Heterogeneity of serum, egg amylase and egg lysozyme in the interspecific hybrids of ducks

Polymorphism of serum and egg amylase by means of horizontal agarose gel electrophoresis and egg lysozyme by means of horizontal starch gel electrophoresis in Pekin, Muscovy ducks and their interspecific hybrids was studied. In the interspecific hybrids of ducks the codominant type of heredity of serum, egg yolk and egg white amylase isozymes, as well as egg white lysozyme, were found.     

A New Method for the Analysis of Fatty Acid Mixtures by Gas Chromatography

Deamidation of lysozyme was observed during storage in a buffer solution and in egg white. The peak corresponding to native lysozyme from Bio-Rex 70 column chromatography was gradually decreased, while the peaks corresponding to deamidated lysozyme were increased during storage in 0.1 m carbonate buffer at pH 9.5. A similar change was observed during storage in egg white, but the change in egg white was larger than that in the buffer solution. A detailed analysis of the elution peaks from the Bio-Rex 70 column suggested that one to three residues of amide in lysozyme were mainly deamidated during storage in the buffer solution, and that more than three residues in lysozyme were deamidated during storage in egg white. There were significant differences in lysozyme activity between native and deamidated lysozyme, the activity being decreased in proportion to the degree of deamidation.     

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.     

Purification of the basic protein avidin using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected.     

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.     

Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk

Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.     

Immunocytochemical observations on the organ distribution of exogenous monomeric and dimerized lysozyme.

Using commercial anti-lysozyme antibodies and anti-dimerized lysozyme rabbit serum produced by us we demonstrated by immunohistochemistry, in some organs of rats, the expression of exogenous egg white lysozyme preparations beside the native lysozyme. After oral administration, the egg white lysozyme was detected in intestinal epithelium, proximal and distal tubules of some nephrons, pulmonary alveolar walls and hepatocytes in the 3rd zone of liver acini, whereas native lysozyme was strongly expressed in intestinal and pulmonary macrophages in both the experimental and control animals. However, expression of the dimerized lysozyme released from the intraperitoneally implanted mini-osmotic pumps and detected using specific antisera was evident only on erythrocytes in intestinal blood vessels. It is concluded that the lysozyme preparations administered per os or parenterally are resorbed to blood circulation and distributed among various organs in an active form and maintaining their antigenic specificity. It may speak for their direct anti-inflammatory and immunomodulatory effects in respiratory, urinary, digestive and other systems.     

Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.     

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Biochemical characterization of lysozymes present in egg white of selected species of anatid birds

The isolation of lysozyme from the egg white of several representative species of waterfowl is described. The purified lysozymes were analyzed in order to determine the type and molecular weight of each enzyme. All enzymes found in duck egg whites were found to be of the c-type. In contrast all true geese, the Mute Swan as well as the Northern Blackneck Screamer contain lysozyme g in their egg white.     

Biochemical characterization of lysozymes present in egg white of selected species of anatid birds

The isolation of lysozyme from the egg white of several representative species of waterfowl is described. The purified lysozymes were analyzed to determine the type and molecular weight of each enzyme. All enzymes found in duck egg whites were found to be of the c-type. In contrast all true geese, and the mute swan species as well as the northern blackneck screamer contain lysozyme g in their egg white.     

Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of alpha-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site. G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions. Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme. The variation in the position of the remaining boundary may be related to differences in substrate binding.     

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.     

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Identification of a Component of Crystalline Egg Albumin Bactericidal for Thermophilic Aerobic Sporeformers

During an investigation of the effect of basic and acidic proteins on the growth of thermophilic aerobic sporeformers, crystalline egg albumin was found to be strongly bactericidal. This finding was uncharacteristic of acidic proteins. The bactericidal fraction was heat sensitive and separated from the non-bactericidal albumin fraction during gel filtration on Sephadex G-75. Cells of Micrococcus lysodeikticus and Bacillus stearothermophilus were lysed rapidly by the bactericidal component, leading to its tentative identification as lysozyme. The bactericidal substance possessed an electrophoretic mobility on polyacrylamide gel containing sodium dodecyl sulfate identical to that of crystalline egg white lysozyme. Users of crystalline egg albumin are cautioned that commerical preparations may be contaminated with lysozyme. Destruction of the thermophilic aerobes by lysozyme should be considered when performing counts on egg products.     

A simple technique for the immobilization of lysozyme by cross-linking of hen egg white foam

A novel technique has been described for the immobilization of lysozyme, naturally present in hen egg white by cross-linking the egg white foam with glutaraldehyde. This technique results in a mechanically stable and porous matrix exhibiting about 6-times the lytic activity against Micrococcus lysodeikticus cells, as compared to the unfoamed matrix. Foamed egg white matrix can be used for the continuous lysis of bacterial cells     

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO₅₀PO₅₀/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188 U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.     

Egg white lysozyme is the major protein of the hen's egg vitelline membrane

Lysozyme accounts for 37% of the proteins of the hen's egg vitelline membrane. It can be extracted by salt solutions and purified by gel filtration on Sephadex G-50. There are no differences between the chemical and enzymic properties of egg white and vitelline membrane lysozymes. Vitelline membranes of ovarian eggs do not contain lysozyme. It is thus concluded that lysozyme is localized in the outer layer. Vitelline membranes from fertilized and unfertilized eggs contain the same amount of lysozyme; its percentage decreases after two days of incubation.     

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system. (c) 1995 John Wiley & Sons, Inc.