Purification Of Lysozyme Research Articles

Nanofibrous Membranes with High Mechanical Strength for the Separation and Purification of Lysozyme

Nanofibrous Membranes with High Mechanical Strength for the Separation and Purification of Lysozyme

Development of a glutamate-functionalized macroporous cation-exchange matrix for partial purification of lysozyme from chicken egg white

The polymeric monoliths, called cryogels, arouse interest due to their versatility and to be a good alternative for enzyme purification processes. Lysozyme is an enzyme that stands out for its antimicrobial activity and is being used as a preservative in the food industry. This work aimed to produce a new cationic exchange matrix functionalized with monosodium glutamate for partial lysozyme purification of egg white. The structural characteristics of the support delivered were evaluated, presenting an ion exchange capacity (IEC) of 362×10−3 mol Na+. kg−1dry cryogel, zero charge point (PCZ) around pH 6.0, and porosity more significant than 90 %. The FTIR and TGA analyses proved that the modification of the XRD matrix indicated the material's amorphous nature. The hydrodynamic studies showed asymmetry, indicating the pores' non-uniformity and low resistance to flow. The lysozyme was purified from egg white, and a purification factor of 15.11 was obtained. SDS-PAGE electrophoresis analysis indicated high purity of lysozyme, with no other protein bands appearing in the eluted solution, indicating that the adsorbent has high potential for application in lysozyme purification processes.

UiO66-based molecularly imprinted polymers with water-compatible deep eutectic solvent as functional monomer for purification of lysozyme from egg white.

Protein-templated molecularly imprinted polymers have limitations such as poor mass transfer, slow recognition kinetics, and difficulties in isolation and purification due to their large molecular sizes, complex structures, and flexible conformations. To address these limitations and obtain lysozyme (Lyz)-imprinted polymers, a molecularly imprinted polymer (UiO66@DES-MIPs) was prepared for the first timeby using Lyz as a template molecule, a metal-organic framework (UiO66-NH2) as a matrix, and a water-compatible deep eutectic solvent (DES) as a functional monomer. The introduction of UiO66-NH2 by the solvothermal method with a large specific surface area and favorable stability and resistance to environmental disturbances into the MIPs can reduce the "embedding" phenomenon and acquire a higher binding capacity and fast mass transfer. In addition, a water-soluble binary DES (1:2 molar ratio of choline chloride to 1,3 dimethylurea) prepared by a hydrothermal method as a functional monomer generates multiple forces with Lyz, increasing the hydrophilicity of UiO66@DES-MIPs and contributing to the formation and stabilization of the imprinted sites. Consequently, UiO66@DES-MIPs exhibited good selectivity, water compatibility, and fast adsorption equilibrium (the adsorption equilibrated at 243.87 ± 4.88mgg-1 in 90min). Besides, reusability experiments indicated that the UiO66@DES-MIPs could be recycled six times without obvious loss of adsorption capacity. The imprinting factor of UiO66@DES-MIPs is 3.67. The isolation and purification of Lyz from egg white confirmed the practicability of UiO66@DES-MIPs. The high adsorption capacity and specific recognition make this polymer a promising candidate for the isolation and purification of biological macromolecules.

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria.

In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

Performance of pseudo‐specific cryogel in lysozyme purification from chicken egg white

AbstractThe application of cryogels for biomolecule purification has expanded due to their adsorption efficiency and operational advantages. In this study, polyacrylamide cryogels functionalized with l‐phenylalanine (cryogel‐Phe) via the glutaraldehyde method were designed for lysozyme adsorption. Cryogel functionalization was confirmed by Fourier‐transform infrared spectroscopy and Kjeldahl analysis, indicating the immobilization of 458.65 mgphenylalanine gcryogel−1. Cryogel‐Phe showed high porosity (0.95) and a Young's modulus of 526.71 kPa. Thermogravimetric analysis indicated that thermal degradation occurred above 200°C. Differential scanning calorimetry and X‐ray diffraction confirmed that the cryogel material was amorphous. In addition, the column presented a hydraulic permeability of 4.15 × 10−13 m2, axial dispersion ranging from 10−7 to 10−6 m2 s−1, and a height equivalent to a theoretical plate ranging from 0.10 to 0.21 cm. The highest adsorption of lysozyme (67.65 mg g−1) was obtained using sodium thiocyanate saline solution (0.025 mol L−1, pH 5.0). The ability of the cryogel‐Phe column to capture and purify lysozyme was confirmed by high enzymatic activity (1294.17 U ml−1), purity (87.92%), purification factor (11.49), and sulphate‐polyacrylamide electrophoresis gel (SDS‐PAGE) electrophoresis gel.

Reactive Green 19 dye-ligand immobilized on the aminated nanofiber membranes for efficient adsorption of lysozyme: Process development and optimization in batch and flow systems

The performance of lysozyme adsorption by the aminated nanofiber membrane immobilized with Reactive Green 19 (RG19) dyes was evaluated in batch and flow systems. The physicochemical properties of the dye-immobilized nanofiber membrane were characterized. The parameters of batch-mode adsorption of lysozyme (e.g., pH, initial dye concentration, and lysozyme concentration) were optimized using the Taguchi method. In a flow process, the factors influencing the dynamic binding performance for lysozyme adsorption in the chicken egg white (CEW) solution include immobilized dye concentration, adsorption pH value, feed flow rate, and feed CEW concentration. The impact of these operating conditions on the lysozyme purification process was investigated. Under optimal conditions, the recovery yield and purification factor of lysozyme achieved from the one-step adsorption process were 98.52% and 143 folds, respectively. The dye-affinity nanofiber membrane also did not exhibit any significant loss in its binding capacity and purification performance after five consecutive uses.

