Activity Of Hen Egg White Lysozyme Research Articles

Pharmacological Modulation of Host Immunity with Hen Egg White Lysozyme (HEWL)-A Review.

In the 100 years since its discovery, lysozyme has become an important molecule, both as model for studies in different fields and as a candidate for the therapy of various pathological conditions. Of the dozens of known lysozymes, in this review we focus on one in particular, lysozyme extracted from hen egg white (HEWL), and its interaction with the immune system when it is administered orally. Experimental data show that there is an axis that directs immune system activation from GALT (gut-associated lymphoid tissue) and the intestinal lymphocyte clusters. Although a contribution of peptidoglycans from digestion of the bacterial cell wall in the intestinal lumen cannot be excluded, immune stimulation is not dependent on the enzymatic activity of HEWL. The immune responses suggest that HEWL is able to recover from immunodepression caused by tumor growth or immunosuppressants, and that it also improves the success of chemotherapy. The positive results obtained in a small Phase 2 study in patients, the ease of oral administration of this protein, and the absence of adverse effects suggest that HEWL may play an important role in all diseases where the immune system is weakened or where its enhancement plays a critical role in the resolution of the pathology.

Binding of bioactive esculin and esculetin with hen egg white lysozyme: Spectroscopic and computational methods to comprehensively elucidate the binding affinities, interacting forces, and conformational alterations at molecular level

The binding of bioactive esculin and esculetin with lysozyme have been studied using multi-spectroscopic and computational methods. Intrinsic fluorescence of lysozyme was quenched via static quenching mechanism by these ligands and binding was observed to be moderate in nature. Structural alteration of the protein was noted upon ligand binding and enzymatic activity of lysozyme was also influenced in vitro. • Coumarin derivatives, esculin and esculetin quenched intrinsic fluorescence of HEWL through a static quenching mechanism. • Trp residue occupied a more hydrophobic environment upon interactions with esculin and esculetin. • The α-helical content of HEWL decreased in presence of ligands which could have affected the enzymatic activity of HEWL. • Δ G ° values were observed to be negative which indicated complex formation occurred spontaneously. Multi-spectroscopic and computational methods were employed to investigate the binding mechanism of esculin and esculetin with hen egg white lysozyme (HEWL). UV–vis and fluorescence spectroscopy proved static quenching mechanism was involved in the fluorescence quenching process. The binding constant ( K b ) determined for HEWL-esculin/esculetin complex was in the order of 10 4 M −1 . A negative Δ H ° [-(14.19 ± 2.56) kJ mol −1 ] and positive Δ S ° [+(40.98 ± 3.10) J K −1 mol −1 ] for HEWL-esculin complex suggested the presence of hydrophobic forces and hydrogen bonding. Whereas positive values of both Δ H° [+(9.96 ± 4.26) kJ mol −1 ] and Δ S ° [+(120.05 ± 14.14) J K -1 mol −1 ] for HEWL-esculetin complex indicated the presence of hydrophobic forces in the binding process. Blue shifts were observed for HEWL-esculin/esculetin complexes on carrying out synchronous and three-dimensional (3-D) fluorescence. A red edge excitation shift (REES) of 3 nm was observed for HEWL, whereas 6 and 5 nm for HEWL-esculin and HEWL-esculetin complexes, respectively, were also measured. Circular dichroism (CD) studies revealed that the % α-helical content of HEWL (25.56 ± 2.24) decreased in the presence of esculin (17.96 ± 1.54) and esculetin (19.90 ± 2.14). Amide I peak of HEWL, determined from Fourier transform infrared (FTIR) studies, shifted from 1657 cm −1 to 1654 cm −1 and 1652 cm −1 upon complexation with esculin/esculetin, respectively. The binding distance ( r ) for HEWL-esculin (4.40 ± 0.14 nm) and HEWL-esculetin (4.66 ± 0.11 nm) complexes were determined from fluorescence resonance energy transfer (FRET) theory. Thermal analysis resulted in a lower T m value for the protein–ligand complexes (71.8 °C) compared to the native protein (73.3 °C). Molecular docking and molecular dynamic (MD) simulations provided insight into the binding affinity and flexibility of HEWL upon complexation, respectively. In the presence of esculin and esculetin, a change in enzyme activity of HEWL was observed.

