Activity Of Egg White Lysozyme Research Articles

Kosmotropic and chaotropic effect of biocompatible Fe3O4 nanoparticles on egg white lysozyme; the key role of nanoparticle-protein corona formation

Accurate understanding of the interaction between different types of nanoparticles and proteins is very important in various fields, especially in Nano-biotechnology. Concentration-dependent nanoparticles can cause structural changes in proteins. There are several procedures to increase the efficiency of nanoparticles in these interactions. In this study, the effect of magnetic nanoparticles (MNPs) including Fe3O4 and Fe3O4@RA (Rosmarinic acid coated with Fe3O4) was estimated on the stability and activity of egg white Lysozyme (EWL). The synthesis of MNPs was done using co-precipitation method; then they were characterized by X-ray diffraction (XRD), Fourier-transform infrared (FT-IR), vibrating sample magnetometer (VSM), CHNS elemental analysis, dynamic light scattering (DLS) and field emission scanning electron microscopy (FE-SEM). The results of fluorescence analysis showed that both MNPs can have two opposite effects on the tertiary structure of EWL, depending on their concentration. Accordingly, at the concentrations below a concentration threshold (0.0083 and 0.0067 for Fe3O4 NPs and Fe3O4@RA, respectively), they improve the structure of the EWL (kosmotropic-like effect) and at the concentrations above it, they gradually cause its degradation (chaotropic-like effect). Changes in the protein activity against Micrococcus luteus bacteria based on UV analysis data were also fully consistent with the fluorescence analysis. In order to study the secondary structure of EWL, CD Spectra showed an increase in the helicity of EWL after interaction with both MNPs (3.7% and 1.1% increase in the helicity for 0.02 and 0.1 mg.mL−1 of Fe3O4 respectively, and also 5.5% and 2% for 0.02 and 0.1 mg.mL−1 of Fe3O4@RA respectively) and a decrease in this factor after interaction with pure RA (0.9% and 4.1% decrease in the helicity for 0.02 and 0.1 mg.mL−1 of pure RA respectively).

ANALYSIS OF THE COMPONENTS OF TABLET MIXTURES WITH IMMOBILIZED LYSOZYME

Aim. Study of the effect of the tablet mixtures components with immobilized lysozyme on the determinationactive substances for dietary supplementcreation. Methods. Activity of egg white lysozyme was determined bythe bacteriolytic method with Micrococcus lysodeikticus2665 as substrate. Protein content was determined by the Lowry-Hartree method, quercetin using zirconium (IV) chloride, chlorhexidinedigluconate by the reaction with α-naphtholand chromato-mass-spectrometrically. Tablet mixtures were prepared using wet granulation technology. Results. Quercetin and lysozyme are quantified in the composition of tablet mixtures by the spectrophotometry methods. It was shown that chlorhexidine digluconate is determined quantitatively in the absence of other components, while their addition complicates its quantitative analysis. So, in the presence of lysozyme, 80.22% of analyte is determined, quercetin – 90.52%, mannitol – 89.82%, lactose – 30.87%, sucrose by 40.97%, polyvinylpyrrolidone – 91.34%, calcium citrate – 52.99%. Conclusions. The methods for the quantitative analysis of the active components of tablet mixtures: lysozyme, quercetin and chlorhexidine digluconate are determined. It was found that lysozyme, quercetin, mannitol, polyvinylpyrrolidone, sucrose and lactose interfere with the quantitative determination of chlorhexidine digluconate by spectrophotometry and the chromatographic mass spectrometry methods in both one-factor and multivariate experiments.

