Lysozyme Layers Research Articles

Phase-transitioned lysozyme-mediated fabrication of zeolitic imidazole framework-8 hybrid membrane with enhanced separation and antifouling properties

Metal-organic framework (MOF)-based membranes have been considered a promising candidate for the separation membrane due to their high porosity and controlled pore structure. However, the fabrication of a robust MOF-based membrane is commonly subject to the limitations of MOF particle agglomeration and interface defects. Herein, we propose a facile and effective approach for the preparation of a robust zeolite imidazole framework-8 (ZIF-8)/protein hybrid membrane by introducing a phase-transited lysozyme (PTL) layer to mediate the growth of ZIF-8. The PTL layer was self-assembly on the ultrafiltration membrane and then ZIF-8 was in-situ growth to form a uniform ZIF-8/PTL hybrid layer. The prepared ZIF-8/PTL membrane possessed water permeance up to 39.5 L m−2 h−1 bar−1 and high rejections (>99.0%) for heavy metal ions. Furthermore, the fabricated ZIF-8/PTL hybrid membrane was verified to have excellent antimicrobial activity and antifouling performance. These results indicate that as a simple and robust approach, PTL modification can be employed to construct new interfaces for mediating MOF growth, and the prepared MOF-based membranes have great potential for water treatment.

Facile surface treatment strategy to generate dense lysozyme layer on ultra-high molecular weight polyethylene enabling inhibition of bacterial biofilm formation

Medical plastics such as those found in endotracheal tubes are widely used in intensive care units for the treatment of critically ill patients. Although commonplace in hospital environment, these catheters are at a high risk of bacterial contamination and have been found responsible for numerous health-care-associated infections. Antimicrobial coatings that can prevent harmful bacterial growth are required to reduce the occurrence of such infections. In this study, we introduce a facile surface treatment strategy that could form antimicrobial coatings on the surface of average medical plastics. The strategy involves treatment of activated surfaces with lysozyme, a natural antimicrobial enzyme presenting in human lacrimal gland secretions which is widely used for wound healing. Using ultra-high molecular weight polyethylene (UHMWPE) as the representative surface, oxygen/argon plasma treatment for 3 min led to the increase of surface roughness and the generation of negatively charged groups, with the zeta potential measured as −94.5 mV at pH 7. The activated surface could accommodate lysozyme with a density of up to 0.3 nmol/cm2 through electrostatic interaction. Antimicrobial activity of the resulting surface (UHMWPE@Lyz) was characterized with Escherichia coli and Pseudomonas sp. strains, and the treated surface significantly inhibited the bacterial colonization and the formation of biofilm compared to the untreated UHMWPE. This method of constructing an effective lysozyme-based antimicrobial coating is a generally applicable, simple and fast process for surface treatment with no adverse solvent and wastes involved.

Two-stage optical trapping and assembling of protein at air/solution interface

Optical trapping at interfaces has recently gained relevance due to the expansion of optical potential far away from the focus, especially for proteins where submillimeter structures have been described. Initially, lysozyme clusters are trapped as a shallow layer at the surface, becoming thicker with irradiation time. Nonetheless, overcoming a concentration threshold, lysozyme clusters inside the solution are collected and transported toward the focus, invading the lysozyme layer, which results in a border between them, although no concentration jump is detected. This two-stage optical trapping occurs due to the long-range interaction originating from the focus.

DNA Penetration into a Lysozyme Layer at the Surface of Aqueous Solutions.

The interactions of DNA with lysozyme in the surface layer were studied by performing infrared reflection–absorption spectroscopy (IRRAS), ellipsometry, surface tensiometry, surface dilational rheology, and atomic force microscopy (AFM). A concentrated DNA solution was injected into an aqueous subphase underneath a spread lysozyme layer. While the optical properties of the surface layer changed fast after DNA injection, the dynamic dilational surface elasticity almost did not change, thereby indicating no continuous network formation of DNA/lysozyme complexes, unlike the case of DNA interactions with a monolayer of a cationic synthetic polyelectrolyte. A relatively fast increase in optical signals after a DNA injection under a lysozyme layer indicates that DNA penetration is controlled by diffusion. At low surface pressures, the AFM images show the formation of long strands in the surface layer. Increased surface compression does not lead to the formation of a network of DNA/lysozyme aggregates as in the case of a mixed layer of DNA and synthetic polyelectrolytes, but to the appearance of some folds and ridges in the layer. The formation of more disordered aggregates is presumably a consequence of weaker interactions of lysozyme with duplex DNA and the stabilization, at the same time, of loops of unpaired nucleotides at high local lysozyme concentrations in the surface layer.