Recombinant expression of hen egg white lysozyme with the assistance of xylanase fusion partner in Pichia pastoris

ABSTRACT Due to its bacteriolytic activity, hen egg white lysozyme (HEWL) is widely used in the feed, food, and pharmaceutical industries. However, its application is hindered by low protein expression levels in microbial expression systems. In this work, a novel fusion protein expression strategy was proposed for increasing the expression level of HEWL. First, HEWL, fused with a highly expressed fusion protein partner xylanase XynCDBFV, is expressed in Pichia pastoris. Secondly, a linker including endogenous protease cleavage sites was introduced between two fusion proteins in order to separate them directly during the secretion process. Finally, the results show that the supernatant of XynCDBFV-HEWL has a higher HEWL expression level and activity compared with HEWL only. It should be noted that the expression of HEWL reaches to about 3.5 g/L, and the activity of HEWL against Micrococcus lysodeikticus reaches to 1.50 × 105 U/mL in a fed-batch fermentation, which is currently the highest level of recombinant expression of an egg white-derived lysozyme. Taken together, we acquired bioactive HEWL for large-scale recombinant production in Pichia pastoris using a novel fusion protein expression strategy, which could then be used for a variety of applications.

Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Molecular recognition of two bioactive coumarin derivatives 7-hydroxycoumarin and 4-methyl-7-hydroxycoumarin by hen egg white lysozyme: Exploring the binding mechanism, thermodynamic parameters and structural changes using multispectroscopic and computational approaches

Multispectroscopic and computational methods of exploring the interaction between a carrier protein and therapeutic compounds provide a preliminary investigation into establishing the efficacy of such compounds. Here, two coumarin derivatives, 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC), were selected to carry out numerous biophysical interaction studies with a model carrier protein, hen egg white lysozyme (HEWL). Fluorescence spectroscopy studies conducted between HEWL and 7-HC/4-Me-7-HC revealed the binding constants (K b) were in the range of 104 M−1, indicating a moderate nature of binding. The quenching mechanism observed during complexation process was an unusual static quenching due to the effect of temperature on the rate constant. Thermodynamic parameters revealed a positive ΔH and ΔS for HEWL-7-HC/4-Me-7-HC, indicating hydrophobic forces played a dominant role in the interaction process. FRET studies suggested a possible non-radiative energy transfer from the donor (HEWL) to the acceptor (coumarins). Molecular docking studies revealed the interaction of 7-HC/4-Me-7-HC with intrinsic fluorophores, Trp63 and Trp108, Trp108 being an essential residue for binding as proven by molecular dynamic (MD) simulation. MD simulation studies also indicated conformational stability gained by HEWL upon interaction with 7-HC and 4-Me-7-HC. The microenvironment surrounding the Trp residues showed a significant Stoke’s shift on carrying out 3-D fluorescence. CD studies revealed a decrease in the alpha helical content of HEWL upon interacting with the ligands. Enzymatic assay conducted for HEWL in the presence of 7-HC/4-Me-7-HC saw an increase in the activity of HEWL, suggesting a change in structural conformation and stability of the protein, altering its activity. Communicated by Ramaswamy H. Sarma

Mechanistic study of lysozyme glycation by fructose and modulation by curcumin derivatives

Glycation can affect the protein conformational changes and direct involvement of advanced glycation end products (AGEs). These products are major cause of many diseases and disorders, such as diabetes and its complications. Curcumin (diferuloymethane) and its derivatives have been exhibited an inhibitory effect on modulation of glycation and oxidative stress. In this work, it is attempted to study the effects of fructose (Fru) on structure and activity of Hen Egg-White Lysozyme (HEWL) as a model protein and modulating by curcumin (Cur) and its derivative, dimethyl curcumin (DMC) via various techniques, such as enzymatic activity assay, fluorescence spectroscopy and ThT assay, circular dichroism spectropolarimetry, transmission electron microscopy (TEM) as well as molecular dynamic simulation and docking studies. The results indicate that Cur and DMC preserve secondary and tertiary structures of lysozyme and they prevent upper and lower loop locking and promote enzymatic activity somewhat by prohibiting the binding of lysine-97 to the lower movable loop of the active site. Moreover, simulation results show a good correlation with the experimental data.

