Native Lysozyme Research Articles

Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme).

Hydrophobic interaction is important for protein conformation. Conjugation of a hydrophobic group can introduce intermolecular hydrophobic contacts that can be contained within the molecule. It is possible that a strongly folded state can be formed in solution compared with the native state. In this study, we synthesized cholesteryl conjugated lysozyme (CHLysozyme) using lysozyme and cholesterol as the model protein and hydrophobic group, respectively. Cholesteryl conjugation to lysozyme was confirmed by nuclear-magnetic resonance. Differential-scanning calorimetry suggested that CHLysozyme was folded in solution. CHLysozyme secondary structure was similar to lysozyme, although circular dichroism spectra indicated differences to the tertiary structure. Fluorescence measurements revealed a significant increase in the hydrophobic surface of CHLysozyme compared with that of lysozyme; CHLysozyme self-associated by hydrophobic interaction of the conjugated cholesterol but the hydrophobic surface of CHLysozyme decreased with time. The results suggested that hydrophobic interaction changed from intramolecular interaction to an intermolecular interaction. Furthermore, the relative activity of CHLysozyme to lysozyme increased with time. Therefore, CHLysozyme likely forms a folded state with an extended durability of activity. Moreover, lysozyme was denatured in 100% DMSO but the local environment of tryptophan in CHLysozyme was similar to that of a native lysozyme. Thus, this study suggests that protein solution stability and resistance to organic solvents may be improved by conjugation of a hydrophobic group.

Novel lysozyme–mannooligosaccharide conjugate with improved antimicrobial activity: preparation and characterization

Lysozyme is a well-known natural antimicrobial agent commonly used in the food industry. However, the lack of antimicrobial activity against Gram-negative bacteria limits its application. In order to extend the antimicrobial spectrum of lysozyme, a lysozyme–mannooligosaccharide (MOS) conjugate was prepared in this study via the Maillard reaction under optimal conditions, i.e., the lysozyme–MOS ratio of 1:7.5, pH 7.5 and reaction duration of 6 days. The prepared conjugate was subjected to a series of assays, including the free amino group content assay, intrinsic fluorescence spectrometry, surface hydrophobicity assay and FT-IR spectrometry. During conjugation, 55.76% of the free amino groups on lysozyme were consumed, suggesting better reactivity of MOS. Both intrinsic fluorescence spectra and surface hydrophobicity index indicated that more hydrophobic residues embedded in lysozyme would be exposed during conjugation, but the conjugation of MOS lowered both parameters, making the lysozyme–MOS conjugate more hydrophilic and soluble. Furthermore, the antimicrobial activity demonstrated that the lysozyme–MOS conjugate possessed improved activity against Gram-negative bacteria, including Escherichia coli and Salmonella enterica, with almost 50% reduction of log CFU, compared to the native lysozyme. The extended antimicrobial spectrum of lysozyme will broaden its applications as a preservative and provide an effective alternative to existing antibiotics in the food industry.

In Situ Absorption and Fluorescence Microspectroscopy Investigation of the Molecular Incorporation Process into Single Nanoporous Protein Crystals.

Protein crystals exhibit distinct three-dimensional structures, which contain well-ordered nanoporous solvent channels, providing a chemically heterogeneous environment. In this paper, the incorporation of various molecules into the solvent channels of native hen egg-white lysozyme crystals was demonstrated using fluorescent dyes, including acridine yellow G, rhodamine 6G, and eosin Y. The process was evaluated on the basis of absorption and fluorescence microspectroscopy at a single-crystal level. The molecular loading process was clearly visualized as a function of time, and it was determined that the protein crystals could act as nanoporous materials. It was found that the incorporation process is strongly dependent on the molecular charge, leading to heterogeneous molecular aggregation, which suggests host–guest interaction of protein crystals from the viewpoint of nanoporous materials.

Direct Detection of Lysozyme in Viscous Raw Hen Egg White Binding to Sodium Dodecyl Sulfonate by Reactive Wooden-tip Electrospray Ionization Mass Spectrometry.