LYSOZYME-CONTAINING PREPARATION “ARTIFICIAL TEARS”: OBTAINING, PROPERTIES

Aim. The development and study of the chemical and physical properties of eye drops with lysozyme as a perspective drug for the treatment of the syndrome of “dry eye.” Methods. Activity of egg white lysozyme was determined by bacteriolytic method (with Micrococcus lysodeikticus ATCC № 4698 as substrate). Protein content was determined by the Lowry-Hartree method, determination of osmolality of the drug was carried out by osmometer – Tear Lab Osmolarity System. Results. A perspective preparation “artificial tears” for dry eye therapy in form of eye drops with lysozyme was developed. There were noted the complete preservation of bacteriolytic activity of the included enzyme (38200 ± 2000 U/ml). The developed drops have the same values of lacrimal fluid viscosity, density and osmolality. Dextran and hydroxypropylmethyl cellulose stabilized lysozyme in the formulation, with usage of sterilizing filtration stored at low temperatures (0–4 °C) for 12 months with 96.4% retention of the original bacteriolytic activity. Conclusions. We propose a perspective preparation “artificial tears” for dry eye therapy in form of eye drops with lysozyme as a natural antibacterial component of tears. We have noted the high bacteriolytic activity of the included lysozyme (38200 ± 2000 U/ml). It was shown, that developed drops have the same values of lacrimal fluid viscosity, density and osmolality. It is found that dextran stabilized lysozyme in the formulation and hydroxypropylmethylcellulose, with usage sterilizing filtration stored at low temperatures (0–4 °C) for 12 months with 96.4% retention of the original bacteriolytic activity.

Stability of Egg White Lysozyme in Combination with Other Antimicrobial Substances

The effects of pH, ionic strength (I), and selected antimicrobial substances on the lytic activity of egg white lysozyme over an extended storage period of 30 d were studied by observing the rate of clearing of a cell suspension of Micrococcus lysodeikticus. During prolonged storage, lysozyme activity remained relatively stable at pH 7 and ionic strength <0.10, whereas lower activity (P < 0.05) was observed at pH 9 and ionic strength >0.14. Lysozyme activity was highly stable (i.e., maintained over 90% of lytic activity from day 1 to day 30) in solutions of 1.0% sodium chloride, 100 ppm sodium nitrite, 4.0% ethanol, and 100 ppm butylated hydroxytoluene. Lysozyme activity also was stable (i.e., retained over 80% of lytic activity) in solutions of 0.1% sodium benzoate and 100 ppm butylated hydroxyanisol. The lysozyme activity was relatively stable (i.e., retained over 70% of lytic activity) in Solutions of 0.3% calcium propionate, 0.1% potassium sorbate, and 0.1% propyl paraben. About half (50%) of the lysozyme activity was retained in solution of 0.5% EDTA. Activity was lost when lysozyme was combined with 0.5% lactic acid, 4% acetic acid, and 100 ppm chlorine water.

Evidence for the Presence of N-unsubstituted Glucosamine Residues in the Peptidoglycan of Sarcobium cytolyticum — an Intracellular Bacterial Parasite of Amoebae

Summary The chemical structure of peptidoglycan of the cell wall of Sarcobium cytolyticum , an intracellular bacterial parasite of small amoebae, was investigated. The bag-shaped peptidoglycan was isolated from intact cells by a procedure involving treatments with sodium dodecyl sulfate and digestion with pronase. The final preparation was virtually free from proteins and neutral sugars. Hydrolysis of peptidoglycan with 4 N HCl at 100°C for 12 hours yielded, in addition to glucosamine, muramic acid, alanine, diaminopimelic acid, glutamic acid and glycine (molar ratiios 0.4 : 0.4 : 1.4 : 1 : 1 : 0.3), significant amounts of a disaccharide composed of glucosaminyl-β (1–4) muramic acid. Peptidoglycan peracetylated with acetic anhydride produced on acid hydrolysis only monomeric subunits represented by glucosamine, muramic acid, alanine, diaminopimelic acid, glutamic acid and glycine in a molar ratio of 1 : 1 : 1.4 : 1 : 1 : 0.3. Estimation of free NH 2 groups in the native peptidoglycan by dinitrophenylation revealed that about 80% of the glucosamine residues were not substituted by acetyl groups and that only 44% of the diaminopimelic acid residues were involved in cross-linkage of peptide subunits. N-acetylation of peptidoglycan did not completely restore its sensitivity to the hydrolytic action of egg white lysozyme.

Modulation of egg-white lysozyme activity by viscosity intensifier additives

The activity of egg-white lysozyme was measured in the presence of carbohydrate additives in the reaction medium. These additives show a significant affinity for water. They depress water activity and increase the viscosity of the medium. Solute-solvent interactions in aqueous solutions of the additives are characterized by properties such as the intrinsic viscosity, Huggins constant apparent molar volume and hydration number. It was found that, despite the lowering of enzyme activity when the concentration of additive is increased, the behavior remains Michaelian and neither modification of iK m nor inhibition by excess substrate is observed. On the other hand, the effect of the viscosity of the medium on enzyme activity was determined. This effect is independent of the nature of the additive at high viscosities (4 mPa −1) for which enzyme activity is very low and appears to vary according to the kind of additive in dilute solution at low viscosities (< 2 mPa s −1).

