose > glycerol.

Investigation of The Stability of The Lysozyme in Magnetic Particles for Drug Delivery

This research investigated lysozyme stability in the presence of magnetic particles for drug delivery. Lysozyme can mostly be found in the hen egg albumin, and it also acts as an essential agent, which is antibodies to fight harmful bacteria or shield the human body. The problems in this research are magnetic particles that can cause toxicity and reduce the therapy's efficiency due to the degradation of carriers (drugs). To minimise this problem, the stability and activity of the lysozyme and Fe2O3 were investigated. The structure of lysozyme and Fe2O3 before and after adding acid or alkaline was defined, and the suitability of Fe2O3 as a drug delivery was determined. The stability of the lysozyme and Fe2O3was identified using an Ultraviolet-Visible (UV-Vis) Spectrophotometer, and the structure was investigated using a Field Emission Scanning Electron Microscope (FESEM) and Scanning Electron Microscope- Energy Dispersive X-Ray (SEM-EDX). Different concentrations become the parameters of this research as a comparison between the samples. This research found that the combination of the lysozyme alkaline material was suitable for the body at a pH of 12.2. It also found that Fe2O3 is ideal for drug delivery in the body when the lysozyme can be carried by Fe2O3 and dissolved in the Fe2O3.

Hydrogel microspheres encapsulating lysozyme/MXene for photothermally enhanced antibacterial activity and infected wound healing

The high mortality and enormous economic burden of bacterially infected wounds remains a huge challenge for human health. The development of ideal wound dressings with desirable antibacterial and good wound healing properties still remains a major problem affecting the regeneration of bacterially infected wound tissue. Herein, we present novel alginate-based hydrogel microspheres containing lysozyme and MXene (i-Lyso@Alg), in which the positively charged lysozyme is immobilized on the negatively charged MXene by electrostatic interaction. Due to the presence of MXene, i-Lyso@Alg exhibits good thermal effect, drug release behavior and strong antibacterial activity under near-infrared (NIR) irradiation. The synthesized i-Lyso@Alg can realize not only improvement of lysozyme stability but also photothermal responsive up-regulation for biocatalysis of lysozyme. The excellent antibacterial activities of i-Lyso@Alg were attributed to the photothermally enhanced lysozyme activity, assisted by bacterial death caused by local thermal effect of photothermally activated MXene and the physical damage due to the MXene. In addition, in the infected skin wounds of rats, i-Lyso@Alg + NIR significantly accelerates the wound healing process by inhibiting the expression of inflammatory factors and bacterial (Staphylococcus aureus) infection, and inducing the expression of pro-angiogenic factors and tissue remodeling. Overall, the results of this study introduce a pioneering approach by integrating the unique photothermal properties of MXene with the enzymatic action of lysozyme within an alginate-based hydrogel microsphere. This synergistic system not only advances the frontier of antibacterial wound dressings but also represents a significant step towards effective management of infected wounds, which possesses great potential in clinical treatment of infected wounds.

Exploring the underlying mechanism of interaction of Sulindac with Human hemoglobin and Lysozyme: Multi-spectroscopic, molecular docking, DFT and MD simulation approaches

Contrasting behavior of Nonsteroidal anti-inflammatory drug Sulindac (SDC) towards two different proteins Human hemoglobin (HHb) and Lysozyme (LYS) has been explored by multispectroscopy, molecular docking, DFT and MD Simulation approaches. The fluorescence quenching experiments showed that SDC strongly binds with both proteins in a static manner. Alteration in the secondary structure of HHb and LYS in presence of SDC was further investigated by CD and UV–visible spectroscopy. It is observed that on addition of SDC, the % α-helix of native HHb decreases whereas it increases for LYS-SDC system substantiated the fact SDC stabilized the secondary structure of LYS and destabilized the HHb structure thus, showing the contrasting traits. Experimental results further validated by computational techniques. Molecular docking and DFT were conducted to study the binding sites, nature of interactions and HOMO-LUMO energy gap in both the systems which revealed that the binding of SDC with both proteins resulted in the considerable change in the molecular structure of the former. Additionally, we performed MD simulations to study the structural stability of Lysozyme and Hemoglobin, both unbound and bound to the Sulindac molecule. Sulindac binding showed distinct impacts on protein stability in both proteins, supported by global and essential dynamics.This study not only provides important insights into the binding of SDC with HHb and LYS but also it provides a useful direction in the pharmacology and clinical medicine fields.