Biogenic synthesis, in-vitro cytotoxicity, esterase activity and interaction studies of copper oxide nanoparticles with lysozyme

Herein, we have shown the eco-friendly and low-cost synthesis of biogenic copper oxide nanoparticles (CuONPs) from Delonix regia (D. regia) leaf extract in aqueous medium. The formation of biogenic CuONPs was characterized by ultraviolet (UV)-visible, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) as well as transmission electron microscopy (TEM). Characterization of green synthesized nanoparticles demonstrated a UV–visible peak at 273 nm. Particle size was analysed from TEM monograph in the range of 69–108 nm which validates formation of nanoparticles. The in-vitro anticancer activity was done against A549 (IC50 = 4.70 μg/mL) and MCF7 (IC50 = 3.77 μg/mL) cell line. The biocompatibility assay of these nanoparticles was done on HEK–293 cell line (normal non-cancerous cells). Moreover, the effect of CuONPs on the lytic activity of HEWL was evaluated against Micrococcus lysodeikticus. Further, the interaction of CuONPs with hen egg white lysozyme (HEWL) at different temperature was investigated by steady state fluorescence spectroscopy. UV–visible and far ultra violet-circular dichroism (far UV-CD) spectroscopies were also employed to study the interaction of CuONPs with HEWL. All the results supported each other and show efficient binding between HEWL and CuONPs. The fluorescence emission study revealed that CuONPs quenches the fluorescence of HEWL involving dynamic quenching. The various binding parameters and structural disparity of HEWL in the presence of synthesised CuONPs were also calculated.

A physicochemical, structural, microbiological and kinetic study of hen egg white lysozyme in complexes with alginate and chitosan

Complexes of hen egg white lysozyme (HEWL) with alginate (ALG) and chitosan (CS) (natural biodegradable polysaccharides) are important as antimicrobial agents in oral, intranasal and buccal administration. The HEWL–ALG, HEWL–ALG–Ca, [HEWL–ALG]–CS, and [HEWL–ALG–Ca]–CS complexes have been investigated using a combination of physicochemical methods (fluorescent spectroscopy, circular dichroism, microscopy, turbidimetry, microbiology, in vitro cytotoxicity assay). The complexes obtained were non-toxic negatively charged microparticles with high HEWL entrapment efficiency. The [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes were found to have the altered tertiary structure of HEWL (and/or changes in the polarity of the microenvironment of Trp residues in HEWL molecules). The HEWL entrapment in all studied complexes was carried out with retention of the HEWL activity. Under the long–term storage conditions (1.5 years at 4 °C), Ca++ cations and CS stabilized HEWL. Under physiological conditions, the [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes demonstrated sustained release of HEWL and resistance to protease action. The [HEWL–ALG]–CS and [HEWL–ALG–Ca]–CS complexes were highly active against Gram-negative Escherichia coli cells. The data obtained are important in the development of antibacterial drugs based on lytic enzymes.

Enhancement of Lytic Activity of HEWL Adsorbed on Biochar by the Optimization of Adsorption Conditions

We have found that HEWL (hen egg white lysozyme) is effectively immobilized on biochar through adsorption, and the amount of HEWL adsorbed on biochar is strongly dependent on the pH value of adsorption solutions. The structure of HEWL adsorbed on biochar changed by the pH value of adsorption solutions and became similar to that of native HEWL at acidic pH value, although the structure of HEWL solubilized in the solution was the same in the range between pH 4 and 9. On the other hand, the lytic activity of biochar-adsorbed HEWL toward Micrococcus lysodeikticus was markedly influenced by the pH value of adsorption solutions and increased with decreasing the pH value. Moreover, the addition of KCl to adsorption solutions enhanced the lytic activity of biochar-adsorbed HEWL.

