Fraction Of Lysozyme Research Articles

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

Rapid and efficient sprayed multilayer films for controlled drug delivery

ABSTRACTThe speed and scalability of film fabrication can dictate the translation of technologies from the laboratory scale to industrial level mass production. Spray‐assisted layer‐by‐layer (LbL) film assembly enables the rapid coating of geometrically complex and porous substrates with functional polyelectrolyte multilayers. Unfortunately, the encapsulation efficiency can be as low as one percent, making this technique prohibitively costly with even modestly priced materials. Herein, we used containment chambers to separately capture the aerosolized solutions for each step in the spray‐LbL process and analyzed the effect of recycling on multilayer film assembly. With potential biomedical applications, we studied the controlled release films of (Poly 2/heparin/lysozyme/heparin)n films and tracked the distribution of lysozyme after film assembly. In a “Conventional” Spray‐LbL protocol, only 6% of the aerosolized lysozyme is incorporated into the film. By collecting and returning the expended solutions to their respective reservoirs (Recycle Spray‐LbL), we increased this efficiency to 15%. We also tuned the final distribution of lysozyme by adjusting the spray times (Optimized Spray‐LbL), which minimized the amount of lysozyme lost to non‐specific adsorption and reduced the fraction of lysozyme lost to the wash step from 30% and 75% (Conventional and Recycle Spray‐LbL, respectively) to 13%. Despite the changes in film assembly parameters, each film demonstrated similar controlled release properties. With the inherent limitations of time and cost facing Dip and Conventional Spray‐LbL technologies, respectively, the implementation of recycling to the latter demonstrates improvements in efficiency and time that may make it more attractive for the manufacture of biomedical coatings. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43563.

Foam fractionation of protein: Correlation of protein adsorption onto bubbles with a pH-induced conformational transition

A foam fractionation apparatus was prepared to aid protein separation at the gas–liquid interface. Using lysozyme as a model protein, we investigated the alteration of enzymatic and optical activities through foaming. The lysozyme transferred to the gaseous nitrogen phase after 5 min of bubbling with no exogenous detergent. The bacteriolytic and optical activities of lysozyme from the foamate were nearly equivalent to those of the original lysozyme. This result indicated that lysozyme did not irreversibly denature during foam fractionation. We then performed protein separation using binary mixtures of lysozyme and α-amylase. When the two proteins were dissolved in bulk solution of pH 10.5, which is close to the isoelectric point (p I) of lysozyme (10.7), selective fractionation of lysozyme from the foam was observed. Indeed, this fractionation was identical to that from a single component solution of lysozyme. Similarly, selective fractionation of α-amylase was achieved in pH 3.0 buffer. Furthermore, circular dichroism (CD) and subsequent model fitting revealed that the protein had a reduced or nearly complete absence of α-helical content, whereas the amount of β-sheet structure and random coil was elevated in the buffer conditions that promoted protein adsorption. These results indicate that a pH-induced conformational transition might correlate with protein foaming.

Rejection amplification in the ultrafiltration of binary protein mixtures using sandwich configurations

Protein fractionation of lysozyme and myoglobin mixtures was investigated using sandwich arrangements of ultrafiltration membranes under controlled permeate flux. Both proteins have close molecular weights with values of 14300 g mol −1 for lysozyme and 17400 g mol −1 for myoglobin. The aim was to improve the selectivity of the separation by masking the pore size distribution of ultrafiltration membranes by having one membrane on top of another. Two membranes were arranged together in sandwich configurations. Two possible sandwich configurations were compared by assessing the transmission and selectivity for the protein mixture. For both sandwich configurations membrane fouling was reduced compared with a single membrane. Overall the S-sandwich with Biomax PES membranes was superior.