Spread Layers of Lysozyme Microgel at Liquid Surface.

The spread layers of lysozyme (LYS) microgel particles were studied by surface dilational rheology, infrared reflection–absorption spectra, Brewster angle microscopy, atomic force microscopy, and scanning electron microscopy. It is shown that the properties of LYS microgel layers differ significantly from those of ß-lactoglobulin (BLG) microgel layers. In the latter case, the spread protein layer is mainly a monolayer, and the interactions between particles lead to the increase in the dynamic surface elasticity by up to 140 mN/m. In contrast, the dynamic elasticity of the LYS microgel layer does not exceed the values for pure protein layers. The compression isotherms also do not exhibit specific features of the layer collapse that are characteristic for the layers of BLG aggregates. LYS aggregates form trough three-dimensional clusters directly during the spreading process, and protein spherulites do not spread further along the interface. As a result, the liquid surface contains large, almost empty regions and some patches of high local concentration of the microgel particles.

Metal-organic framework based nanozyme hybrid for synergistic bacterial eradication by lysozyme and light-triggered carvacrol release

Pathogenic bacteria seriously endanger human health. Metal-organic framework (MOF)-enzyme composites are novel antibacterial materials for effective bacterial inactivation. In this study, we develop a MOF-based nanozyme hybrid for synergistic bacterial eradication, which integrates features of bacterial capture, magnetic assembly, lysozyme hydrolysis, and light-triggered thermal generation and carvacrol release. Core-shell nanoparticles (Fe3O4@PVP@NH2-MIL-88B(Fe), FPM; PVP = polyvinylpyrrolidone) are synthesized by loading PVP-modified Fe3O4 to NH2-MIL-88B(Fe), and then covalently immobilizing lysozyme onto the FPM surface through enzymatic reaction by microbial transglutaminase. The lysozyme-covered FPM is further employed as a host matrix for loading antibacterial carvacrol to synthesize Fe3O4@PVP@MIL-88B(Fe)–NH-lysozyme/carvacrol (FPMLC) nanozyme hybrid. This FPMLC can capture bacteria by electrostatic attraction and the FPMLC-bacteria composite is then assembled by an extra magnet. The lysozyme layer can degrade peptidoglycan of the bacterial cell wall, and the released carvacrol can disrupt the bacterial cell membrane under near-infrared (NIR) irradiation. Systematic antibacterial results demonstrate that the developed FPMLC nanozyme hybrid at a low dose of 100 μg/mL completely inactivates (100%) both Escherichia coli and Staphylococcus aureus at cell density of 106 CFU/mL. This FPMLC exhibits high-efficiency antibacterial efficacy but no significant cytotoxicity in vitro, and therefore provides promising antibacterial applications in biomedical, environmental, and food fields.

Lysozyme-based composite membranes and their potential application for active packaging

Barrier packaging materials are significant in protecting the packaged contents from damage caused by oxygen, fogging, fats and oils in the environment. In this study, an active barrier packaging composite based on lysozyme and plastic films including polyethylene (PE) and biaxially oriented polypropylene (BOPP) were designed. The barrier packaging composites were prepared using a layer-by-layer self-assembly process to deposit lysozyme onto the surface of the plastic films. The bulk structure and morphology of the composites were investigated using X-ray photoelectron spectroscopy and atomic force microscopy. The barrier properties of the packaging composites were analyzed by measuring water vapor permeability, oil permeability, oxygen permeability, light transmittance and chromaticity. The results showed that the lysozyme coating greatly reduced the water vapor permeability, oxygen permeability and oil permeability of the packaging without affecting the appearance of the original packaging films. The barrier properties of the packaging composites were strengthened by increasing the number of coated lysozyme layers. The composite films showed effective antibacterial activity against E. coli and S. aureus during a pasteurized milk storage experiment, indicating a promising application prospect.

