Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.     

Influence of Acidic Exopolysaccharide of Xanthomonas campestris IBPM 124 on the Kinetic Parameters of Extracellular Bacteriolytic Enzymes

Interactions of a negatively charged exopolysaccharide of Xanthomonas campestris IBPM 124 with its extracellular enzymes (muramidase, endopeptidase, and neutral phosphatase) and also with egg lysozyme, lysostaphin, muramidase of Streptomyces globisporus, and a bacteriolytic enzyme complex of Streptomyces albus were studied. All these enzymes were positively charged under the conditions of their maximal activity. It was shown that interaction of the acidic exopolysaccharide from X. campestris with these enzymes changed their kinetic parameters. The change was either positive (increase in reaction rate) or negative (decrease in reaction rate) and depended on the enzyme and type of substrate cleaved. Due to such interactions, the acidic exopolysaccharide secreted by X. campestris into the environment not only retained and transported positively charged exoenzymes into the near-cellular space, but also regulated their activity.     

Electrophoretic Forms of Chitinolytic and Lysozyme Activities in Ruminal Protozoa

Homogenates from a mixed ruminal protozoal population and a ruminal protozoon Entodinium caudatum were analyzed for chitinolytic and lysozyme activities by sodium dodecyl sulfate polyacrylamide gel electrophoresis. For chitinase activity, up to eight bands in mixed protozoa and seven bands in E. caudatum were detected. Estimated molecular mass ranged from 70 to 110 kDa. These enzymes did not display lysozyme activity. N-Acetyl-β-glucosaminidase activity was also detected in both samples with an estimated molecular mass of 37 kDa. Lysozyme activity in mixed protozoa was present in two major and three minor bands, where one major band displayed the same motility as chicken egg white (CEW) lysozyme, and the other had an approximate molecular mass of 17.5 kDa. The latter remained active even when denatured in the presence of dithiothreitol and renatured under anaerobic conditions. Entodinium caudatum presented one major band coincident with that of CEW lysozyme and a minor band at the 17.5-kDa point. This study showed that protozoal chitinase and lysozyme activities are originated from several enzymes and that none of these enzymes exhibited both activities.     

Murein transglycosylase from phage lambda lysate. Purification and properties

Lysates of induced E. coli (lambda) lysogens contain two enzymes acting on murein: endopeptidase and murein transglycosylase. The transglycosylase was separated from the endopeptidase and purified to homogeneity. Its bacteriolytic activity was 200-fold higher than of hen egg lysozyme. The bacteriolytic activity of the lysate depends on the presence of the enzyme. The endopeptidase alone not lyse the cells, but it enhances the extent of lysis. The properties of the transglycosylase (molecular weight 17 500, pH optimum at 6.6, inactivation by Zn2+), show that it is entirely different from the bacterial enzyme of the same specificity described by others. Data are presented, which suggest that this enzyme is the phage lambda R-gene product.     

Stimulation of spreading of trypsinized human fibroblasts by lysozymes fromStaphylococcus aureus,hen egg white,and human urine

Summary The effect of lysozyme from three different sources—Staphylococcus aureus, hen egg white, and human urine—on adhesion to substrate and spreading of trypsinized human fibroblasts was studied. Several fibroblast strains were tested under various conditions. It was found that the different cell strains did not show the same capability of spreading and stably attaching to substrates when resuspended in media not containing serum. Some strains did not spread, whereas others spread even in the absence of serum. Cell spreading in these strains did not occur when the cells were pregrown for 5 weeks in media supplemented with 1% fetal bovine serum. Lysozyme fromS. aureus allowed stable adhesion to substrate and spreading of all the fibroblast strains unable to elongate in nonsupplemented minimal essential medium. This enzyme accelerated and augmented spreading of the strains capable of elongating in the absence of serum.S. aureus lysozyme also allowed spreading and stable adhesion to substrates of all these strains when they were pregrown for 5 weeks in the presence of 1% fetal bovine serum. Furthermore, hen egg white lysozyme and the lysozyme purified from human urine were both capable of stimulating anchorage to substrate and spreading of trypsinized fibroblasts although their effect was less pronounced than that of theS. aureus lysozyme. Some tentative hypotheses for the mechanism of cell spreading in the presence of lysozyme are made. The possibility that lysozymes, virtually ubiquitous enzymes, may play a specific role in nature in the regulation of cell differentiation and tissue development is finally raised and discussed in light of several previous observations and findings. This work was supported by Grant 79.00677.96 of Piano Finalizzato Tumori by the Consiglio Nazionale delle Ricerche of Italy.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.     