A small angle X-ray scattering study comparing protic ionic liquid solvent effects on the solution behaviors of hen egg white and human lysozyme

Proteins demand customized solvents for maintaining their stability. However, the specific impact of different ions on different proteins remains unclear. This study explores the effects of protic ionic liquid (PIL)-based solvents on two structurally similar proteins, namely hen egg white and human lysozyme. By employing size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and capillary SAXS, the effects of protein concentration (3 to 60 mg/mL), IL type (five PILs) and concentration (1, 10 and 20 mol% with water) and protein type on the aggregation behavior and radius of gyration (Rg) of the two lysozymes were studied. HEWL was mostly monomeric in buffer and solubilized beyond 20 mg/mL, while HL displayed slight aggregation in buffer but had increased aggregation at over 10 mg/mL. Further capillary SAXS data show that both proteins became more aggregated at concentrations exceeding 20 mg/mL and over 1 mol% PILs. The nitrate anion generally showed the “salting in” effect, while formate and glycolate anions induced the “salting out” effect on the two proteins. Overall, anion selection and protein solution behaviors at protein concentration of over 20 mg/mL and at 20 mol% ILs can be critical for maintaining the lysozyme stability.

Sustainable combination of ionic liquid and deep eutectic solvent for protecting and preserving of the protein structure: The synergistic interaction of enzymes and eco-friendly hybrid ionic fluids

Hybrid ionic fluids (HIFs) are one of the emerging and fascinating sustainable solvent media, a novel environment-friendly solvent for biomolecules. The HIFs have been synthesized by combining a deep eutectic solvent (DES), an ionic liquid (IL) having a common ion. The stability and activity of hen's egg white lysozyme (Lyz) in the presence of a recently designed new class of biocompatible solvents, HIFs have been explored by UV–visible, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) along with dynamic light scattering (DLS) measurements. This work emphasizes the effect of DES synthesized by using 1:2 choline chloride and glycerol [Glyn], ILs (1-butly-3-methylimidazolium chloride [BMIM]Cl and choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moving forward, we also studied the secondary structure, thermal stability and enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5) M of [BMIM]Cl, [Chn][Ac] ILs, [Glyn] DES and [Glyn][BMIM]Cl (hybrid ionic fluid1) as well as [Glyn][Chn][Ac] (hybrid ionic fluid2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz, whereas the stability and activity are increased by DES and are maintained by HIFs at all the studied concentrations. Overall, the experimental results studied elucidate expressly that the properties of Lyz are maintained in the presence of hybrid ionic fluid1 while these properties are intensified in hybrid ionic fluid2. This work has elucidated expressly biocompatible green solvents in protein stability and functionality due to the alluring properties of DES, which can counteract the negative effect of ILs in HIFs.

Analyzing synergy in combined influence of sorbitol and glycine betaine on protein Stability: Thermodynamic and mechanistic insights

