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.     

Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11 degrees and 25 degrees C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS-PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. Copyright 1999 Academic Press.     

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.     

Co-extraction of egg white proteins using ion-exchange chromatography from ovomucin-removed egg whites

Efficient isolation of egg white components is desired due to its potential uses. Existing methods mainly targeted on one specific protein; an attempt has been made in the study to co-extract all the valuable egg white components in a continuous process. Ovomucin was first isolated by our newly developed two-step method; the resultant supernatant obtained after ovomucin isolation was used as the starting material for ion-exchange chromatography. Anion-exchange chromatography of 100 mM supernatant yielded a flow-through fraction and three other fractions representing ovotransferrin, ovalbumin and flavoproteins. The flow-through fraction was further separated into ovoinhibitor, lysozyme, ovotransferrin and an unidentified fraction which represents 4% of total egg white proteins. Chromatographic separation of 500 mM supernatant resulted in fractions representing lysozyme, ovotransferrin and ovalbumin. This co-extraction protocol represents a global recovery of 71.0% proteins.     

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.     

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.     

Novel isoelectric precipitation of proteins in a pressurized carbon dioxide-water-ethanol system

A novel isoelectric precipitation of proteins in a pressurized carbon dioxide-water-ethanol system was developed where carbon dioxide was used as a volatile acid. The pH-pressure curves of the system with the absence and presence of proteins were investigated. By introducing the pressurized carbon dioxide to a solution containing protein, the pH value in the solution was decreased to the isoelectric region of the model protein BSA. Addition of ethanol could lower the buffer capacity of the protein, which made the precipitation concentration of protein go beyond the limits in a system without ethanol and well exploited the application field of the technique. In addition, ethanol in solution played the role of aiding precipitation in the process. Another model protein, hen egg white lysozyme, was also studied but could not be precipitated in the above system. All of these phenomena prove that isoelectric precipitation is the key point in the pressurized carbon dioxide-water-ethanol system.     

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.     

Protein refolding at high concentration using size-exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. (c) 1996 John Wiley & Sons, Inc.     

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.     

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.     

Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.     

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.     

Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification

Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.     

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.     

The influence of operational parameters on lysozyme refolding using size-exclusion chromatography

The influence of several parameters on the gel filtration refolding of hen egg white lysozyme from a starting concentration of 40 mg/ml was investigated. Refolding was found to be unaffected by temperature between 30 and 50°C, giving 100% recovered specific activity. At 10°C a 20% reduction in refolding yield was observed. Refolding was carried out successfully with both acrylamide (Sephacryl S100)- and dextran (Superdex 75)-based gel media. At the isoelectric pH of lysozyme, aggregation was suppressed in the column method, whereas protein aggregates were formed during dilution-based refolding. A number of compounds (carboxymethyl cellulose, dextran, sucrose) were added to the mobile phase to reduce the relative viscosity between the sample and mobile phase. Only sucrose, up to 20% (wt), was found not to interfere with lysozyme refolding.     

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.     

Improved method for rapid purification of protein kinase from streptomycetes

Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, ∼ 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylaminde gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues.     

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.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.