Purification and Properties of Pectinesterase from Acrocylindrium

The crude enzyme fraction of precipitates resulting from the addition of 70% alcohol to the culture filtrate of A. lunatus was separated by CM-Sephadex and Sephadex G-75 chromatography into 13 fractions having lytic activity for M. radiodurans, M. lysodeikticus and P. radiora. Five of the fractions showed similar lytic activity spectra, but the other fractions were separated by the specificities of their lytic activities. This result indicates that the wide lytic spectrum of the crude enzyme against microorganisms is attributable to the action of many lytic enzymes. All fractions, except for P2-2 fraction (designated as the P2-2. enzyme), contained at least two proteins as determined by disc gel electrophoresis. The P2-2 enzyme was purified 34-fold by rechromatography on Sephadex G-75, and appeared to be homogeneous on disc gel electrophoresis. The enzyme was able to lyse intact cells of M. radiodurans and M. lysodeikticus without detergent, and those of P. radiora with detergent, but was not able to digest casein.     

Expression of Recombinant Human Lysozyme in Egg Whites of Transgenic Hens

Chicken egg lysozyme (cLY) is an enzyme with 129 amino acid (AA) residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV) infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY) cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G₁ transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY) at 57.66 ± 4.10 μg/ml in the egg white and the G₂ transgenic hens of the G₁ transgenic cock A011 expressed rhLY at 48.72 ± 1.54 μg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Development of an eco-friendly antibacterial textile: lysozyme immobilization on wool fabric

Lysozyme, a type of natural enzyme, has been widely used for bacteriostatic functionalization of various materials due to its efficient and selective antibacterial properties. Herein, we report the preparation and characterization of an eco-friendly antibacterial textile based on the immobilization of lysozyme from chicken egg white onto wool fibers. Tris(hydroxymethyl)phosphine (THP) was employed as the cross-linker for the immobilization of lysozyme on the surface of wool fiber. The mechanism of THP cross-linking was investigated via phosphorus test, energy-dispersive spectroscopy (EDX) and Fourier transform infrared spectroscopy (FT-IR). The surface staining, optimization of immobilization parameters, morphology, antibacterial properties, and durability of wool fibers with immobilized lysozyme were also assessed. The results show that hydroxymethyl groups of THP reacted with amino groups of wool fiber and lysozyme through Mannich reaction, which successfully immobilized lysozyme on the wool fiber. The wool fibers incorporated with lysozyme had better antibacterial properties and durability compared with the untreated wool fabric. This facile immobilization approach of lysozyme provides an effective strategy for environmentally benign modification and functionalization of keratin and keratin-containing materials.

     

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein–protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the β domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein–protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.     

Activity of bacteriolytic enzymes adsorbed to clays

Myxococcus virescens is able to produce extracellular bacteriolytic enzymes that are rapidly adsorbed on montmorillonite. These adsorbed enzymes are active and can be assayed by measuring the release of UV-absorbing materials in mixtures containingMicrococcus luteus cells. The activity of the clay-adsorbed enzymes is, however, considerably lower than that of the unadsorbed enzymes. Both unadsorbed and adsorbed enzymes have their maximum activity at approximately the same pH. At lower clay-enzyme concentrations, the activity is proportional to the concentration. If, however, increasing amounts of clay are added to a fixed volume of clay-enzyme suspension, the activity remains almost unchanged until a definite limit is reached, then the activity decreases rapidly. This limit was dependent only on the ratio of the amounts of enzyme and clay and not on the absolute concentration of the enzyme. The montmorillonite-adsorbed bacteriolytic enzymes fromM. virescens were not active against gram-negative bacteria, and no activity against purified cell walls fromM. luteus could be measured. Montmorillonite-adsorbed egg white lysozyme was not active onM. luteus cells.     