The cells of many organisms have mixtures of organic osmolytes, which raises the question of whether the action of the osmolytes on protein’s stability is dependent or independent in nature. To resolve this query, it is essential to formulate a system that allows the investigation of the ability of osmolytes to fold or unfold a protein. Such stabilizing osmolytes include numerous amino acids and their derivatives, polyols, and methylamine molecules. Polyols and Glycine Betaine (GB) are the most efficient osmo protectants reported till date. The influence of sorbitol and GB on the stability of lysozyme has not been elucidated at low pH conditions. Further, the combined effect of sorbitol and GB under low pH conditions is also unexplored. To elucidate the mechanism of action of GB, sorbitol, and their equimolar combinations, we have studied their effect on lysozyme at different concentrations of the additives at pH 2.1, 2.4, 2.7, and 3.2. Lysozyme's thermal denaturation curves were obtained in the presence of different concentrations of sorbitol, glycine betaine, and their equimolar combinations at different pH. In their combined presence, there is no significant enhancement or decrease in the values of thermodynamic parameters including temperature (T1/2), enthalpy of transition (ΔH), constant-pressure heat capacity change (ΔCp), free energy at 25 ℃ (ΔG0), and preferential solvation (ΔΓ23) at different pH conditions which indicates that there is no effective competition or synergy amongst the osmolytes and each one interacts independently within the studied range of osmolyte concentration and pH. Furthermore, fluorescence spectra and CD spectra demonstrated that the secondary and tertiary structure of lysozyme are unperturbed in the case of glycine betaine and sorbitol at different concentrations and pH.

Investigation of the stability of lysozyme in the presence of immidazolium ionic liquids

Investigation of the stability of lysozyme in the presence of immidazolium ionic liquids

The inhibition of fibril formation of lysozyme by sucrose and trehalose.

The two disaccharides, trehalose and sucrose, have been compared in many studies due to their structural similarity. Both possess the ability to stabilise and reduce aggregation of proteins. Trehalose has also been shown to inhibit the formation of highly structured protein aggregates called amyloid fibrils. This study aims to compare how the thermal stability of the protein lysozyme at low pH (2.0 and 3.5) is affected by the presence of the two disaccharides. We also address the anti-aggregating properties of the disaccharides and their inhibitory effects on fibril formation. Differential scanning calorimetry confirms that the thermal stability of lysozyme is increased by the presence of trehalose or sucrose. The effect is slightly larger for sucrose. The inhibiting effects on protein aggregation are investigated using small-angle X-ray scattering which shows that the two-component system consisting of lysozyme and water (Lys/H2O) at pH 2.0 contains larger aggregates than the corresponding system at pH 3.5 as well as the sugar containing systems. In addition, the results show that the particle-to-particle distance in the sugar containing systems (Lys/Tre/H2O and Lys/Suc/H2O) at pH 2.0 is longer than at pH 3.5, suggesting larger protein aggregates in the former. Finally, the characteristic distance separating β-strands in amyloid fibrils is observed for the Lys/H2O system at pH 2.0, using wide-angle X-ray scattering, while it is not clearly observed for the sugar containing systems. This study further shows that the two disaccharides stabilise the native fold of lysozyme by increasing the denaturation temperature. However, other factors, such as a weakening of hydrophobic interactions and hydrogen bonding between proteins, might also play a role in their inhibitory effect on amyloid fibril formation.

Characterization of caffeic acid-induced changes in the structure and stability of lysozyme: insights from spectroscopy and molecular dynamics simulations

Characterization of caffeic acid-induced changes in the structure and stability of lysozyme: insights from spectroscopy and molecular dynamics simulations

Molecular insights into the interaction between lysozyme and cephalosporins: From multi-spectral experiments to computational simulations

In this study, the interaction mechanism, binding mode, and structural changes between lysozyme and the third-generation cephalosporins (cefoperazone, cefazoxime, and ceftriaxone) were studied by using multi-spectral experiments combined with computational simulations (molecular docking and molecular dynamics). The results of ultraviolet and fluorescence spectra verified that the three kinds of cephalosporins were able to interact with lysozyme by static quenching, among which ceftriaxone had the strongest effect. Thermodynamic parameters and molecular docking results indicated that the hydrophobic force and hydrogen bond played a major role in the bonding process. By circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) analysis, the conformational structure of lysozyme was changed, and its structural stability was reduced after combination with cephalosporins. MD simulation further verified that the structural stability and compactness of lysozyme decreased after the combination with three cephalosporins, while the flexibility and microenvironment hydrophobicity increased. Among them, the stability of lysozyme-ceftriaxone was the worst, the reaction was the most intense, and the microenvironment changed sharply, while the reaction of cefoperazone and cefzoxime was mild, and the microenvironment changed little. Moreover, this property can provide a theoretical basis for further drug synergy.