Investigating the structural and conformational behavior of HEWL in the presence of iron metallosurfactant and sodium oleate metallo-catanionic aggregates

Surface-active enzymes and surface-active aggregates developed from metallosurfactant (MS), are under investigation for their complexation in an aqueous medium with the aim of development of mixtures that have potential applications in industries. Herein, the surface-active enzyme, hen egg white lysozyme (HEWL) and metallo-catanionic aggregates of hexadecyltrimethylammonium iron (II) trichloride, FeCTAC I and sodium oleate (SO) have been used. Before going for the investigation for the structural integrity of the protein, we have focused to deduce the morphology of metallo-aggregates at two different molar ratios of FeCTAC I:SO namely 70:30 and 30:70. These two molar ratios of FeCTAC I:SO show very interesting behavior by developing different morphologies namely, micelles in 70:30 and vesicles in 30:70 molar ratio, as reported from SAXS data. Further, the changes induced in the integrity of the protein by the metallo-catanionic aggregates have been potentially studied. The protein's integrity has been untangled by SDS-PAGE, UV/Visible, fluorescence spectroscopy and HEWL activity. The forces that are responsible for all the changes in the integrity of the protein are examined by extrinsic fluorescence study. Further, the effect of temperature on the activity of HEWL in the presence and absence of metallo-catanionic aggregates has also been elucidated.

Optical characterization and tunable antibacterial properties of gold nanoparticles with common proteins

The interaction of three proteins, viz. Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hen Egg White Lysozyme (HEWL) with gold nanoparticles (GNPs) is investigated using surface plasmon resonance (SPR) spectroscopy, fluorescence spectroscopy and circular dichroism (CD). Size and morphology of the samples was established using Transmission Electron Microscopy (TEM) and stability studies was established using zeta potential analysis. The stability of protein-GNP complex was found to be greater than that of individual protein as well as individual GNPs. Also HEWL-GNP complex was more stable compared to the other protein complexes. Absorbance of proteins increases with increase in gold nanoparticle concentration due to the extension of peptide strands of protein and decrease in hydrophobicity of gold nanoparticles. A ground state complex is also formed which is evident from the moderate shift observed in the absorbance peaks. Apparent association constant was also determined from the absorption spectra and was found to be maximum for HEWL and minimum for HSA. Gold nanoparticles were found to act as quenchers and reduced the protein fluorescence intensity. Binding constant and number of binding sites were found to be maximum for HEWL and minimum for HSA. The temperature dependent fluorescence studies were also performed to calculate the thermodynamic parameters and to determine the nature of interaction between the proteins and gold nanoparticles. The circular dichroism studies elucidate the reason behind the maximum binding for HEWL and minimum binding for HSA. TGA analysis determined the thermal stability of the samples. Fluorescence lifetime studies indicate static quenching of proteins. Antibacterial activity of protein-gold nanoparticles was studied against four pathogens, viz. Bacillus pumilus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. HEWL exhibits a tunable antimicrobial activity against Pseudomonas aeruginosa due to the maximum binding of HEWL with gold nanoparticles. The study proposes a novel method for adjusting the antibacterial activity of HEWL against Pseudomonas aeruginosa when the resistance of this pathogen is a major issue in the chemotherapy of many infectious diseases. Thus the combination therapy of protein-gold nanoparticles could prove to be a new approach in medical field in the near future.