A proteolytic artifact associated with the lysis of bacteria by egg white lysozyme.

Polyacrylamide gel electrophoresis of cell-free extracts of Escherichia coli that had been grown in a medium containing 32Pi disclosed the presence of several 32P-labeled proteins. Comparison of the electrophoretic patterns obtained in the presence of carrier unlabeled purified E. coli glutamine synthetase before and after treatment with trypsin, subtilisin, or snake venom phosphodiesterase showed that most of the 32P was present in the adenylyl moieties of adenylylated glutamine synthetase. Low molecular weight 32P-labeled degradation products of glutamine synthetase were also observed in extracts prepared by treatment of cells with lysozyme but not in extracts prepared by sonic oscillation. The degradation of glutamine synthetase in lysozyme-prepared extracts is likely due to an intrinsic proteolytic activity of egg white lysozyme. Proteolysis probably occurs at the esterase site of lysozyme described by Piszkiewicz and Bruice [Piszkiewicz, D. & Bruice, T.C. (1968) Biochemistry 7, 3037-3047]. Selective carboxymethylation of lysozyme histidine-15 leads to simultaneous loss of esterase and protease activities but only to partial loss of lytic activity. In view of these findings, caution is needed in the interpretation of results obtained with extracts of cells prepared by lysozyme treatment, especially when such extracts are used to investigate the properties of proteolytic enzymes.

Enzymes lytic against Pseudomonas aeruginosa produced by Bacillus subtillis YT-25. II. Lytic action of B-enzyme on Pseudomonas aeruginosa.

B-enzyme was produced by Bacillus subtilis YT-25 and lysed the native cells of Pseudomonas aeruginosa extensively in the presence of NLF (Native Cell-Lytic Factor). NLF was a peptide which was also produced by B. subtilis YT-25. It was found that B-enzyme hydrolyzed the peptidoglycan of P. aeruginosa eventually to disaccharide units. Because the reducing end of the enzymatic digest was muramic acid, B-enzyme seemed to be an endo-N-acetylmuramidase. Whereas, egg white lysozyme which was an endo-N-acetylmuramidase hardly lysed the native cells of P. aeruginosa. Specific activity of B-enzyme for the murein of P. aeruginosa was higher than that of egg white lysozyme in the buffer of low ionic strength and the surface components of P. aeruginosa did not affect the activity of B-enzyme but strongly inhibited the activity of egg white lysozyme. These facts seemed to explain the superiority of B-enzyme to egg white lysozyme in the lysis of the native cells of P. aeruginosa.

Lytic Action of Egg White Lysozyme on the Food Contaminating Microorganisms

Lytic Action of Egg White Lysozyme on the Food Contaminating Microorganisms

Purification and properties of lysozyme produced by Staphylococcus aureus.

A method based on cold ethyl alcohol fractionation at different pH levels and ionic strengths and on gel filtration on a Sephadex G-200 column was used to concentrate and purify lysozyme from the culture supernatant fluid of Staphylococcus aureus strain 524. The final, nondialyzable product exhibited a 163-fold rise in specific activity over that of the starting material. Staphylococcal lysozyme is a glycosidase which splits N-acetylamino sugars from the susceptible substrate. Staphylococcal lysozyme was shown to be similar to egg white lysozyme in its optimal temperature for reaction, optimal pH, activation by NaCl and Ca(++) ions, inhibition by sodium citrate and ethylenediaminetetraacetate, and inactivation by Cu(++) ions and sodium dodecyl sulfate. It differs from the egg white lysozyme in its temperature susceptibility range (staphylococcal lysozyme is inactivated at 56 C). It acts on whole cells and cell walls of Micrococcus lysodeikticus, murein from S. aureus 524, and cell walls of S. epidermidis Zak. The last substrate was not susceptible to the action of egg white lysozyme in the test system used. The mechanism of action of staphylococcal lysozyme seems to be analogous to that of egg white lysozyme; however, the biological specificity of the two enzymes may be different.

Hydrolysis of benzyl 2-acetamido-4- O-(2-acetamido-2-deoxy-β- D-glucopyranosyl)-2-deoxy-β- D-glucopyranoside by egg-white lysozyme

The synthesis of methyl and benzyl 2-acetamido-4- O-(2-acetamido-2-deoxy-β- D-glucopyranosyl)-2-deoxy-β- D-glucopyranoside, and the action of egg-white lysozyme on the latter compound, as well as on tri- N-acetyl-chitotriose, tetra- N-acetylchito-tetraose, and penta- N-acetylchitopentaose, are described.