Direct characterization of native protein binding to ligands in raw biological samples is a challenging task, because the ligand solution might induce proteins to aggregation, flocculation and denaturation. In this work, we developed a reactive wooden-tip electrospray ionization mass spectrometry (ESI-MS) for formation and characterization of protein-ligand complexes upon rapid mixing in electrospray droplets. Raw viscous hen egg white (HEW) was directly loaded onto a wooden tip to induce spray ionization, and sodium dodecyl sulfonate (SDS) solution was directly loaded into the HEW spray by a pipette tip, and thus lysozyme-DS complexes were then formed in the electrospray droplets and were detected subsequently by mass spectrometry. The new approach was successfully applied to investigate interaction of SDS and native lysozyme in electrospray droplets of standard solution and raw egg white. Our results showed that wooden-tip ESI-MS is a promising method to form and characterize protein-ligand complexes.

Sugar Vinyl Sulfoxide Glycoconjugation of Peptides and Lysozyme: Abrogation of Proteolysis at the Lysine Sites.

We describe a glycoconjugation strategy in which a sugar vinyl sulfoxide, acting as Michael donor, reacts efficiently with amine nucleophiles arising from the lysine side chain in peptides and proteins, at physiological pH and temperature. The method permits glycoconjugation of the lysine residues present in lysozyme with the sugar vinyl sulfoxide. The glycoconjugation of the protein abrogates the trypsin-mediated proteolysis at the lysine sites. The modified protein catalyzes digestion of the Gram-negative Escherichia coli cell wall and retains the same antimicrobial property as the native lysozyme.

Bacterial killing is enhanced by exogenous administration of lysozyme in the lungs

ObjectiveLysozyme, a 14-kDa protein, is one of the most abundant antimicrobials in the lungs. Its concentration in airway surface sufficient to kill several bacterial pathogens in vitro. The purpose of this study was to determine if administration of exogenous lysozyme would further enhance bacterial killing in vivo. MethodsTo assess the effect of acute lung infection on endogenous lysozyme protein levels, mice were infected by intratracheal instillation of Pseudomonas aeruginosa and bronchoalveolar (BAL) fluid assessed for lysozyme concentration and for muramidase activity. In order to inform in vivo testing, species-specific bacterial killing efficacy was determined by incubating mucoid P. aeruginosa with 2×105 units of chicken lysozyme, human lysozyme or with vehicle at 37°C for 2hours. Subsequently, mice challenged with intratracheally-administered mucoid P. aeruginosa, were reintubated and injected with 2×105 Units of native human lysozyme, recombinant human lysozyme or with vehicle. Lung bacterial burden was enumerated subsequently. ResultsThe concentration of lysozyme protein in BAL fluid from mice challenged with mucoid clinical isolate of P. aeruginosa was increased 4-fold at 6hours post-infection. Quantitative culture showed that the number of recoverable bacteria was significantly decreased by both chicken and human lysozyme compared to vehicle but human lysozyme was significantly more effective than chicken egg lysozyme. In vivo, 24hours post-infection quantitative culture of lung homogenates showed that the number of viable bacteria recovered from mice treated with either native or recombinant lysozyme was decreased with 0.76±0.25×104 and 0.84±0.16×104, respectively, vs. 7.0±2.52×104 CFU/g protein in mice treated with HBSS, both P<0.05. ConclusionsThese results indicate that endogenous lysozyme is increased during acute lung infection and that early administration of exogenous lysozyme further enhances bacterial killing in vivo.

Lysozyme encapsulated gold nanoclusters for probing the early stage of lysozyme aggregation under acidic conditions