Structure and Stability of Complex Coacervate Core Micelles with Lysozyme

Encapsulation of enzymes by polymers is a promising method to influence their activity and stability. Here, we explore the use of complex coacervate core micelles for encapsulation of enzymes. The core of the micelles consists of negatively charged blocks of the diblock copolymer PAA42PAAm417 and the positively charged homopolymer PDMAEMA150. For encapsulation, part of the positively charged homopolymer was replaced by the positively charged globular protein lysozyme. We have studied the formation, structure, and stability of the resulting micelles for three different mixing ratios of homopolymer and lysozyme: a system predominantly consisting of homopolymer, a system predominantly consisting of lysozyme, and a system where the molar ratio between the two positively charged molecules was almost one. We also studied complexes made of only lysozyme and PAA42PAAm417. Complex formation and the salt-induced disintegration of the complexes were studied using dynamic light-scattering titrations. Small-angle neutron scattering was used to investigate the structures of the cores. We found that micelles predominantly consisting of homopolymer are spherical but that complex coacervate core micelles predominantly consisting of lysozyme are nonspherical. The stability of the micelles containing a larger fraction of lysozyme is lower.

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition.

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution-alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.

A proposed mechanism for detergent-assisted foam fractionation of lysozyme and cellulase restored with β-cyclodextrin

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as beta-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to beta-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.

Adsorption Isotherms and Irreversible Binding of Proteins on Commercially Available Hydrophobic Adsorbents

Measurement of ascending and descending breakthrough curves of two proteins, hen egg white lysozyme and bovine serum albumin, on commercially available hydrophobic-interaction chromatographic columns reveal that only one part, i.e., the reversible adsorbed fraction, of the total bound amount can be desorbed by dilution of the feed solution. The other part is bound irreversibly; its binding behavior follows a hysteresis. Our procedure allows a clear separation of both parts. The reversibly adsorbed fraction of lysozyme can be represented with a Henry-type isotherm. For both proteins and for the reversible as well as the irreversible part, the adsorbed amount increases when the number of charges of the proteins decreases by approaching the isoelectric point.

Fractionation of Lysozyme and Chicken Egg Albumin Using Ultrafiltration with 30-kDa Commercial Membranes

Lysozyme is an important protein in chicken egg white (CEW) and it is feasible to separate lysozyme from other CEW proteins using ultrafiltration. To identify suitable conditions for the fractionation of lysozyme from CEW, we studied with the model binary mixture of lysozyme and chicken egg albumin (CEA) with the composition and concentration of the two proteins similar to those in natural CEW. Two commercial ultrafiltration (UF) membranes of 30-kDa molecular weight cutoff (MWCO) made of regenerated cellulose (RC) and poly(ether sulfone) (PES) membranes were used. By systematically examining the effect of the physicochemical properties of the solution and the hydrodynamics of the membrane systems, the most suitable conditions to achieve maximum transmission and selectivity for lysozyme were identified. The PES UF membranes offer distinct advantages for the fractionation of lysozyme from this binary mixture. With the 30-kDa PES UF membrane, the suitable conditions were identified as pH 10−11, salt concentr...

Effect of cation species on equilibrium of reversed micellar extraction of lysozyme

Ion strength is one of the important conditions to control extraction of lysozyme with AOT reversed micelles. The extraction equilibrium changes largely with a type of the salt, i.e. cation species. For the three salt systems, KCl, BaCl2 and CaCl2, extracted fraction of lysozyme is measured for various pH values. For divalent cation systems, the behavior of the protein-extracted fraction against pH is quite different from that for KCl system. The salt concentration has a strong influence on the extracted fraction and the characteristic trend is explained by the manner of the interaction between divalent cation and anionic surfactant, AOT. Difference of the trend in the extracted fraction between barium and calcium systems is also discussed in terms of hydration of the cations. Because of the larger hydration number, combination of calcium ion to interfacial layer of AOT makes the interface rather flexible, thus the cation exchange occurs more frequently than in barium system.