Structural alterations and inhibition of lysozyme activity upon binding interaction with rotenone: Insights from spectroscopic investigations and molecular dynamics simulation

The pervasive employment of pesticides such as rotenone on a global scale represents a substantial hazard to human health through direct exposure. Therefore, exploring the interactions between such compounds and body macromolecules such as proteins is crucial in comprehending the underlying mechanisms of their detrimental effects. The present study aims to delve into the molecular interaction between rotenone and lysozyme by employing spectroscopic techniques along with Molecular dynamics (MD) simulation in mimicked physiological conditions. The binding interaction resulted in a fluorescence quenching characterized by both dynamic and static mechanisms, with static quenching playing a prominent role in governing this phenomenon. The analysis of thermodynamic parameters indicated that hydrophobic interactions primarily governed the spontaneous bonding process. FT-IR and circular dichroism findings revealed structural alternations of lysozyme upon complexation with rotenone. Also, complexation with rotenone declined the biological activity of lysozyme, thus rotenone could be considered an enzyme inhibitor. Further, the binding interaction substantially decreased the thermal stability of lysozyme. Molecular docking studies showed the binding location and the key residues interacting with rotenone. The findings of the spectroscopic investigations were confirmed and accurately supported by MD simulation studies.

Synergistic Effect of Charged Amino Acids on the Stability and Fibril Formation of Lysozyme

AbstractOsmolytes alter the stability and aggregation of proteins. The effect of mixture of osmolytes is generally not a simple additive of their individual effects. Here, we examine the effect of different mixtures of charged amino acids (AAs) on the stability and fibrillation of lysozyme. Arg, Asp and Glu induce three‐state unfolding. The addition of Lys, Asp and Glu increases the thermal stability of lysozyme, whereas Arg does not alter the stability. The mixture of AAs destabilizes the intermediate, but the overall stability of lysozyme is increased. Arg with other AAs in the ratio of 1 : 0.5 and 1 : 1, and the equimolar ratio of Glu+Asp synergistically increases the stability, whereas the other mixtures counteract the stabilizing effects of each other. The apparent time taken for fibrillation is reduced with lower concentrations of Arg (≤0.75 M) and Glu (0.25 M) and at higher concentrations of Lys (1 M). In the mixed AAs, Arg and Lys with other AAs, and 1 : 1 ratio of Glu+Asp synergistically increases the fibrillation time. These observations suggest that Arg induces more synergistic stabilization and the presence of basic AAs in the mixture increases the fibrillation time compared to acidic AAs which show synergy only at equimolar concentrations.

Exploring the therapeutic potential of recombinant human lysozyme: a review on wound management system with antibacterial.

Lysozyme, a natural antibacterial enzyme protein, possesses the ability to dissolve the cell walls of Gram-positive bacteria, demonstrating broad-spectrum antibacterial activity. Despite its significant potential in treating wound infections and promoting wound healing, its widespread clinical application has yet to be realized. Current research is primarily focused on carrier-based delivery systems for lysozyme. In this review, we discuss four delivery systems that can be employed for lysozyme in wound healing treatment, specifically hydrogels, nanofilms, electrospun fibrous membranes, and modified-lysozyme composite systems. These systems not only enhance the stability of lysozyme but also enable its controlled and sustained release at wound sites, potentially overcoming some of the challenges associated with its direct application. Lastly, we delve into the perspectives and challenges related to the use of these delivery systems, hoping to spur further research and innovation in this promising field.

Lysozyme stability in various deep eutectic solvents using molecular dynamics simulations

The ability of neat deep eutectic solvents (DESs) to influence protein structure and function has gained considerable interest due to the unstable nature of enzymes or therapeutic proteins, which are often exposed to thermal, chemical, or mechanical stresses when handled at an industrial scale. In this study, we simulated a model globular protein, lysozyme, in water and six choline chloride-based DES using molecular dynamics simulations, to investigate the structural changes in various solvent environments, giving insights into the overall stability of lysozyme. Root mean square deviation (RMSD) and root mean square fluctuations (RMSF) of the C-α backbone indicated that most DESs induced a less flexible and rigid lysozyme structure compared to water. The radius of gyration and end-to-end distance calculations pointed towards higher structural compactness in reline and levuline, while the structure of lysozyme considerably expanded in oxaline. Protein-solvent interactions were further analysed by hydrogen bonding interactions and radial distribution functions (RDF), which indicated a higher degree of lysozyme-hydrogen bond donor (HBD) interactions compared to lysozyme-choline hydrogen bonding. Surface area analysis revealed an overall % increase in total positive, negative, donor, and acceptor surface areas in malicine and oxaline compared to water and other DESs, indicating the exposure of a larger number of residues to interactions with the solvent. Reline, levuline, and polyol-based DESs comparatively stabilized lysozyme, even though changes in the secondary/tertiary structures were observed. Communicated by Ramaswamy H. Sarma

The regulation of protein crystallization using choline chloride precipitant and its mechanism

Inorganic salts are the most commonly used precipitants in protein crystallization. However, due to their strong ability to destroy the hydration shell and diffuse electric double layer of proteins, the use of inorganic salts can easily cause protein precipitation. This work studied protein crystallization using organic salt choline chloride (cc) as the precipitant. Compared with NaCl, crystallization, it was found that lysozyme could nucleate at a lower supersaturation and the single-crystal area was significantly enlarged with cc. Even at a high supersaturation of nearly 30, lysozyme crystals could still maintain a complete tetragonal morphology with no urchin-like crystals in cc solution. Furthermore, the average crystal size in cc solution was larger than that in NaCl solution even at a lower supersaturation. In addition, the quantitative efficiency of yield was about equal in cc and NaCl system. Combining Zeta potential, fluorescence spectrum and MD simulation, it was found that cc could interact with lysozyme through hydrogen bond, which weakened the damage of Cl− to diffuse electric double layer of proteins and increased the stability of lysozyme. DLS revealed that the presence of cc could promote the formation of large protein aggregates, which was favorable for crystal growth. In addition, CD spectra and lysozyme activity showed that the presence of cc did not affect the secondary structure and activity of lysozyme. This work proved that cc could be used as a substitute precipitant to grow protein crystals with large size and improve the stability of protein crystallization.

Coarse-grained simulations of lysozyme-silica-nanoparticle corona.

Protein coronas, formed by proteins and nanomaterials, have various applications in the biomedical field. Here, large-scale simulations of protein coronas have been carried out by an efficient mesoscopic coarse-grained method with the BMW-MARTINI force field. The effects of protein concentration, size of silica nanoparticles (SNPs), and ionic strength on the formation of lysozyme-SNP coronas are investigated at the microsecond time scale. Simulations results indicate that (i) an increase in the amount of lysozyme is favorable for the conformation stability of adsorbed lysozyme on SNPs. Moreover, the formation of ringlike and dumbbell-like aggregations of lysozyme can further reduce the conformational loss of lysozyme; (ii) for a smaller SNP, the increase of protein concentration exhibits a greater effect on the adsorption orientation of lysozyme. The dumbbell-like lysozyme aggregation is unfavorable for the stability of lysozyme's adsorption orientation; however, the ringlike lysozyme aggregation can enhance the orientation stability; (iii) the increase of ionic strength can reduce the conformation change of lysozyme and accelerate the aggregation of lysozyme during their adsorption process on SNPs. This work provides some insights into the formation of protein coronas and some valuable guidelines for the development of novel biomolecule-NP conjugates.

Stabilization of lysozyme in aqueous dispersion of graphene oxide sheets

This study examines the effect of surface oxygen groups upon ability of graphene oxide (GO) sheets in suppressing the fibrillation of lysozyme (LYZ). Graphite was oxidized using 6 and 8 wt equivalents of KMnO4, and as produced sheets were abbreviated as GO-06 and GO-08, respectively. Particulate characteristics of sheets were characterized by light scattering and electron microscopic techniques, and their interaction with LYZ was analysed by circular dichroism (CD) spectroscopy. After ascertaining acid-driven conversion of LYZ to fibrillary form, we have shown that the fibrillation of dispersed protein can be prevented by adding GO sheets. Inhibitory effect could be attributed to binding of LYZ over the sheets via noncovalent forces. A comparison between GO-06 and GO-08 samples showed superior binding affinity of the latter. Higher aqueous dispersibility and density of oxygenated groups in GO-08 sheets would have facilitated the adsorption of protein molecules, thus making them unavailable for aggregation. Pre-treatment of GO sheets with Pluronic 103 (P103, a nonionic triblock copolymer), caused reduction in the adsorption of LYZ. P103 aggregates would have rendered the sheet surface unavailable for the adsorption of LYZ. Based on these observations, we conclude that fibrillation of LYZ can be prevented in association with graphene oxide sheets.

Study of stability, kinetic parameters and release of lysozyme immobilized on chitosan microspheres by crosslinking and covalent attachment for cotton fabric functionalization

Chitosan microspheres are used in the biotechnological, biomedical, and enzyme immobilization fields. This study synthesized lysozyme-chitosan microspheres by crosslinking and covalent attachment. The synthesis conditions were adjusted, and spherical particles content lysozyme were obtained and characterized by Scanning electron microscopy (SEM) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Activity and stability assays were conducted to determine the immobilization method's influence, and the kinetics parameters were calculated. The enzymatic activity recovery yields of 65.8% and 20.0% were obtained for crosslinking and covalent attachment, respectively. Surface lysozyme-chitosan microspheres' enzymatic activity was obtained by covalent attachment. By the crosslinking method, surface and into particle enzyme activity were observed. The functionalization of cotton fabric was achieved, and the impregnation slightly modified cotton fabric properties such as resistance and permeability. Finding antimicrobial properties against bacteria S. aureus and P. aeruginosa, with a growth of 49.12% and 65.31%, respectively, were obtained.

Micro-Scale Vacuum Compression Molding as a Predictive Screening Tool of Protein Integrity for Potential Hot-Melt Extrusion Processes.

Hot-melt extrusion (HME) is used for the production of solid protein formulations mainly for two reasons: increased protein stability in solid state and/or long-term release systems (e.g., protein-loaded implants). However, HME requires considerable amounts of material even at small-scale (>2 g batch size). In this study, we introduced vacuum compression molding (VCM) as a predictive screening tool of protein stability for potential HME processing. The focus was to identify appropriate polymeric matrices prior to extrusion and evaluation of protein stability after thermal stress using only a few milligrams of protein. The protein stability of lysozyme, BSA, and human insulin embedded in PEG 20,000, PLGA, or EVA by VCM was investigated by DSC, FT-IR, and SEC. The results from the protein-loaded discs provided important insights into the solid-state stabilizing mechanisms of protein candidates. We demonstrated the successful application of VCM for a set of proteins and polymers, showing, in particular, a high potential for EVA as a polymeric matrix for solid-state stabilization of proteins and the production of extended-release dosage forms. Stable protein-polymer mixtures with sufficient protein stability after VCM could be then introduced to a combination of thermal and shear stress by HME and further investigated with regard to their process-related protein stability.