Lytic Action of Egg White Lysozyme on the Food Contaminating Microorganisms

1) To examine the lytic action of egg-white lysozyme on Bacillus subtilis, one loopful of spore suspensions as reported by Akashi were inoculated to 100ml control peptone medium (pH 7.2) and 100ml sample peptoe media (pH 7.2) to which lysozyme was added in the rate of 0.0125, 0.025 and 0.05% concentration respectively, and these media were cultured at 37°C or 72 hours.After culturing, these were heated at 100°C for one hour. Viable bacteria counts of these control and sample heated cultures were measured after culturing on nutrient agar media at 37°C for 48 hours. No growth of organisms were observed in the control culture, however in the sample culture, bacterial number of 1.7×105-1.5×106 was observed.2) These control and sample cultures were centrifuged at 10, 000G for 20 minutes, and supernatant fluids were lyophilized. Each of 0.3g of these lyophilized materials were dissolved in 10ml of distilled water. As standard solutions, 0.3g of N-acetylglucosamine and glucosamine-HCl were dissolved in distilled water. Paper chromatographic investigation was made by using these solutions.These solutions were chromatographed on Toyo No. 51 filter paper with three kinds of solvent…n-butanol-pyridine-water (6+4+3 by vol.), pyridine-acetic acid-ethyl acetate-water (5+1+5+3 by vol.), and n-butanol-acetic acid-water (4+1+5 by vol.) by use of Elson and Morgan spray reagent. Results obtained are as follows:From control solution, no spots were detected, however from sample solution, spots with the same Rf value as that of N-acetylglucosamine were observed. In paper chromatography developed with the solvent system of pyridine-acetic acid-water (6+4+3 by vol.) by use of the spray reagent containing 0.05M Na2B4O7-95% ethanol (1:1 by vol.) and p-dimethylaminobenzaldehyde-acetic acid (2% w/v), spots from sample solution were identical with those produced from N-acetylglucosamine given by the same treatment. The spots sprayed with Ehrlich reagent were eluted with distilled water and lyophilized.The lyophilized materials were taken up into 10ml of distilled water and hydrolyzed with 2N HCl at 102°C for 72 hours. Hydrolysates were taken up into 10ml of distilled water. Hydrolysates were concentrated under reduced pressure. In the qualitative analysis reported by Ludowieg, these concentrated materials produced the same pink color intensity given by glucosamine HCl showing the peak O. D. value at 538mμ. Hydrolysates examined by one-dimensional chromatography with the solvent of pyridine +ethanol +water (6+4+3 by vol.) gave spot with Ehrlich-reagent, showing the same Rf value as that of glucosamine-HCl.

Lysozyme substrates. Chemical synthesis of [formula omitted] and its reaction with lysozyme

In order to obtain a lysozyme substrate which can be assayed colorimetrically, p- nitrophenyl-O-(2- acetamido-2- deoxy-β- D - glucopyranosyl)-(1 → 4)-O-(2- acetamido-2- deoxy-β- D - glucopyranosyl)-(1 → 4)-2- acetamido-2- deoxy-β- D - glucopyranoside was synthesized. The action of egg-white lysozyme ( N-acetylmuramide glycanohydrolase, EC 3.2.1.17) on this compound was found to be random, hydrolysis occurring by two pathways, namely one which liberates p-nitrophenol and another which liberates p-nitrophenyl-2-acetamido-2-deoxy-β- D-glucopyranoside. The reaction of the substrate with lysozyme was inhibited non-competitively by N, N-diacetylchitobiose or N-acetyllactosamine, whereas N, N, N-triacetylchitotriose inhibited the reaction competitively. These results suggest that at least three monosaccharide units are necessary for binding to the active site of lysozyme.