Experiments with lysis of living cells of Eremothecium ashbyii and of Methanomonas by microbial enzymes

The possibility to use microorganisms as human food is limited by several factors. The intact cell is resistant to digestion, the cell wall is unbalanced in essential amino acids, and the nucleic acids are said to be harmful. For using single cell protein as food it may thus be necessary to disrupt the cell wall and separate the protein from nucleic acid. This paper is concerned with the production and properties of extracellular enzymes able to lyse cell walls of microorganisms. Soil bacteria and actinomycetes have been cultivated and lytic enzymes from these organisms have been used to lyse living cells of the yeast like organism E. ashbyii. Efforts were also made to use these enzymes for lysing cell of a Methanomonas sp.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer     

Radiation resistance ofSalmonella

Summary The ionizing radiation resistances of sixSalmonella species were examined. The experimental variables were the suspending medium, the presence or absence of air, and the temperature during the irradiation process.S. typhimurium ATCC 14028,S. enteritidis ATCC 9186,S. newport ATCC 6962,S. dublin ATCC 15480,S. anatum ATCC 9270, andS. arizonae ATCC 29933 were suspended in phosphate buffer (0.1 M, pH 7.0), brain heart infusion broth (BHI) or mechanically deboned chicken and exposed to gamma radiation from cesium-137 at 0.12 kGy per min. The radiation resistance of theSalmonella increased approximately two-fold when assayed in sterile mechanically deboned chicken rather than in buffer or BHI. The average radiation (0.30 to 1.20 kGy) D-value for all sixSalmonella strains was 0.56 kGy in mechanically deboned chicken.S. enteritidis was significantly more resistant to ionizing radiation than the other five strains ofSalmonella tested on mechanically deboned chicken. The temperature of irradiation but not the presence or absence of air significantly influenced the survival ofS. typhimurium andS. enteritidis in mechanically deboned chicken. Treatment of chicken meat with ionizing radiation would be an effective means for control ofSalmonella contamination.     

A novel thermophilic lysozyme from bacteriophage phiIN93

The lysozyme of bacteriophage φIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a φIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, φIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 °C for 1 h in the presence of β-mercaptoethanol.     

Purification,biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001

The discovery of microbial fibrinolytic enzymes is essential to treat cardiovascular diseases. This study reports the discovery of a fibrinolytic enzyme secreted by Bacillus cereus SRM-001, a microorganism isolated from the soil of a chicken waste-dump yard. The B. cereus SRM-001 was cultured and the secreted fibrinolytic enzyme purified to show that it is a ～28 kDa protein. The purified enzyme was characterized for its kinetics, biochemical and thermal properties to show that it possesses properties similar to plasmin. A HPLC-MS/MS analysis of trypsin digested protein indicated that the fibrinolytic enzyme shared close sequence homology with serine proteases reported for other Bacillus sp. The results show that the B. cereus SRM-001 secreted enzyme is a ～28 kDa serine protease that possesses fibrinolytic potential.     

Homology of lysozymes of bacterial and vertebrate origin

Theoretical analysis of structural and functional organization of vertebrate lysozymes, T4-phage lysozyme, lambda-phage endolysin and extracellular lysozyme of Chalaropsis species suggests a genetic relationship between the enzymes in question. It has been shown that the lysozyme sequences exhibit both inter- and intramolecular homology. The obtained data lend support to the concept postulating a common ancestor for the lysozyme family and subsequent divergent evolution of these proteins. The two-component primary structure of lysozymes can result from structural gene duplication and allows to explain similar catalytic activity and different substrate specificity of these enzymes by the differentiation and specialization of functions of the N- and C-components of the protein chains.     

A large scale enzyme screen in the search for new methods of silicon-oxygen bond formation

Biotransformations make use of biological systems to catalyze or promote specific chemical reactions. Transformations that utilize enzymes as “greener” and milder catalysts compared to traditional reaction conditions are of particular interest. Recently, organosilicon compounds have begun to be explored as non-natural enzymatic substrates for biotransformations. The aims of this study were to screen readily available (approximately eighty) enzymes for their ability to catalyze in vitro siloxane bond formation under mild reaction conditions using a model monoalkoxysilane as the substrate and to make a preliminary evaluation of potential factors that might lead to activity or inactivity of a particular enzyme. Several new hydrolase enzymes were observed to catalyze the formation of the condensation product when compared to peptide controls, or buffer solutions at the same pH, as judged from quantitative analyses by gas chromatography. Aspergillus ficuum phytase, Aspergillus niger phytase, chicken egg white lysozyme, porcine gastric mucosa pepsin, and Rhizopus oryzae lipase all catalyzed the condensation of silanols in aqueous media. Factors involved in determining the activity of an enzyme towards silanol condensation appear to include: the presence of imidazole and hydroxyl functions in the active site; solvent; the presence of water; the surface properties of the enzyme; possible covalent inhibition; and steric factors in the substrate.