Protein aggregation can lead to several incurable amyloidosis diseases. The full aggregation pathway is not fully understood, creating the need for new methods of studying this important biological phenomenon. Lysozyme is an amyloidogenic protein which is often used as a model protein for studying amyloidosis. This work explores the potential of employing Lysozyme encapsulated gold nanoclusters (Ly-AuNCs) to study the protein's aggregation. The fluorescence emission properties of Ly-AuNCs were studied in the presence of increasing concentrations of native lysozyme and as a function of pH, of relevance in macromolecular crowding and inflammation-triggered aggregation. AuNC fluorescence was observed to both redshift and increase in intensity as pH is increased or when native lysozyme is added to a solution of Ly-AuNCs at pH 3. The long (μs) fluorescence lifetime component of AuNC emission was observed to decrease under both conditions. Interestingly it was found via Time-Resolved Emission Spectra (TRES) that both AuNC fluorescence components increase in intensity and redshift with increasing pH while only the long lifetime component of AuNC was observed to change when adding native lysozyme to solution; indicating that the underlying mechanisms for the changes observed are fundamentally different for each case. It is possible that the sensitivity of Ly-AuNCs to native lysozyme concentration could be utilized to study early-stage aggregation.

Thermo-separating polymer-based aqueous two-phase systems for the recovery of PEGylated lysozyme species

Fractionation of native, mono and di-PEGylated lysozyme was performed in 36 different polymer-polymer aqueous two-phase systems using UCON as a phase-forming component. After a discrete partition analysis, dextran 75 kDa-UCON, volume ratio 3, tie-line length 35% w/w; ficoll 70 kDa-UCON, volume ratio 1, tie-line length 45% w/w and a PEG 8 kDa-UCON volume ratio 3, tie-line length 65% w/w systems were selected for optimization via salt addition and to observe the behavior of the lysozyme species in mixtures. The dextran-UCON and the PEG-UCON systems with 75 mM NaCl showed effectiveness in separating 75% and 87% of mono-PEGylated lysozyme from the rest of the lysozyme species in the top and bottom phases, respectively. These results are an advancement in incorporating these extractions in different processes since the use of UCON simplifies the removal of the polymers, providing the opportunity of recycling it to the operation.

Co-aggregation of ovotransferrin and lysozyme

Ovotransferrin (OVT) is the main protein component of egg white responsible for initial gelation due to its high thermal susceptibility. Co-aggregation of lysozyme (LYZ) is involved in OVT aggregate formation at low temperatures during pasteurization. Undesirable formation of aggregates limits the degree of thermal processing that can be applied to egg white products. However, the characteristics of co-aggregates of OVT and LYZ have not been elucidated. Here, we determined the thermal co-aggregation process of OVT and LYZ in terms of protein composition, structure, intermolecular forces, and morphology. The amount of LYZ involved in co-aggregates was dependent on the amount of aggregated OVT regardless of the mixing ratio. Native LYZ had the capability of precipitating soluble OVT aggregates by non-covalent association. The co-aggregates of OVT and LYZ formed colloidal particles with a large network, which was not observed in systems consisting of either protein alone. The hierarchical co-aggregation of OVT and LYZ started from the aggregation of OVT involving LYZ non-covalently, which suppresses electrostatic repulsion between soluble OVT aggregates, followed by the growth of insoluble aggregates by disulfide bond crosslinkage between the soluble aggregates. These results provide information regarding efficient pasteurization and thermal processing of hen egg white.

X-ray crystallographic analysis of time-dependent binding of guanidine hydrochloride to HEWL: First steps during protein unfolding

Time-dependent binding of guanidine hydrochloride (GuHCl) to hen egg-white lysozyme (HEWL), and effects of this binding on the protein structure have been investigated by solving X-ray structures of crystalline complexes. The complexes have been prepared by soaking, for different periods of time, native lysozyme crystals in solutions containing 2.5M GuHCl. In the refined structures, the number of water molecules in the protein's first solvent shell has progressively decreased from 152 to 115, showing protein's preference for guanidinium over water. Guanidinium ions preferentially hydrogen bond with the backbone carbonyl oxygen atoms. In their van der Waals interactions, they do not show any preference for apolar residues. Guanidinium ions have replaced water molecules that form cages around exposed hydrophobic residues. Guanidinium binding has decreased the average length of water-water hydrogen bond by 0.1Å. The hydrogen bonds between main chain atoms have been weakened by GuHCl, and this may be the reason for increased potency of GuHCl compared to urea. Guanidinium binding destabilizes the β-domain by causing loss of hydrogen bonds involving Asn 59 side chain. Interestingly, this loss is almost identical to that observed in structures of amyloidogenic variants of human lysozyme. Compounds preventing this loss could be anti-amyloidogenic.

The antimicrobial spectrum of lysozyme broadened by reductive modification.

In order to broaden the antimicrobial action spectrum of lysozyme against Gram-negative bacteria, different partially reduced lysozyme derivatives were obtained by reducing the R-S-S-R bonds of lysozyme using the reducing agent Na2SO3. The circular dichroism behavior analysis showed that the tertiary structure of the partially reduced lysozyme molecules became more flexible, leading to an obvious increase in the surface hydrophobicity and the tryptophan fluorescence intensity. Zeta potential analysis indicated that the introduction of SO32- led to a decrease in the surface charges of the reduced lysozyme molecules. Compared with the native lysozyme, the reduced lysozymes not only showed a 1 log increase in the antimicrobial activity against Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076 (P <0.05), but also maintained nearly the same antimicrobial activity against Staphylococcus aureus ATCC 29213 (P >0.05). Overall, the reductive modification with the food-friendly, compatible, and safe reducing agent Na2SO3 has broadened the antimicrobial action spectrum of the modified lysozymes against Gram-negative bacteria, with the enhancement extent depending on the reduction degree and the type of bacterial strains. The integrated results suggest that the Na2SO3-reduced lysozyme can be used as a novel safe potential bactericidal additive for food-processing industry.

Fast and accurate data collection for macromolecular crystallography using the JUNGFRAU detector

The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources.

Protein-Based Nanoparticles for the Delivery of Enzymes with Antibacterial Activity.

Proteins represent a versatile biopolymer material for the preparation of nanoparticles due to their biocompatibility, biodegradability, and low immunogenicity. This study presents a protein-based nanoparticle system consisting of high surface PEGylated lysozyme polyethylene glycol-modified lysozyme (LYZmPEG ). This protein modification leads to a solubility switch, which allows a nanoparticle preparation using a mild double emulsion method without the need of surfactants. The method allows the encapsulation of large hydrophilic payloads inside of the protein-based nanoparticle system. Native lysozyme (LYZ) was chosen as payload because of its innate activity as natural antibiotic. The mild particle preparation procedure retains the structure and activity of the enzyme which was successfully tested against the gram-positive bacteria strain M. Luteus. In comparison, the particle system shows no toxicity to human cells. This first report of a full protein-based particle material for the transport of large hydrophilic payloads opens up new therapeutic applications for biopolymer-based delivery systems.

The Effect of Denatured Lysozyme on Human Corneal Epithelial Cells.

During contact lens wear, the amount of lysozyme deposited on contact lenses varies depending on the lens material. The binding of lysozyme to some contact lens materials may result in a conformational change that denatures the protein to an inactive form. This investigation evaluated the effect that denatured lysozyme has on human corneal epithelial cells (HCECs) by measuring cell viability and the release of inflammatory cytokines. HCECs were exposed to lysozyme that was denatured to various activity levels. After 24-hour exposure to the lysozyme (1.9 mg/mL) in growth media, the cells were evaluated for cell viability using confocal microscopy. The metabolic activity of the cells was determined using an alamarBlue assay. Cell supernatants were analyzed for inflammatory cytokines. Using confocal microscopy, there was no detectable change in the viability of the HCECs after exposure to the denatured lysozyme. However, using alamarBlue, a decrease in the metabolic activity of the HCECs exposed to denatured lysozyme was detected. HCECs exposed to lysozyme that was 67%, 47%, and 22% active showed a reduction in metabolic activity when compared with native (100% active) lysozyme and the media controls (P < 0.05). Exposure to the denatured lysozyme also caused an increase in the release of inflammatory cytokines (P < 0.05) from the HCECs. The results of this study show that denatured lysozyme can have a detrimental effect on HCECs. Both a reduction in metabolic activity and an increase in the release of inflammatory cytokines occurred after HCEC exposure to denatured lysozyme.

One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release

The self-organizing complexes with hyaluronic acid (HA) and polydopamine (PDA), an adhesion mediator via hydrogen bonding, were investigated for use as protein drug carriers. The complexes were prepared with HA of different molecular weights (20 kDa and 200 kDa) and various molar ratios of dopamine and lysozyme, a model protein. Dopamine-conjugated HA (HADA)/PDA complexes were prepared by one-pot synthesis by relying on the self-polymerization of dopamine under oxidative, weakly basic conditions. Lysozyme was bound via coacervation and hydrogen bonding into HADA/PDA complexes. Complex diameters were 100–300 nm, based on transmission electron microscopy image and dynamic light scattering findings. Circular dichroism and differential scanning calorimetry showed that a stable protein formulation was obtained without degradation while preserving the thermal characteristics of lysozyme. Transition temperature (Tm) of the HADA/PDA/lysozyme complex (1:10:0.05 ratio) was 72.45 °C, which is close to the Tm of the native lysozyme (72.46 °C). The efficacy of complexes was also evaluated to protect the structural stability of lysozyme. Lysozyme (0.33 mol) was complexed with HA monomer; consequently, lysozyme activity in the HADA/PDA complex was not affected from short-term degradation. Protein encapsulation and efficacy of the formulations showed successful complexation as protein carriers, thus suggesting an effective combinatory protein delivery system.

NOVEL CYANINE DYES AS POTENTIAL AMYLOID PROBES: A FLUORESCENCE STUDY

The applicability of the novel heptamethine cyanine dyes AK7-5 and AK7-6 to the detection and characterization of one-dimensional protein aggregates (amyloid fibrils) associated with numerous pathologies has been evaluated using the method of fluorescence spectroscopy. It was found that both the monomeric and aggregated forms of these dyes can bind to amyloidogenic protein lysozyme, but the concomitant changes in the electronic structure of H-aggregates render them capable of fluorescing. The growth of the hypsochromic bands with negligible changes of the monomeric peaks induced by the native protein and the opposite effects induced by the lysozyme fibrils suggest that the native lysozyme has more binding sites for the dye aggregates than fibrillar protein, while the fibril grooves represent specific binding site for the dyes monomers. The observed spectral behavior of the cyanine dyes, viz. significant distinctions in the fluorescence responses produced by the monomeric and fibrillar forms of lysozyme, suggest the possibility of recruiting these compounds as fluorescent amyloid markers along with the classical amyloid marker Thioflavin T.

Molecular Dynamics Study of Amyloidogenic Mutants of Human Lysozyme

The mutants of human lysozyme are capable of fibril formation implicated in the etiology of familial systemic or renal amyloidosis. A series of 100 ns thermal unfolding molecular dynamics (MD) simulations with WT human lysozyme and its seven amyloidogenic variants (I56T, D67H, F57I, W64R, Y54N, F57I/T70N and T70N/W112R) have been performed at 500 K. The molecular dynamics simulations were performed with GROMACS software (version 5.1) using the CHARMM36m force field. The MD results have been analysed in terms of the parameters characterizing both the global and local protein structure, such as the backbone root mean-square deviation, gyration radius, solvent accessible surface area, the root mean-square fluctuations and the secondary structure content. Depending on the observed effects, the examined variants of human lysozyme have been roughly divided into three groups comprising of mutants with faster (Y54N and F57I/T70N), similar (D67H and I56T) or slower (W64, F57I and T70N/W112R) unfolding rate compared to the wild-type counterpart. The analysis of the protein fluctuational behavior revealed that in most mutants the β-domain displays stronger fluctuations (except the W64R and F57I) and higher flexibility of the C- and D-helices relative to the native lysozyme with the exception of W64R and Y54N which show marked decrease (W64R) or increase (Y54N) in mobility of almost all residues. The analysis of secondary structure evolution provided evidence for higher stability of α-domain compared to β-domain. The results obtained reinforce the idea that mutation-induced global structural destabilization is not the only factor contributing to protein misfolding, the modifications in conformation and dynamics of selected protein regions may also play significant role in amyloid fibril formation.

Simulation of mono‐PEGylated lysozyme separation in heparin affinity chromatography using a general rate model

AbstractBACKGROUNDThe bioavailability of therapeutic proteins is improved through PEGylation. This chemical modification involves the production of isomers with different numbers and sites of attached PEG chains, which are difficult to separate efficiently. Their purification with chromatography requires an understanding of the operation and the evaluation of different operational conditions. The General Rate Model (GRM) was applied for modelling the linear salt gradient elution of mono‐PEGylated and native lysozyme in Heparin Affinity Chromatography (HAC) considering mass balance equations for proteins in the bulk‐fluid phase, in the particle phase and the kinetic adsorption.RESULTSThe model was able to simulate the individual proteins and the separation of these in a PEGylation reaction using as proof‐of‐concept a mono‐PEGylated and native lysozyme mixture under changes of operational parameters such as the gradient length (5, 13, 25 column volumes) and flow (0.8 and 1.2 mL min‐1) with a relative error in retention times of less than 6% and correlation coefficients greater than 0.78.CONCLUSIONSimulation of the elution curves of PEGylated lysozyme in HAC was performed in this work and the diverse information generated by the model is explained through the physicochemical protein properties. This simulation represents a tool for optimization, prediction and future scale‐up of PEGylated proteins purification, which would reduce the investment in time and resources to test several operating conditions. © 2017 Society of Chemical Industry

Recovery of PEGylated and native lysozyme using an in situ aqueous two‐phase system directly from the PEGylation reaction

AbstractBACKGROUNDPurification of PEGylated proteins from reactions is still a challenge due to the formation of isomers. In this work, the recovery of PEGylated lysozyme was studied in a novel integrative approach called in situ aqueous two‐phase systems (ATPS). The excess of PEG in the lysozyme PEGylation reaction (LPR) was used as part of the phase forming chemicals with different polymer (UCON, ficoll, dextran) and salt (sodium phosphate, potassium sulphate, sodium sulphate, ammonium sulphate, sodium carbonate) solutions.RESULTSThe best option for the in situ ATPS formation was the addition of a 4 mol L−1 ammonium sulphate in 20 mmol L−1 Tris–HCl (pH 7.0) solution to the PEGylation reaction. PEGylated conjugates of lysozyme exhibited a preferential partition to the top (PEG‐rich) phase while native protein was partitioned to the bottom (salt‐rich) phase. Di‐PEGylated lysozyme partitioned entirely to the top phase (recovery of 100%), while the mono‐PEGylated protein presented a recovery yield of 56% in the top phase. On its part, 97.7% of the native lysozyme was concentrated in the bottom phase.CONCLUSIONThe proof‐of‐concept presented in this work allows the recovery of PEGylated proteins directly from a PEGylation reaction. It is the first time that PEGylated proteins have been fractionated using a PEG–ammonium sulphate system taking advantage of the unreacted PEG left in a PEGylation reaction. Based on reaction engineering, in situ ATPS are a potential mechanism for PEGylation control, a good alternative for ‘packed‐bed’ or on‐column PEGylation processes and a pioneer strategy in the development of phase transfer catalysis in PEGylation. © 2017 Society of Chemical Industry

Physicochemical and functional properties of ultrasonic-treated tragacanth hydrogels cross-linked to lysozyme

The purpose of this study was to prepare, characterize and investigate physiochemical and functional attributes of hen egg white lysozyme (LZM) cross-linked with ultrasonic-treated tragacanth (US-treated TGC) under mild Maillard reactions conditions. FT-IR spectroscopy together with OPA assay revealed that covalent attachment of LZM with TCG's. Under optimum condition (pH=8.5, 60°C, RH=79%, 8 days), only one of the free amino group of LZM was blocked by TGC whereas under the same condition, US treated-TGC's blocked about three amino groups. The thermal stability of the LZM-TGC conjugates differed depending on the lengths of the main and branch chains. The microstructure of LZM-TGC conjugates was characterized by scanning electron microscopy. US-treated TGC-LZM exhibited improved solubility, emulsion properties, foam capacity and stability as compared with the native LZM. Since this gum is extensively used in food industry and application of LZM as a natural antimicrobial agents in different food systems is recommended and practiced in some countries, the results of this study indicates that a conjugated product of these two polymers combines different properties into one macromolecule and improves the property of each. These properties may make the conjugate an attractive food ingredient.