Tritium-Probe Method in a Study of Adsorption Layers of Lysozyme on the Surface of Detonation Nanodiamonds

Tritium-labeled lysozyme was used to determine its adsorption on the surface of nanodiamonds produced by detonation synthesis. It was found that the amount of the adsorbed protein depends on the charge of the nanodiamond surface. A multilayer coating is formed on the originally positively charged nanodiamonds, with the adsorption becoming 4–5 times stronger on negatively charged nanodiamonds produced from the original nanodiamonds via their annealing in air. According to IR spectroscopic data, the adsorption of lysozyme results in that the number of loops and turns in the protein structure decreases in the adsorption on two types of nanodiamonds. The use of a tritium probe did not find any significant difference between the structures of protein molecules in their adsorption on nanodiamonds with different functional surface compositions. The orientation of molecules in the surface layer was found to be the same: Protein parts containing amino acid residues of phenylalanine are contacting with the sorbent, and the amino acid residues of proline, which are contained in the loops, are on the surface of the adsorption layer.

Mechanism of lysozyme adsorption onto gold surface determined by quartz crystal microbalance and surface plasmon resonance

In this study, the physicochemical characterization of lysozyme adsorbed on gold was investigated. Through the use of MP-SPR it was possible to establish that the orientation of molecules changes from side-on to between or end-on with increasing surface coverage. The data confirms that the process of adsorption is driven primarily by electrostatic interactions but also by hydrophobic forces. MP-SPR data was compared with the Random Sequential Adsorption model for a molecule with an ellipsoidal shape. Contact angle measurements showed that higher surface coverage also translates in more hydrophilic properties of obtained lysozyme layer. Comparison of CD and PM-IRRAS spectra in solution and adsorbed state respectively showed changes in the secondary structures of lysozyme. These changes are dependent on pH, but fundamentally they go in the direction of the increase of β-turn/random content with a simultaneous decrease in β-sheet fraction, which suggests that aggregation is not occurring. The combination of MP-SPR and QCM-D measurements allowed the estimation of the number of water molecules associated with the lysozymes films. It has been observed that hydration decreases from 70% in pH = 4 to 30% in pH = 11. This data indicates that hydration is driven mainly by the degree of protonation of lysozyme molecules.

Investigating the Influence of Polysorbate 20/80 and Polaxomer P188 on the Surface & Interfacial Properties of Bovine Serum Albumin and Lysozyme.

To provide new insights into how protein-surfactant competitive adsorbtion and corresponding surface tension reduction properties at the air-water and oil-water interface are impacted by the type of protein and the associated protein surface rheology. Interfacial Rheology was utilized to obtain surface G' and G" as a function of frequency. Force tensiometry was utilized to obtain changes in surface tension as a function of surfactant concentration. The impact on surface properties of two different proteins i.e. BSA and Lysozyme was investigated as a function of surfactant concentration i.e. polysorbates PS 20, PS 80 and Poloxomer (Kolliphor P188). Surface tension and interfacial tension measurements for BSA showed that in mixed BSA/polysorbate surfactant systems, BSA dominates the interfacial behavior at both the air-water and oil-water interfaces, until a high polysorbate concentration of 0.1mg/ml. At these high polysorbate concentrations a mixed BSA-Polysorbate interfacial layer is formed as corroborated by the surface elasticity values being lower than that of pure BSA but higher than that of pure Polysorbate. For Kolliphor, it was observed that Kolliphor was unable to displace BSA at any concentration. This is corroborated by the high surface elasticity of the BSA which is maintained in the presence of Kolliphor. Surface and interfacial tension measurements for lysozyme show that for mixed lysozyme/polysorbate surfactant systems, the surface tension values are lower than that exhibited by either the lysozyme or the polysorbate surfactants. This potentially indicates the formation of a mixed layer of lysozyme and polysorbate. At the high polysorbate concentrations probed, the surface elasticity values are however closer to that of pure polysorbates, indicating that the mixed layer may be more heavily polysorbate dominated, especially at high polysorbate concentrations. For Kolliphor, the response was similar to that seen in the Kolliphor-BSA system in which the Kolliphor was not able to displace the protein i.e. Lysozyme. In conclusion, it was seen that competitive adsorption between proteins and common excipient surfactants is dictated by the type of protein and its effective structuring/rigidity at the surface as reflected through surface elasticity and surface tan delta values. BSA was seen to exhibit a higher surface elasticity than lysozyme, and therefore has a more rigid structure and is more competitive at the interface.