Effect of cation species on equilibrium of reversed micellar extraction of lysozyme

Abstract Ion strength is one of the important conditions to control extraction of lysozyme with AOT reversed micelles. The extraction equilibrium changes largely with a type of the salt, i.e. cation species. For the three salt systems, KCl, BaCl2 and CaCl2, extracted fraction of lysozyme is measured for various pH values. For divalent cation systems, the behavior of the protein-extracted fraction against pH is quite different from that for KCl system. The salt concentration has a strong influence on the extracted fraction and the characteristic trend is explained by the manner of the interaction between divalent cation and anionic surfactant, AOT. Difference of the trend in the extracted fraction between barium and calcium systems is also discussed in terms of hydration of the cations. Because of the larger hydration number, combination of calcium ion to interfacial layer of AOT makes the interface rather flexible, thus the cation exchange occurs more frequently than in barium system.

Lytic Activity and Biochemical Properties of Lysozyme in the Coelomic Fluid of the Sea UrchinStrongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchinStrongylocentrotus 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° and 25°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.

Hollow-Fiber Affinity Partitioning Chromatography Using Affinity-Based Reversed Micelles as Stationary Phase

An organic phase containing reversed micelles of Cibacron Blue F-3GA modified lecithin (CB-lecithin) was attached to microporous polypropylene hollow-fiber membranes. This led to the formation of a hollow-fiber column with immobilized dye-affinity reversed micelles within the membrane pores as the stationary phase. This column for chromatographic separation of proteins was called hollow-fiber affinity partitioning chromatography (HFAPC). With a column of 3 mL in net hollow fiber volume, lysozyme and bovine serum albumin (BSA) could be completely separated, and HFAPC experiments with overloaded lysozyme/BSA mixtures yielded a lysozyme fraction with a purity of 93.5% and a high yield. Furthermore, lysozyme was purified by the HFAPC from a crude chicken egg-white solution containing 9.56 mg proteins with a recovery yield of 98.2%. The purity of the lysozyme fraction was increased by 47.4-fold, reaching 85.3% of the total proteins.

Extraction of Proteins by Reversed Micellar Solution in a Packed Column.

Protein extraction using reversed micelles is carried out in a packed column. The extracted fraction of lysozyme and the overall capacity coefficient, Kca, are measured for various flow rates and different packing materials. The same column without packings is also used as a spray column. By using a flat interface stirred cell, mass transfer coefficients for the aqueous and the organic phases are measured as well as the protein transfer rate. The results indicate that the resistance of protein solubilization at the interface controls protein transfer from the aqueous into the organic phase. The extracted fraction in the packed column is about three times larger than that for the spray column due to the larger holdup of dispersed phase in the packed column. Since the drops hardly break or coalesce in the packed bed and the protein transfer coefficient is constant, the overall capacity coefficient is controlled by the holdup of dispersed phase. On the assumption that the drop size and the transfer coefficient are constant, the correlation between holdup and flow rates is obtained. Values of Kca and the extracted fraction are calculated from the holdup correlation. Effects of packing materials on the values of Kca are small. A high protein activity is retained in the packed column operation, which indicates that the packed column does little harm to proteins due to moderate mixing.

Timing, targeting and sorting of azurophil granule proteins in human myeloid cells.