Bacopa monnieri inhibit hen egg white lysozyme fibrillation and help in retaining its activity at acidic condition

Inhibiting protein misfolding and aggregation is crucial for the treatment of several amyloidoses. Though various types of synthetic drugs are being explored as therapeutic agents, herbal extracts are better alternative owing to their natural origin, higher bioavailability and improved biosafety characteristics. In the present study, we demonstrate that night long (∼12 hr) preincubation of hen egg white lysozyme (HEWL) with Bacopa monnieri (brahmi) at neutral pH, impede the aggregation and fibrillation of protein at pH 2.0. Employing different biophysical techniques such as static and dynamic light scattering, Thioflavin T (ThT) assay, sedimentation assay and atomic force microscopy (AFM), we show that brahmi inhibit the HEWL aggregation in concentration dependent manner. 8-anilino-1-naphthalene sulfonate (ANS) fluorescence reveals that brahmi masks the exposure of hydrophobic domain of lysozyme. Significant recovery of enzymatic activity of HEWL in the presence of brahmi at pH 2.0 is salient feature of this work. Nearly 90% recovery of catalytic activity of lysozyme after 216 hr (9 days) of incubation indicate that interaction of HEWL-brahmi stabilizes the native structure of protein thus enhancing the activation energy barrier for protein misfolding and subsequent aggregation. Our findings show that brahmi could be promising alternative for the therapies of several protein misfolding disorders.Communicated by Ramaswamy H. Sarma

HEAVY METAL-INDUCED INHIBITION OF ENZYMATIC ACTIVITY IN HEN EGG WHITE LYSOZYME

Objective: The objective of this study was to determine the content of chromium (Cr), nickel (Ni), cadmium (Cd), boron (B), mercury (Hg), and lead (Pb) in chicken hen eggs in hen’s eggs and to evaluate their influence on the enzymatic activity of hen egg white lysozyme (HEWL).Methods: In this study, we have determined the content of Cr, Ni, Cd, B, Hg, and Pb in chicken hen eggs using absorption atomic (AA) method. The residual enzymatic activity of HEWL, HEWL was evaluated using spectrophotometric method.Results: The levels of Cr, Ni, Cd, B, Hg, and Pb in hen eggs were 1.8300, 1.1300, 0.5350, 0.5200, 0.0025, and 0.0021 mg/kg, respectively. The inhibition of enzymatic activity of HEWL, by exposure to heavy metals was studied. We found that Cr, Ni, Cd, B, Hg, and Pb produced a loss of enzymatic activity. Cr III was the metal with the highest reduction of HEWL enzymatic activity. 0.01 M of Cr III was able to reduce HEWL enzymatic activity levels up to 49% after 24 h of exposure to this metal. An exposure of 0.05 M of Cr III reduced HEWL enzymatic activity to 1%. 24 h exposure to 0.01 M of Cd presented a reduction of 37% of HEWL enzymatic activity and 0.05 M of Cd resulted in only 75% of HEWL enzymatic residual activity.Conclusions: Content of heavy metals in hen egg from Tungurahua region was high. Cr, Ni, Cd, B, Hg, and Pb produced a loss of enzymatic activity of HEWL.

Effect of cations and anions of ionic liquids on the stability and activity of lysozyme: Concentration and temperature effect

Herein, we have shown the effect of cations and anions of three different ionic liquids (ILs); 1-Decyl-3-methylimidazolium chloride [DMIM][Cl], 1-Butyl-1-methylpyrrolidinium tetrafluoroborate [BMP][BF4] and 1-Butyl-1-methylpyrrolidinium bromide [BMP][Br] on the stability and activity of hen egg white lysozyme (HEWL). The spectroscopic techniques; fluorescence, time resolved fluorescence, UV–visible, CD spectroscopy and molecular docking method were applied to investigate the differential behavior of HEWL in the aqueous solution of these ILs. Fluorescence results showed that imidazolium-based IL binds more strongly than pyrrolidinium based ILs with HEWL. The docking results suggested that these ILs bind at the active site of HEWL mainly through electrostatic, hydrophobic and van der Waals interactions. Results revealed that depending upon cations and anions, these ILs exert concentration dependent effect on HEWL. The conformational stability of HEWL is maintained in [BMP][BF4] and [BMP][Br] ILs while it decreases with increasing concentration of [DMIM][Cl]. A slight increase of 1 °C in melting temperature (Tm) of HEWL is obtained in presence of [BMP][BF4] and [BMP][Br] ILs while a much decrease of about 16.9 °C in Tm is obtained in presence of [DMIM][Cl]. Surprisingly, different effects of these ILs were observed on the activity of HEWL against Gram-positive bacteria; Micrococcus lysodeikticus. The activity of HEWL increases with increasing concentration of [BMP][BF4] and [DMIM][Cl] up to certain concentration range while it decreases with increasing concentration of [BMP][Br]. Comparatively, higher activity is obtained in presence of [DMIM][Cl] than [BMP][BF4].