Fabrication of Multilayer Films on the Basis of Lysozyme Protein and Precipitant (Iodide and Potassium) Ions on a Silicon Substrate by the Modified Langmuir–Schaefer Method

Multilayer structures, consisting of successive lysozyme layers and layers of iodide and potassium ions, have been formed on a silicon substrate by the modified Langmuir–Schaefer method; the use of this technique implies preliminary preparation of a crystallization protein solution containing an oligomeric phase. The multilayer structure has been characterized by methods of X-ray reflectivity and total external reflection X-ray standing waves (TER XSW). The data obtained indicate the formation of a dense lysozyme film (3 nm thick) and layers of potassium and iodide ions, adjacent to this film.

Functionalization of titanium implants with phase-transited lysozyme for gentle immobilization of antimicrobial lysozyme

This work deals with the implementation of a gentle method for the coating of titanium surfaces with lysozyme in order to prevent microorganisms adhesion and proliferation and, consequently, implant failure. Titanium medical grade 5 (Ti6Al4V) discs having either polished surface or biomimetic surface (BAS) were pretreated with oxygen plasma to clean the surface and, at the same time, increase its hydrophilicity. Functionalization consisted in a first layer of phase-transited lysozyme (PTL) for durable anchorage. This layer was treated with citric acid which was used as a spacer for, in a final step, ionically bind bioactive lysozyme without altering its enzymatic activity. Functionalized discs were characterized using a variety of techniques (profilometry, SEM-EDX, FTIR-ATR, water contact angle, mechanical friction) and their enzymatic activity was demonstrated against M. lysodeikticus. Lysozyme functionalization prevented Staphylococcus aureus adhesion while maintaining the high cytocompatibility of the discs with relevant cell lines. Relevantly, stability of the coating was demonstrated after prolonged storage in PBS, freeze-drying cycles and conventional γ-ray sterilization protocols; the disc maintaining the enzymatic activity of lysozyme. Even after friction against bone-mimicking material resembling implantation screwing, the functionalized discs still retained more than 70% enzyme activity. Overall, this affordable approach could be useful to endorse titanium implants with antimicrobial properties.

Investigation of loosely and firmly bound albumin and lysozyme layers on dental materials

The total amount of albumin and lysozyme adsorbed on different dentally relevant materials like titanium, gold, ceramics, poly(methyl methacrylate) (PMMA) and polytetrafluoroethylene (PTFE) was measured by bicinchoninic acid assay (BCA assay) after detachment of the proteins from the samples. The total amount of protein was separated into the amount of loosely and firmly adsorbed protein. The parts of loosely and firmly bound protein differ remarkably depending on the material. For albumin, the loosely bound protein dominates the total adsorption. In contrast, for lysozyme the amount of loosely and firmly bound protein is in some cases equal and sometimes there is more firmly than loosely bound lysozyme. In general, the firmly bound protein masks the electrostatics of the surface. The amount of loosely bound protein follows no strict tendency, whereas the amount of firmly bound protein is mostly governed by entropic and hydrophobic interactions, but modulated by electrostatics and thus by the isoelectric points of the surface and of the proteins. Most protein adsorbs on the metallic surfaces, if compared to the investigated non‐metallic surfaces. For the adsorption on gold, the number of cysteines in the amino acid sequence influences the amount of protein on the gold surface. Least albumin adsorbs on PMMA, least lysozyme adsorbs on PMMA and PTFE. For albumin, pH depending conformational changes play a key role for the adsorption at pH 9.0.