Lytic Action of Egg White Lysozyme on the Microorganisms Contaminating Food

There are a lot of papers concerning the lytic action of lysozyme on several strains. Most of these works, however, have been carried out in the biochemical field to clarify the mechanism of lysis by lysozyme, and in these studies, the test strains were generally used for the experiment after being killed by exposing to ultraviolet ray.Since the microorganisms contaminating food generally grow well on food, it seems reasonable that in a study of the lytic action of lysozyme on the microorganisms contaminating food, iiving test strains should be used.A new method for determining the lytic activity of lysozyme on living microorganisms has been developed in our laboratory. By using this method, the lytic action of egg white lysozyme on Proteus vulgaris and Escherichia coli was investigated.According to the results, the lytic activity of egg white lysozyme on these two strains was the highest when. they were treated with 0.05% lysozyme, and this activity became lower in the order treated with 0.025, 0.0125, 0.1, and 0.2% lysozyme.It was also fond that egg white lysozyme exhibited a fairly highem lytic activity on Escherichia coli as compared with that on Proteus vulgaris.

Acetylhexosamine compounds enzymically released from micrococcus lysodeikticus cell walls : II. Enzymic sensitivity of purified acetylhexosamine and acetylhexosamine-peptide complexes

Free di-saccharide (N-acetylglucosamine-N-acetylmuramic acid) is released from a purified amino sugar complex, probably tetra-saccharide, by the action of egg-white lysozyme and of a similar enzyme secreted by a Streptomyces. The di-saccharide is also released from a purified poly-acetylamino sugar-peptide-di-saccharide compound by the action of the same enzymes on its poly-acetylamino sugar moiety. Differences in the affinity of egg-white lysozyme and of the Streptomyces enzyme for their substrates are discussed. A second bacteriolytic enzyme, also secreted by the Streptomyces, liberates free disaccharide from the purified peptide-di-saccharide and poly-acetylamino sugar-peptide-di-saccharide complexes by splitting the bond between the carboxyl group of muramic acid and the amino group of the peptide moiety.

Factors affecting the activity of egg white lysozyme

A turbidimetric assay for lysozyme which employs Sarcina lutea as substrate is described. The method is sensitive to 0.03 μ M lysozyme solutions (0.5 μg./ml.). In 0.025 M citrate, lysozyme exhibits a broad pH optimum in the range pH 5.9 – 6.3. One-tenth molar citrate and 0.03 M trichloroacetate markedly inhibited lysozyme. The activity of lysozyme is affected by the ionic strength of the solution. An activation was observed up to an ionic strength of 0.2; above this value an inhibition of lysozyme occurred. Urea and glycerol reversibly inhibit lysozyme. A photoinactivation of lysozyme in the presence of riboflavin was observed.

Studies on the defense mechanisms of the mucous membranes with particular reference to the oral cavity

It can be seen from the experiments described that submaxillary mucoid can inhibit the activity of egg white lysozyme against the test organisms, Micrococcus lysodeikticus. This inhibition is probably a nonspecific one, being the result of the formation of a mucoid-lysozyme salt which is removed from solution by precipitation. The inhibition can be reversed to a considerable degree by the addition of sodium chloride. This reversal effect is probably due to the dissociation of the mucoid-lysozyme complex by the neutral salt. The inhibition of lysozyme activity by submaxillary mucoid is greatest at low pH (3.5 and 4.5); at the higher pH levels of 5.5 and 6.8, there was some slight activity, particularly after long incubation periods. This may possibly be a result of the dissociating effect that higher pH levels would have on the mucoid-lysozyme complex. It was also shown that the lytic activity of whole saliva is greater than that of centrifuged saliva against Micrococcus lysodeikticus. This is probably a result of the lytic agent being sedimented with the insoluble mucins of the saliva. Quaternary ammonium salts such as “Octab” and “Cetab” are excellent reagents for the precipitation of submaxillary mucoid as well as other acid mucopolysaccharides. When these reagents were used to precipitate the mucins from saliva, the lytic activity of the supernatant saliva was increased from five-to tenfold. The addition of “Cetab” to centrifuged saliva, from which the mucin and much of the lytic activity is sedimented, did not increase the lytic activity of the supernatant fluid. This would tend to support the concept that the lytic agent in saliva is in complex with another constituent of the saliva, probably a mucoid or mucopolysaccharide.

The action of egg white lysozyme on ovomucoid and ovomucin

1. 1. No increase in reducing substances, decrease in viscosity or production of amino-sugars was observed when ovomucoid preparations were incubated with egg white lysozyme. 2. 2. Addition of lysozyme to swollen ovomucin resulted in the separation of a compact precipitate of mucin plus lysozyme. 3. 3. The data suggest that the thinning of the thick white gel which occurs during storage of eggs may, in part, be due to the action of lysozyme on mucin.