Biosynthesis of 3 human granule proteins, myeloperoxidase, defensin and lysozyme, all present in azurophil granules, was investigated in normal bone marrow cells and in the promyelocytic cell line HL-60 to see whether differences in timing of biosynthesis could explain the well established differences in their subcellular localization in the mature neutrophil (targeting), and whether differences exist in the efficiencies by which granule proteins are retained in cells (sorting). Normal human bone marrow cells were separated into three bands by density gradient centrifugation. Band 1 contains band and segmented cells, band 2 mainly myelocytes, metamyelocytes and some band cells, and band 3 myeloblasts and promyelocytes in addition to megakaryocytes and proerythroblasts. Cells from these bands, as well as undifferentiated HL-60 cells, were pulsed with radiolabeled cysteine and methionine, and biosynthesis of granule proteins was subsequently evaluated by immunoprecipitation and quantified by phosphorimaging. Myeloperoxidase synthesis was maximal in cells from band 3 while defensin biosynthesis was maximal in cells from band 2. Lysozyme was synthesized in cells from all bands but was maximal in cells from band 2. These results are in agreement with our hypothesis that timing of biosynthesis determines the localization of individual granule proteins. While myeloperoxidase and defensins were efficiently retained in immature cells (band 3), a significant fraction of lysozyme was routed out of the cells, showing that differences exist in the sorting of granule proteins between constitutive and regulated secretion. In addition, defensin was less efficiently retained in cells from band 2 than from band 3, indicating that sorting mechanisms may depend on the stage of cell maturation.

Nonoxidative antimicrobial effects of human polymorphonuclear leukocyte granule proteins on Chlamydia spp. in vitro.

Proteins from isolated granules of human polymorphonuclear leukocytes were assessed for their nonoxidative microbicidal effect on chlamydiae by two different methods: a radioisotope assay for elementary body integrity and a biological assay for inclusion development. Crude granule extract, which consisted of a mixture of all granule proteins, caused a 20 to 30% decrease in infectivity and a 52% decrease in infectivity when incubated with Chlamydia psittaci CAL-10 and Chlamydia trachomatis serovar E, respectively. To define more specifically the components that were damaging to chlamydiae, crude granule extract was subjected to Sephadex G-75 column chromatography and isolated granule fractions were obtained. Only fractions containing lysozyme as the major component consistently caused reductions in infectivity of C. trachomatis elementary bodies. In contrast, fractions collected after the lysozyme fraction, containing proteins with molecular masses of 13,000 daltons or less, had detrimental effects on C. psittaci infectivity. Additional experiments using highly purified human polymorphonuclear leukocyte lysozyme confirmed its infectivity-reducing action upon C. trachomatis but not upon C. psittaci.

The Relationship of the Lysozyme Fraction in Thick Egg White to Fertility and Hatchability of Eggs

INTRODUCTIONCONSIDERABLE variation exists in fertility and hatchability of eggs among hens within individual breeder flocks as well as between different flocks. Likewise the height of the thick albumen of fresh eggs differs greatly among hens within a given flock. Forsythe and Foster (1950) reported that differences in the electrophoretic components of egg white from six strains of chickens were statistically significant. Cotterill and Winter (1954) reported significant differences between hens in lysozyme level of the egg white. The level of lysozyme was determined by the degree of lysis of a cell suspension of M. lysodeikticus. The same authors (1955) postulated that the interaction of lysozyme and ovomucin was responsible for the firm gel of thick egg white. Wilcox (1955) accounted for the increased thick albumen of eggs having a high lysozyme content by increased complexing between lysozyme and ovomucin. Studies by Wilcox and Cole (1954) indicate that the lysozyme content…

Incorporation of 14 C-Amino Acids into Egg Proteins by the Laying Hen , ,

LAYING hens have been fed and injected with various radioactive compounds in order to investigate the synthesis of egg constituents as well as to produce labeled egg materials for use in subsequent experimentation. Mandeles and Ducay (1962) injected laying hens with 14 C-lysine and 14 C-glutamic acid and examined the radioactivity in the conalbumin, ovalbumin, and lysozyme protein fractions of the egg white. Following a single injection of the two amino acids, peak activities in the egg white proteins were observed in the second egg produced after injection. The rates at which labeled amino acids were incorporated into the conalbumin and ovalbumin fractions were similar, but higher than for the lysozyme fraction. These observations in conjunction with results obtained with oviduct minces (Mandeles and Ducay, 1962) suggested that the time or rate of synthesis for these proteins varied during the egg laying cycle and that the oviduct was the common site of…