Deciphering the Interaction of 5,7‐Dihydroxyflavone with Hen‐Egg‐White Lysozyme through Multispectroscopic and Molecular Dynamics Simulation Approaches

AbstractIn recent times, research based on the bio‐active flavonoid, chrysin has shown its potential therapeutic applications such as anti‐diabetic, neuroprotective, anti‐asthmatic, anti‐depressant, etc. In order to understand its molecular recognition process, we studied its interaction with hen egg white lysozyme (HEWL) at physiological conditions. Chrysin (5,7‐Dihydroxyflavone) was able to quench the intrinsic fluorescence of HEWL through static quenching mechanism and the binding constant (Kb) was found to be (4.390±0.088 ×104) M−1 at 298 K and it decreased with the increase in temperature. The value of thermodynamic parameters such as ΔH (‐17.154±0.872) kJ mol−1 and ΔS (+31.267±3.151) J K−1 mol−1 indicated that hydrogen bonding and hydrophobic forces played a central role in the complexation process. Alteration in the Trp micro‐environment was induced due to the incorporation of chrysin in the binding site as indicated by synchronous and three dimensional (3D) fluorescence studies. FT‐IR and CD studies revealed that the secondary structure of HEWL was altered on chrysin binding, which resulted in an increase in the α‐helical stability of the protein. Chrysin inhibited the enzymatic activity of HEWL against M. lytus. Molecular docking and molecular simulation studies revealed that hydrogen bonding and hydrophobic forces played a vital role in complexation process. This study provides substantial insight into the binding of a bio‐active flavonoid (chrysin) with a carrier protein (HEWL).

Exploring the non-covalent binding behaviours of 7-hydroxyflavone and 3-hydroxyflavone with hen egg white lysozyme: Multi-spectroscopic and molecular docking perspectives

The interactions of bio-active flavonoids, 7-hydroxyflavone (7HF) and 3-hydroxyflavone (3HF) with hen egg white lysozyme (HEWL) have been established using differential spectroscopic techniques along with the help of molecular docking method. The characteristic dual fluorescence of 3HF due to the excited intramolecular state proton transfer (ESIPT) process is altered markedly upon binding with HEWL. Both the flavonoids quenched the intrinsic fluorescence of HEWL through static quenching mechanism while the binding affinity of 7HF was found to be greater than 3HF under experimental conditions. The binding constant (Kb) values were estimated to be in the order of 104 M−1 and decreased with the rise in temperature. The contributions of the thermodynamic parameters (ΔH° and ΔS°) revealed that hydrophobic forces along with hydrogen bonding played a crucial role in the interaction of HEWL with 7HF and 3HF respectively and this finding was aptly supported by the molecular docking studies. The donor (HEWL) to acceptors (7HF and 3HF) binding distances were calculated using the Föster's theory. The phenomena of blue shifting of the emission maxima of the residues indicated the increase in hydrophobicity around the Trp micro-environment upon addition of the flavonoids was observed from synchronous and 3D fluorescence measurements whereas REES study indicated the decrease in mobility of the Trp residues upon addition of the ligands. The CD, FTIR and thermal melting studies indicated the alteration in the structural stability of HEWL on ligand binding and it was found that the % α-helical content decreased on complexation with 7HF and 3HF respectively as compared to native state. The flavonoids were found to inhibit the enzymatic activity of HEWL. The molecular docking results and accessible surface area (ASA) calculations revealed that the flavonoids bind within the active site of HEWL. The negative ΔG° values obtained from experimental and molecular docking studies indicate the spontaneity of the interaction processes.

