Lysozyme in egg whites of tortoises and turtle. Purification and properties of egg white lysozyme of Trionyx gangeticus Cuvier.

Egg white proteins of three species of tortoises and turtle and of hen have been compared by electrophoretic and immunochemical methods. The proteins lacked similarity in electrophoresis, but tortoise and turtle egg white proteins which did not crossreact with those of the hen showed some cross-reaction among themselves. The occurrence of lysozyme as two allelic variants which were distinguishable in electrophoresis was noted only in the egg white of one of the species of tortoise, namely, Trionyx gangeticus Cuvier. Tortoise lysozyme which showed strong lytic activity toward cell walls of Micrococcus lysodeikticus did not exhibit any cross-reaction with hen lysoyzme. It was purified, crystallized, and found to be homogeneous in sodium dodecyl sulfatepolyacrylamide gel electrophoresis, immunochemical tests, and sedimentation. The physicochemical and enzymatic properties of tortoise lysozyme were found to be strikingly similar to those of hen lysozyme with minor differences which could be due to differences in their primary structure. Its average molecular weight of 15,400 was determined from sedimentation and diffusion coefficient values, Archibald experiment, and amino acid composition. The molecule appeared to undergo pH-dependent expansion at pH 2 and dimerization above pH 5.7. In enzymatic properties, tortoise lysozyme showed a specific activity of 29,000–31,000 units and gave a pH optimum at pH 7.5 and an apparent K_a value of 250 mg· liter^?1. Like hen lysozyme, its activity showed strong ionic strength dependence, weak chitinase activity, susceptibility to inhibition by N-acetyl-glucosamine, and stability toward heat.     

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Summary Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

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-acetyl-muramidase. Whereas, egg white iysozyme 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 Iysozyme 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 Iysozyme. These facts seemed to explain the superiority of B-enzyme to egg white Iysozyme in the lysis of the native cells of P. aeruginosa.     

Intracellular Peptidase of Bac. subtilis

A peptidase was isolated from the cells of amylase-producing Bac. subtilis by means of cell lysis with egg white lysozyme, followed by freezing and thawing, salting out, dialysis and ion-exchanger column chromatography. The enzyme required manganese ion to show the enzyme activity. Also the enzyme was stable in the presence of magnesium ion. The enzyme hydrolyzed various synthetic peptides by stepwise removal of the amino terminal amino acid of peptides and thus the peptidase was found to be aminopeptidase.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

Synthesis of (±)-Xanthoxins

The enzymatic properties of P2-2 enzyme were determined by using cells of M. radiodurans. The enzyme was: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16,000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.     

Digestive function of lysozyme in synanthropic acaridid mites enables utilization of bacteria as a food source

The activity of lysozyme, the enzyme that hydrolyzes peptidoglycan in G⁺ bacterial cell walls, was detected in whole mite extracts (WME) and in spent growth medium extracts (SGME) of 14 species of synanthropic mites (Acari: Acaridida). The adaptation of lysozyme for digestive activity and bacteriophagy was based on: (i) high lysozyme activity in SGME, and (ii) the correlation of maximum lysozyme activity at acidic pH values, corresponding to pH in the ventriculus and caeca. We show that the digestion of fluorescein-labeled Micrococcus lysodeikticus cells began in ventriculus and continued during the passage of a food bolus through the gut. The fluorescein was absorbed by midgut cells and penetrated to parenchymal tissues. Eight species showed a higher rate of population growth on a M. lysodeikticus diet than on a control diet. The lysozyme activity in SGME was positively correlated to the standardized rate (r _s) of population growth, although no correlation was found between r _s and lysozyme activity in WME. The lysozyme activity in WME was negatively correlated to that in SGME. The highest activity of digestive lysozyme was found in Lepidoglyphus destructor, Chortoglyphus arcuatus and Dermatophagoides farinae. All of these findings indicate that lysozyme in acaridid mites possesses both defensive and digestive functions. The enzymatic properties of mite lysozyme are similar to those of the lysozymes present in the ruminant stomach and in the insect midgut.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

New variant of quail egg white lysozyme identified by peptide mapping

Two lysozymes were purified from quail egg white by cation exchange column chromatography and analyzed for amino acid sequence. The enzymes showed the same pH optimum profile for lytic activity with broad pH optima (pH 5.0-8.0) but had difference in mobility on native-PAGE. The native-PAGE immunoblot showed one or two lysozymes present in individual egg whites. The established amino acid sequence of quail egg white lysozyme A (QEWL A) was the same as quail lysozyme reported by Kaneda et al. [Kaneda, M., Kato, I., Tominaga, N., Titani, K., Narita, K., 1969. The amino acid sequence of quail lysozyme. J. Biochem. (Tokyo). 66, 747-749] and had six amino acid substitutions at position 3 (Phe to Tyr), 19 (Asn to Lys), 21 (Arg to Gln), 102 (Gly to Val) 103 (Asn to His) and 121 (Gln to Asn) compared to hen egg white lysozyme. QEWL A and QEWL B showed one substitution, at the position 21, Gln replaced by Lys, plus an insertion of Leu between position 20 and 21, being the first report that QEWL B had 130 amino acids. The amino acid differences between two lysozymes did not seem to affect antigenic determinants detected by polyclonal anti-hen egg white lysozyme, but caused them to separate well from each other by ion exchange chromatography.     

Amino acid sequence of a lysozyme (B-enzyme) from Bacillus subtilis YT-25

The amino acid sequence of a lysozyme, (B-enzyme), from Bacillus subtilis YT-25 was determined by conventional methods. B-Enzyme comprised 117 amino acid residues and had a heterogeneous sequence in the amino-terminal region. The amino acid sequence of B-enzyme was different from those of all other lysozymes the sequences of which are known. However, the partial amino acid sequence of Ser(74) to Ser(97) of B-enzyme was homologous with that of the active-site region of hen egg-white lysozyme (Ser(36) to Ser(60], which includes one of the catalytic amino acids, Asp(52). It is interesting that B-enzyme has an amino acid sequence homologous with that of the gag protein p25 of the AIDS virus ARV-2.     

Purification and characterization of mono-and diacylglycerol lipase isolated from Penicillium camembertii U-150

Summary A novel enzyme hydrolysing mono- and diacylglycerol was found in the culture filtrate of an isolated fungus, Penicillium camembertii. The enzyme was separated into two forms (A- and B-enzyme) with almost the same molecular weight (37,000–39,000), amino acid composition and identical N-terminal amino acid sequence. B-Enzyme, a major component, was purified approximately 210-fold with an activity yield of 2.6%. The B-enzyme was specific to mono- and diacylglycerols and hydrolysed long-chain monoacylglycerols most efficiently. Triacylglycerols were completely inert as substrates for the enzyme. The B-enzyme preferred to attack -position to -position of monoacylglycerol, but showed no stereospecificity on mono- and diacylglycerol. Both Fe³⁺ and Hg²⁺ inhibited B-enzyme activity significantly.Offprint requests to: S. Yamaguchi     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Cause of the abnormal acidity of ionogenic groups of the lysozyme active center in cell wall lysis <Emphasis Type="Italic">Micrococcus lysodeicticus</Emphasis>

The influence of pH within the range 6.9–10.0 on the kinetic parameters of Micrococcus lysodeicticus cell lysis catalyzed by hen egg lysozyme has been studied at 25°C and 37°C. The effective pK _b values have been calculated for the group determining lysozyme catalytic activity. The ΔH _ion value indicates that this group is a carboxyl, although its pK (9.15 at 25°C) is far beyond the range characteristic of carboxylic groups. The cause of this abnormal pK _b value is supposed to be the strong negative charge of the bacterial cell wall. As a result, the enzyme, which catalyzes the hydrolysis of N-acetylglucosamine-N-acetylmuramic acid copolymer, operates in a highly acidic microenvironment.     

Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus

Lysozyme represents the best characterized enzyme involved in the self-defense from bacteria. In this study we analysed the effects of zinc on the lysozyme-like activity of the seastar Marthasterias glacialis mucus. This activity, detected by measuring the cleared lysis area of dried Micrococcus lysodeikticus cell walls on Petri dishes, was significantly reduced in presence of zinc. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in seastars due to the decrease of antibacterial protection. The benefits of using the test of lysozyme activity to monitoring environmental pollution are highlighted.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Crystallographic determination of the mode of binding of oligosaccharides to T4 bacteriophage lysozyme: Implications for the mechanism of catalysis

Phage lysozyme has catalytic activity similar to that of hen egg white lysozyme, but the amino acid sequences of the two enzymes are completely different.The binding to phage lysozyme of several saccharides including N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) and (GlcNAc)₃ have been determined crystallographically and shown to occupy the pronounced active site cleft. GlcNAc binds at a single location analogous to the C site of hen egg white lysozyme. MurNAc binds at the same site. (GlcNAc)₃ clearly occupies sites B and C, but the binding in site A is ill-defined.Model building suggests that, with the enzyme in the conformation seen in the crystal structure, a saccharide in the normal chair configuration cannot be placed in site D without incurring unacceptable steric interference between sugar and protein. However, as with hen egg white lysozyme, the bad contacts can be avoided by assuming the saccharide to be in the sofa conformation. Also Asp20 in T4 lysozyme is located 3 Å from carbon C₍₁₎ of saccharide D, and is in a position to stabilize the developing positive charge on a carbonium ion intermediate. Prior genetic evidence had indicated that Asp20 is critically important for catalysis. This suggests that in phage lysozyme catalysis is promoted by a combination of steric and electronic effects, acting in concert, The enzyme shape favors the binding in site D of a saccharide with the geometry of the transition state, while Asp20 stabilizes the positive charge on the oxocarbonium ion of this intermediate. Tn phage lysozyme, the identity of the proton donor is uncertain. In contrast to hen egg white lysozyme, where Glu35 is 3 Å from the glycosidic DOE bond, and is in a non-polar environment, phage lysozyme has an ion pair, Glull … Arg145, 5 Å away from the glycosidic oxygen. Possibly Glull undergoes a conformational adjustment in the presence of bound substrate, and acts as the proton donor. Alternatively, the proton might come from a bound water molecule.     

Lysis of Micrococcus lysodeikticus cells by lysozyme covalently immobilized on the lumen of hollow fibers

Summary A lytic enzyme reactor for microbial cell lysis is described in which lysozyme is immobilized on the lumen of hemodialyzer hollow fibers using epichlorohydrin as a coupling agent. The cell suspension flows through the lumen without any hindrance where the cells are lyzed by the immobilized lysozyme efficiently. Micrococcus lysodeikticus cells at concentrations of 0.25 g/L and 5 g/L were successfully lyzed without clogging the hollow fiber. In comparison with lysozyme immobilized on submicron particles, the activity retention was at least 8 times higher.     

Expression,characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> A16

Lysozyme is an enzyme that is essential for protection against bacterial infections. In this study, a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H. polymorpha chromosome. Recombinant T4 lysozyme was successfully expressed in the yeast H. polymorpha A16; 0.49 g L⁻¹ secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0. Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria, Micrococcus lysodeikticus, and the gram negative bacteria Xanthomonas campestris pv. malvacearum and Xanthomonas oryzae pv. oryzae. The zone of inhibition assay was used to evaluate antimicrobial activity. Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme. SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme. SDS-PAGE without 0.2 mol L⁻¹ dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed inter- and intra-molecular disulfide bonds which resulted in loss of enzyme activity.     

Partial characterization of the cell walls ofMycobacterium leprae

Cell walls were prepared fromMycobacterium leprae (separated and purified from experimentally infected armadillo),M. tuberculosis, M. smegmatis, andMicrococcus lysodeikticus. The purity of the above wall preparations was confirmed after negative staining and shadow-casting and subsequent observation under the electron microscope. As judged from the electron microscopic observations, the bacteria were of different fragility in the following increasing order:M. tuberculosis, M. smegmatis, M. leprae, andMicro. lysodeikticus. The cell walls were hydrolyzed with 6N HCl, and the amino acids were identified by thin-layer chromatography compared with the authentic standards. With the same purification procedures, it was not possible to obtain satisfactorily pure peptidoglycan fromM. leprae. In leprosy bacilli,meso-DAP was found to be present, ín the walls; however, contamination by nonwall amino acids did not allow the confirmation of previous results, a finding that suggests that glycine completely replaced L-alanine inM. leprae cell walls.