Thermo-sensitive poly(DEGMMA-co-MEA) microgels: Synthesis, characterization and interfacial interaction with adsorbed protein layer

The novel microgels, poly[di(ethylene glycol) methyl ether methacrylate-co-2-methoxyethyl acrylate] poly(DEGMMA-co-MEA) microgels, were synthesized. The poly(DEGMMA-co-MEA) microgels were thermo-sensitive and exhibited a volume phase transitive temperature (VPTT) of 14–22 °C. The incorporation of hydrophobic comonomer MEA shifted the VPTT of poly(DEGMMA-co-MEA) microgels to lower temperatures. The interfacial interaction of poly(DEGMMA-co-MEA) microgels and three model proteins, namely fibrinogen, bovine serum albumin and lysozyme, was investigated by quartz crystal microbalance (QCM). An injection sequence of “microgel-after-protein” was then established for the real-time study of the interaction of proteins and the microgels at their swollen and collapsed states by using QCM technique. The results indicated that the interfacial interaction of poly(DEGMMA-co-MEA) microgels and adsorbed protein layers was mainly determined by the electrostatic interaction. Because poly(DEGMMA-co-MEA) microgels were negatively charged in Tris-HCl buffer solution (pH = 7.4), the microgels did not adsorb on negatively charged fibrinogen and bovine serum albumin layers but strongly adsorbed on positively charged lysozyme layer. Stronger interaction between lysozyme and the microgels at collapsed state (i.e. at 37 °C) was observed. Furthermore, the incorporation of MEA might weaken the interaction between poly(DEGMMA-co-MEA) microgels and proteins.

Soft landing of cell-sized vesicles on solid surfaces for robust vehicle capture/release.

Based on a concept of a smooth and steady landing of fragile objects without destruction via a soft cushion, we have developed a model for the soft landing of deformable lipid giant unilamellar vesicles (GUVs) on solid surfaces. The foundation for a successful soft landing is a solid substrate with a two-layer coating, including a bottom layer of positively charged lysozymes and an upper lipid membrane layer. We came to a clear conclusion that anionic GUVs when sedimented on a surface, the vesicle rupture occurs upon the direct contact with the positively charged lysozyme layer due to the strong coulombic interactions. In contrast, certain separation distances was achieved by the insertion of a soft lipid membrane cushion between the charged GUVs and the lysozyme layer, which attenuated the coulombic force and created a mild buffer zone, ensuring the robust capture of GUVs on the substrate without their rupture. The non-covalent bonding facilitated a fully reversible stimuli-responsive capture/release of GUVs from the biomimetic solid surface, which has never been demonstrated before due to the extreme fragility of GUVs. Moreover, the controllable capture/release of cells has been proven to be of vital importance in biotechnology, and similarity the present approach to capture/release cells is expected to open the previously inaccessible avenues of research.

Adsorption of lysozyme on silver and its influence on silver release.

Silver is increasingly used in antimicrobial coatings of biomedical devices and implants to hinder infections. As proteins have been shown to largely influence the extent of released metals from various metal surfaces at biological conditions, silver may also be influenced in the same way. The aim of this study is to relate the structure of adsorbed lysozyme (LSZ) to the release of silver from metallic silver surfaces. Simultaneous adsorption measurements were performed in real time on the same surface using combined ellipsometry and quartz crystal microbalance with dissipation monitoring measurements to provide a more comprehensive understanding on the adsorption kinetics and the layer structures. The concentration of LSZ in 0.15 M NaNO3 solution (pH 7, 25 °C) influences the structure of the adsorbed layer. Monolayer coverage is obtained at concentrations ≤0.1 g/L, while a bilayer structure with a rigid inner layer and a relatively loosely adsorbed outer layer is formed at 1 g/L. The inner layer of LSZ is assumed to bind firmly to silver via disulfide bridges, which makes it irreversibly adsorbed with respect to dilution. The amount of released silver is further influenced by the structure of the LSZ layer. At low LSZ concentrations (≤0.1 g/L) the amount of released silver is not significantly different compared with non-protein-containing NaNO3 solutions; however, noticeable reduction was observed at higher concentrations (1 g/L). This reduction in silver release has several possible explanations, including (i) surface complexation between LSZ and silver ions that may result in the incorporation of silver in the irreversible adsorbed layer and, hence, reduce the amount of released silver into solution, and (ii) net charge reversal at the protein/solution interface to slightly positive surface potentials. Any release of silver will therefore exhibit an electrostatic repulsion during transportation through the protein layer results in a reduced amount of silver in solution.

Interaction between lysozyme and humic acid in layer-by-layer assemblies: Effects of pH and ionic strength

The interaction between protein and soluble organic matter is studied through layer-by-layer assembly of lysozyme (LSZ) and purified Aldrich humic acid (PAHA) at a solid surface (2-D) and in solution (3-D). By bringing a silica surface in alternating contact with solutions of LSZ and PAHA a layer-by-layer LSZ–PAHA assembly is formed. At pH 5 the negative charge density of PAHA is about 3 times that of the positive LSZ; the layers of LSZ and PAHA are stable and the adsorbed amounts decrease with increasing ionic strength. The mass ratios PAHA/LSZ in the layers depend on the ionic strength; K+ incorporation is relatively large (∼25%) when PAHA is the outer layer of the assembly. At pH 6 and 8, and moderate ionic strength (0–100mmolL−1 KCl) the assembly is accompanied by partial solubilization of positive LSZ by the much more negative PAHA followed by desorption of the complex. The solubilization increases with increasing pH, and decreases with increasing KCl concentration. At 400mmolL−1 KCl the electrostatic interactions are so well screened that the assembly is no longer accompanied by layer erosion. Assembly of PAHA and LSZ in solution is also investigated at pH 5 and 5mmolL−1 KCl. The PAHA/LSZ mass ratio at the iso-electric point of the assembly depends on the order of the addition. When LSZ is added to the negative assembly K+ is incorporated in the complex, but when PAHA is added to the positive assembly PAHA and LSZ neutralize each other.

Linear and nonlinear microrheology of lysozyme layers forming at the air-water interface.

We report experiments studying the mechanical evolution of layers of the protein lysozyme adsorbing at the air-water interface using passive and active microrheology techniques to investigate the linear and nonlinear rheological response, respectively. Following formation of a new interface, the linear shear rheology, which we interrogate through the Brownian motion of spherical colloids at the interface, becomes viscoelastic with a complex modulus that has approximately power-law frequency dependence. The power-law exponent characterizing this frequency dependence decreases steadily with increasing layer age. Meanwhile, the nonlinear microrheology, probed via the rotational motion of magnetic nanowires at the interface, reveals a layer response characteristic of a shear-thinning power-law fluid with a flow index that decreases with age. We discuss two possible frameworks for understanding this mechanical evolution: gelation and the formation of a soft glass phase.

Physical Characterisation of an Alginate/Lysozyme Nano-Laminate Coating and Its Evaluation on ‘Coalho’ Cheese Shelf Life

This work aimed at the characterisation of a nano-laminate coating produced by the layer-by-layer methodology and its evaluation on the preservation of ‘Coalho’ cheese. Initially, five alternate layers of alginate and lysozyme were assembled in an aminolysed/charged polyethylene terephthalate (A/C PET) and physically characterised by UV/VIS spectroscopy, contact angle, water vapour (WVTR) and oxygen (OTR) transmission rates and scanning electron microscopy. Afterwards, the same methodology was used to apply the nano-laminate coating in ‘Coalho’ cheese and its shelf life was evaluated during 20 days in terms of mass loss, pH, lipid peroxidation, titratable acidity and microbial count. UV/VIS spectroscopy and contact angle analyses confirmed the layers’ deposition and the successful assembly of nano-laminate coating on A/C PET surface. The coating presented WVTR and OTR values of 1.03 × 10−3 and 1.28 × 10−4 g m−2 s−1, respectively. After 20 days, coated cheese showed lower values of mass loss, pH, lipidic peroxidation, microorganisms’ proliferation and higher titratable acidity in comparison with uncoated cheese. These results suggest that gas barrier and antibacterial properties of alginate/lysozyme nanocoating can be used to extend the shelf life of ‘Coalho’ cheese.

Effect of salt ions on protein layers at the air–water interface under a crystallization condition

The density profiles of lysozyme layers at the air–water interface before and after adding salt were determined by X-ray reflection. After adding salt, the density of the lysozyme (LSZ) layers decreases, whereas the thickness drastically increases. The salt ions are considered to increase the LSZ–LSZ interaction, and consequently decrease the surface activity of LSZ, allow adsorbed lysozyme molecules crystallize.