Unraveling the novel effects of aroma from small molecules in preventing hen egg white lysozyme amyloid fibril formation.

This study investigated for the first time the molecular effectiveness of 'aroma' from three small molecules including a phenol (phenyl ethyl alcohol; PEA) and an aldehyde (cinnamaldehyde; Cin) both containing an aromatic ring, and a diamine (N,N,N,N'- Tetramethylethylenediamine; TEMED) at two different amounts (small; S and large; L) in preventing hen egg white lysozyme (HEWL) amyloid fibril formation using Thioflavin T and Nile red fluorescence assays, circular dichroism spectroscopy, SDS-polyacrylamide gel electrophoresis, atomic force microscopy, dynamic light scattering and HEWL activity test. Interestingly, the results revealed that (1) the aroma of PEA, identified as an active constituent of Rosa damascena, prevented fibril formation since PEA-L was able to trap the oligomeric form of HEWL in contrast to PEA-S where protofibrils but not mature fibrils were formed; (2) Cin, previously shown to prevent fibril formation in the liquid form, was also shown to do so in the aroma form by producing protofibrils and not mature fibrils in both Cin- L and Cin-S aroma forms and (3) the aroma of TEMED-L was able to retain HEWL’s native structure completely and prevented both aggregation and fibril formation, while TEMED-S prevented HEWL fibril formation and instead directed the pathway towards amorphous aggregate formation. Furthermore, the ability to trap oligomeric species (by PEA-L aroma) is of great importance for further research as it provides routes for preventing the formation of toxic oligomeric intermediates along the fibrillation pathway. Last but not least, the novelty of this in vitro study on the effect of aroma at the molecular level with a unique experimental set-up using HEWL as a model protein in assessing amyloid fibril formation paves the way for more and detailed studies on the importance of aroma producing molecules and their effects.

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV–vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300K, the value of binding constant (Kb) of HEWL-NG complex (5.596±0.063×104M−1) was found to be greater than that of HEWL-NR complex (3.404±0.407×104M−1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2+, Cu2+ and Fe2+) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

Effects of metal oxide nanoparticles on the structure and activity of lysozyme

We investigated the effects of nanoparticles (NPs) on the structure and activity of hen egg-white lysozyme (HEWL) using CeO2 and ZnO NPs. Our results showed that CeO2 NPs triggered the transition of lysozyme secondary structure from α-helix to β-sheet. CeO2 NPs also induced the hydrophobic region of lysozyme to become exposed to the solvent. In contrast, the secondary structure content and hydrophobic region of lysozyme were only slightly changed in the case of ZnO NPs. In addition, the activity of the lysozyme was observed to decrease upon adsorption on CeO2 NPs, whereas the effect of ZnO NPs on activity was negligible. The glutaraldehyde crosslinking results indicated that the percentage of the dimeric form of lysozyme was greatly enhanced by the addition of both NPs. Furthermore, the adsorption capacity, degree of favorability of adsorption, and surface heterogeneity for CeO2 NPs were found to be greater than those on ZnO NPs. Given that CeO2 NPs exhibit a higher surface area/mass than ZnO NPs, the surface concentration of lysozyme on CeO2 NPs was lower than that on ZnO NPs. This result suggested that more direct interactions were involved between CeO2 NPs and lysozyme, thereby leading to a more significant effect. Moreover, higher surface curvatures may also cause destruction of lysozyme’s structure and thus affect its activity. In addition, taking into account the surface properties and protein properties, the Toth adsorption model along with the generated site energy distribution was further used to exaplain the difference between the results (e.g., structure, stability, and activity) of lysozyme adsorption on CeO2 and ZnO NPs. The results reported here may aid in better understanding the beneficial or harmful impacts of nanoparticles on the biological systems.

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities

ObjectiveTo investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. DesignXylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. ResultsWhile xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. ConclusionsXylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities.