Lytic Activity Of Lysozyme Research Articles

Structural basis for the O-acetyltransferase function of the extracytoplasmic domain of OatA from Staphylococcus aureus

Many bacteria possess enzymes that modify the essential cell-wall polymer peptidoglycan by O-acetylation. This modification occurs in numerous Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, a common cause of human infections. O-Acetylation of peptidoglycan protects bacteria from the lytic activity of lysozyme, a mammalian innate immune enzyme, and as such is important for bacterial virulence. The O-acetylating enzyme in Gram-positive bacteria, O-acetyltransferase A (OatA), is a two-domain protein consisting of an N-terminal integral membrane domain and a C-terminal extracytoplasmic domain. Here, we present the X-ray crystal structure at 1.71 Å resolution and the biochemical characterization of the C-terminal domain of S. aureus OatA. The structure revealed that this OatA domain adopts an SGNH-hydrolase fold and possesses a canonical catalytic triad. Site-specific replacement of active-site amino acids revealed the presence of a water-coordinating aspartate residue that limits esterase activity. This residue, although conserved in staphyloccocal OatA and most other homologs, is not present in the previously characterized streptococcal OatA. These results provide insights into the mechanism of acetyl transfer in the SGNH/GDSL hydrolase family and highlight important evolutionary differences between homologous OatA enzymes. Furthermore, this study enhances our understanding of PG O-acetyltransferases, which could guide the development of novel antibacterial drugs to combat infections with multidrug-resistant bacterial pathogens.

Denaturation of Lysozyme with Visible-light-responsive Photocatalysts of Ground Rhodium-doped and Ground Rhodium-antimony-co-doped Strontium Titanate.

Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO3 (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO3 (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.

Cleavable carbamate linkers for controlled protein delivery from hydrogels

The reversible attachment of proteins to polymers is one potential strategy to control protein release from hydrogels. In this study, we report the reversible attachment of lysozyme to poly(ethylene glycol) (PEG) by degradable carbamate linkers. Phenyl groups with different substituents were used to control the rate of carbamate hydrolysis and the resulting protein release. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed modification with 1–3 PEG chains per lysozyme molecule. Protein PEGylation and PEG chain elimination occurred without changes in secondary protein structure, as demonstrated by circular dichroism spectroscopy. The lytic activity of lysozyme was restored to 73.4±1.7%–92.5±1.2% during PEG chain elimination. Attached PEG chains were eliminated within 24h to 28days, depending on the used linker molecule. When formulated into hydrogels, a maximum of about 60% of the initial dose was released within 7days to 21days. Linker elimination occurs ‘traceless’, so that the protein is released in its native, unmodified form. Altogether, we believe that tethering proteins by degradable carbamate linkers is a promising strategy to control their release from hydrogels.

Dominant structural factors for complexation and denaturation of proteins using carboxylic acid receptors

Complexation accompanied by denaturation of protein with synthetic carboxylic acid receptors was investigated, to evaluate the key factors for recognition of proteins. The synthetic receptors used were tetraphenylporphyrin (TPP) derivatives and receptors bearing multiple (2–8) carboxylic acid groups. The complexation behavior was quantified from the absorption in the far UV CD spectrum attributed to the secondary structure of the protein. TPP derivatives bearing multiple carboxylic acid groups in the side chains exhibited higher affinity than other receptors that were smaller and had fewer carboxylic acid groups. As the degree of complexation was influenced by the pH and ionic strength in aqueous solution, electrostatic interaction was one of the most important factors for the recognition of proteins. Complexation was also estimated by observation of fluorescence quenching of the TPP derivatives. The stoichiometry of the complexes between lysozyme and the porphyrins was investigated by quantitative analysis of the denaturation using CD spectra. From the results of Job plots and slope analysis for the amount of denatured protein, formation of 1:1 complexes was confirmed. The equilibrium association constants ( K ass) for lysozyme and the TPP receptors ranged from 0.6 × 10 6 to 1.1 × 10 6 M −1. The lytic activity of lysozyme was partially lost in the presence of anionic TPP derivatives, due to complexation and denaturation.

INTERACTION OF VIRAL AND BACTERIAL LYSOZYMES WITH ERWINIA AMYLOVORA AND THEIR INHIBITION BY A BACTERIAL PROTEIN

Viral lysozymes can interact with Erwinia amylovora, and their genes may control fire blight when expressed in the pathogen. We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species. With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes. Fusion proteins with the (phi)Ea1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly. For large fusions at the N- or C-terminus lytic activity was completely lost. When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored. Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme. Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with (phi)Ea1h lysozyme and T4 lysozyme. The cellular expression of a viral lysozyme gene can be toxic for a bacterial host. The gene was therefore expressed in yeast as safe environment for its secretion. Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast. When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen. The (phi)Ea1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage (phi)Ea104 and (phi)Ea116 was not affected by the enzyme inhibitor.

Effect of Enzyme Modification by Well-Defined Multi-Armed Poly(Ethylene Glycol) Synthesized Using Polyamidoamine Dendron

Egg white lysozyme was chemically modified by PEGylated PAMAM 1st, 2nd and 3rd generation dendrons, which had 2, 4 and 8 PEG arms, respectively. The number of PEG chains introduced to the lysozyme molecule drastically increased with an increase in dendron generation, although the number of PEGylated PAMAM dendrons introduced decreased due to steric repulsion. The lytic activity of lysozyme to Micrococcus luteus cells was effectively inhibited by conjugating PEGylated PAMAM dendron to the lysozyme, indicating steric stabilization of PEG chains at the surface of lysozyme molecule. In addition, the enzymatic reaction of the lysozyme with oligosaccharide substrate was apparently accelerated by a substrate condensation effect due to the multi-armed structure of PEG.

Turbidimetric and HPLC assays for the determination of formulated lysozyme activity.

In several studies lysozyme has been employed as a model protein to investigate the effects of formulation factors upon biological activity. The aim of this work was to develop and validate an HPLC technique to assay lysozyme and to compare the results with biological activity determined from a validated turbidimetric assay. The turbidimetric assay was based upon the lytic action of lysozyme on Micrococcus lysodeikticus cells, whilst the reverse-phase HPLC assay employed an acetonitrile gradient in 0.1% trifluoroacetic acid. The limits of detection and quantification were 3.84 and 6.24 microg mL(-1) for HPLC assay, whilst the corresponding values for turbidimetric assay were 1.94 and 3.86 microg mL(-1). The methods were used to monitor the loss of enzyme activity after heating. Lysozyme concentrations determined from HPLC peak height were found to correlate (r2 = 0.9963) with those obtained from turbidimetric assay.

Stabilization of Micrococcus lysodeikticus cells towards lysis by lysozyme using glutaraldehyde : application as a novel biospecific ligand for the purification of lysozyme

Micrococcus lysodeiekticus was stabilized against the lytic action of lysozyme by cross-linking with 5% (v/v) glutaraldehyde for 24 h but still retained its ability to bind lysozyme. An immobilized, biospecific ligand was prepared by covalently binding the cells to glutaraldehyde activated amino-Sepharose followed by stabilization of the cells with glutaraldehyde. Lysozyme bound specifically to this column and could be eluted by glycine/NaOH buffer (50 mM, pH 10.0) containing 2 M KCl.

Humoral factors of natural resistance of Bos indicus cattle selected for antibody titre to Brucella abortus.

The concentration of lysozyme, total immunoglobulin and bactericidal activity were measured in sera of Bos indicus cattle, retrospectively screened for specific antibodies to Brucella abortus and classified as being positive reactors or negative reactors. In addition, the effect of complement in the sera was studied to demonstrate complement dependence of antibody-mediated bacterial killing. It was observed that, under the test conditions, serum bactericidal activity and concentration of total immunoglobulin were associated with high specific antibody levels (P less than 0.001). Furthermore, there was a slight decrease in the lytic activity of lysozyme in the sera of animals with high antibody titres.

Histidine-15 and lytic activity of lysozyme

The literature data on the activity of histidine-15 modified hen egg white lysozyme are conflicting: the modified enzyme is reported to have more activity, similar activity or less activity by different authors. Amino acid analysis had shown modification of the single His-15. Detailed activity studies on His-15-modified (by iodoacetic acid or diethyl pyrocarbonate) lysozyme have shown that the contradicting reports are due to the specific choices of ionic strengths and cell wall substrate concentrations and can be attributed to the substrate being negatively charged. Our analysis suggests that even though histidine-15 is far removed from the active site of lysozyme, its chemical modification or binding of the negatively-charged substrate near it, changes the conformation around the active site. However, the change in the optimum activity on chemically modifying His-15 is small.

Lytic Activity of Lysozyme Relevant to Preservatives – Enzyme Interactions

AbstractAt physiological hydrogen-ion concentration, p-hydroxybenzoate esters have been found to exert a synergistic effect on the lytic activity of lysozyme; the effect being increased with increasing the size of the aliphatic chain. Although the investigated parabens were proved to bind in significant quantities with lysozyme, however, such additives fail to mask the formation of substrate-lysozyme complexes. The observation was further extended to some organic mercurials with essentially similar results.At different enzyme concentration levels, phonylmercuric acetate was proved to be the most superior potentiator, whereas, methyl paraben displayed a negligible role.in the light of the findings obtained, the synergistic effect was interpreted on the basis of the preferential orientation of the substrate on the enzyme binding sites. Such an orientation seems to be more accessible in presence of preservatives.

Lethal effect of protamine and histone on competent Bacillus subtilis cells. Inhibition of genetic transformation by protamine in sublethal concentration.

Under experimental conditions of genetic transformation, protamine and total histone were bactericidal for Bacillus subtilis cells. The abilities to cause lethality were very similar for both, either protamine or histone, with no antagonistic effects amongst these natural polycations. With both basic proteins acting simultaneously the enhancement was higher than a summation of the separate lethal effects. Sublethal concentration of protamine added at the beginning of transformation time, produced a strong inhibition of transforming efficiency. The same concentration added later than 10 min from the start of transformation had no inhibitory effect. These facts together with the absence of inhibition by simple pretreatment of DNA alone as well as the cell protection by protamine against lytic activity of lysozyme, suggest a protamine-cell surface interaction which impedes DNA uptake events.

Mechanism of Action of Tetracaine Hydrochloride against Pseudomonas aeruginosa

The mode of action of tetracaine hydrochloride in vitro on Pseudomonas aeruginosa was investigates. The inhibitory and bactericidal action of tetracaine hydrochloride in vitro was adversely affected by magnesium ions. Observation of cellular lysis, leakage of intracellular materials, dehydrogenase activity, and a higher sensitivity of spheroplasts than of whole cells to tetracaine led to the conclusion that tetracaine acts by damaging the cell membrane. The lytic action of lysozyme was potentiated by tetracaine; this finding indicates enhanced permeability of the cell wall. Alteration of cell wall permeability was demonstrated by the finding that the cells exposed to subinhibitory amounts of tetracaine became susceptible to otherwise ineffective erythromycin.

Further Studies on the Reactions of Phenylglyoxal and Related Reagents with Proteins1

1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3.1.4.22] and its derivatives, alpha-chymotrypsin [EC 3.4.21.1], trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], rennin [EC 3.4.23.4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal alpha-amino groups was observed with each of these reagents. 2. Two arginine residues out of four in ribonuclease A, two out of three in alpha-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of alpha-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of alpha-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.

Lytic Action of Egg White Lysozyme on Food Poisoning Microorganism

The lytic action of lysozyme on Vibrio para-haemolyticus which is one of food poisoning organisms was studied by using young stadium vegetative cells from cultures in 1, 3 and 5% NaCl pepton at 37°C for 24 hours.These cultures were inoculated in pepton media in which lysozyme was added in the rate of 0, 0.0125, 0.025, 0.05, 0.1 and 0.2% respectively.Transmittance values and viable counts of bacteria were determined in 1, 24, 48 and 72 hours after incubation at 20°C. In 1% NaCl pepton water, remarkable differences of transmittance value and viable counts of bacteria were found between control and each lysozyme concentration solution. These differences, however, decreased in number in the order treated with 3 and 5% NaCl pepton medium. It was found that the lytic activity of lysozyme became higher in the medium of low NaCl concentration in which the growth activity of this organism decreased, and that this activity was inhibited by the optimum growth of this organism in 3 and 5% NaCl media.Electro-micrographic studies made by the use of each solution in 72 hours cultures showed similar tendency to the results of transmittance values and viable counts of bacteria.

Preliminary investigation of the behavior of lysozyme in urea solutions

The intrinsic viscosity, optical rotation, and lytic activity of lysozyme have been investigated in urea solutions, within the concentration range of from 0 to 10 M urea. Viscosity data show that the protein molecule is a compact and fairly symmetrical unit in its native state, but that it undergoes swelling at urea concentrations higher than 5–6 M. Optical rotation data point to the presence of a very stable folded configuration in the peptide chain of lysozyme. Unfolding occurs only above 7 M urea, and the extent of unfolding in 10 M urea is less than the corresponding increase in swelling of the molecule. Lytic activity was measured by means of two sensitive microorganisms, B. megatherium and M. lysodeikticus. Lysis by lysozyme is somewhat obscured by the marked effect of urea upon killed cells. It is clear, however, that concentrated urea inhibits the enzymic properties of the molecule; this inhibition is fully reversible. It is concluded that at least part of the tertiary structure of the molecule is necessary for some step in the course of the lytic process.

Effects of Azo-Sulfonamides upon Lysozyme

Evidence is presented which indicates that certain soluble sulfonamide-azo dyes interfere with the lytic activity of lysozyme upon M. lysodeikticus. One of these compounds, sulfathiazole-disazo-symmetrical-carbamide-2R acid, completely suppresses the lytic effects of the enzyme in a concentration of 1:10,000 of the drug. To our knowledge, these are the first sulfonamides found to exhibit anti-lysozyme properties.

Action of Sulfamido Compounds upon M. lysodeikticus and Lytic and Bactericidal Activities of Lysozyme

Summary(1) Sulfanilamide in high concentration (1000 mg %) exerts bacteriostatic activity toward small numbers of M. lysodeikticus. (2) Sulfanilamide in concentrations up to 1000 mg % and sulfapyridine and sulfathiazol in concentrations up to 100 mg °/c do not interfere with the lytic activity of lysozyme (egg white). (3) The combined use of lysozyme (egg white) and sulfamido compounds (sulfanilamide and sulfapyridine) exerts somewhat greater bactericidal effects upon M. lysodeikticus than either agent alone.