Extraction and Characterization of Lysozyme from Salted Duck Egg White.

Salted duck egg white (SDEW), as the main by-product in the production process of salted egg yolk, has not been effectively used as a food resource because of its high salt concentration. This study creatively used isoelectric point precipitation, ultrafiltration, and cation exchange to separate and purify lysozyme from SDEW and preliminarily explored the enzymatic properties of lysozyme. The results showed that the relative molecular weight of lysozyme was about 14 KDa, and the specific activity of lysozyme reached 18,300 U/mg. Lysozyme had good stability in the temperature range of 30 °C to 60 °C and pH of 4 to 7. Metal ions, Fe2+, Cu2+, and Zn2+, strongly inhibited lysozyme activity. Different surfactants showed certain inhibition effects on lysozyme from SDEW, among which glycerin had the strongest inhibitory effect. This study aimed to provide a theoretical reference for industrial purification and production of lysozyme from SDEW.

Purification of Egg White Lysozyme Determines the Downstream Fibrillation of Protein and Co-assembly with Phytochemicals to Form Edible Hydrogels Regulating the Lipid Metabolism.

Although the synthetic chemistry or synthetic biological systems have already shown the power of biomaterials engineering, natural bioresource matter is still a valuable library of raw ingredients for the production of biomaterials, in particular, the edible ones. However, the influence of upstream isolation and purification of the raw materials on their performance in the downstream processing procedures is still unexplored, which is essential for the engineering of biomaterials. Based on the comparison of conventional techniques, heating-induced precipitation combined with resin-blending ion exchange was developed as a simple and cheap method for the utilization of egg whites to produce the lysozyme that is found to be exclusively feasible for fibrillation. Even with similar purities, only the lysozyme prepared by this method could be utilized to form ordered linear aggregate fibrils. Fibrillation was recently pursued as a new approach to utilize bioresource mass for high-tech end-products. Phytochemicals, totally replacing salts, induced the lysozyme fibrils to form hydrogels spontaneously, which was further demonstrated in an in vivo study to prevent obesity induced by a high-fat diet (HFD) by reducing lipid absorption and lipogenesis, promoting energy expenditure, and inhibiting inflammation. The agri-food bioresource was successfully employed as a proof of concept in edible biomedical materials for the regulation of lipid metabolism.

Mono-PEGylated lysozyme purification with increased productivity and isomer differentiation through heparin monolith chromatography

PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.

Fast and high-resolution fractionation of positional isomers of a PEGylated protein using membrane chromatography

The fractionation of positional isomers of a PEGylated protein is quite challenging as these have similar molecular weight, and only very slightly different surface charge. In this study, cation exchange z2 laterally-fed membrane chromatography (z2LFMC), which has been shown to be suitable for high-speed, high-resolution protein purification, was used to fractionate positional isomers of mono-PEGylated lysozyme. The performance of the z2LFMC device was compared with a commercial preparative cation exchange column having the same volume and ligand. PEGylated lysozyme purification experiments showed that while the positional isomers of mono-PEGylated lysozyme could not be satisfactorily resolved using the preparative commercial cation exchange column, almost baseline resolution of these could be achieved using the z2LFMC device. Moreover, the z2LFMC device-based process was faster by an order of magnitude. The results discussed in this paper demonstrate that z2LFMC is a superior alternative to column-based chromatography for challenging protein separations, such as the one discussed here, both in terms of speed and resolution.

Biomimetic rigid cryogels with aligned micro-sized tubular structures prepared by conventional redox-induced cryopolymerization in a freezer

To adapt to nature, wood forms an oriented tubular porous structure that is feasible for transporting water and nutrition. Inspired from this adaptability, we here demonstrate a universal approach to obtain rigid cryogels of aligned micro-sized tubular structures by a redox-induced cryopolymerization conducted in a freezer. In contrast to the classical directional freezing method, our method free of any special instrument leads to the aligned porous structures that are dependent on gravity due to the tubular porous structures parallel to gravity direction regardless of the sample placement direction. The versatility of our method is proven in the fabrications of a series of cryogels varying different hydrophilicity. Among the resultant samples, the three-function crosslinked cryogenic sample containing carboxyl group (polyMAA-TRIM, 40/60) was tested to have an unprecedented property integrated by high mechanical strength (9.0 MPa of compressive moduli), good hydrophilicity, and efficient purification of lysozyme (purity > 90 %) from egg white solutions only driven by gravity.

Bisphosphonated-immobilized porous cellulose monolith with tentacle grafting by atom transfer radical polymerization for selective adsorption of lysozyme

Here, a m-xylene bisphosphonate immobilized tentacle-type cellulose monolith (BP-PCM) is prepared by atom transfer radical polymerization for lysozyme purification. In the preparation, the m-xylene bisphosphonate was anchored glycidyl methacrylate and then polymerized to enhance the flexibility of the ligands to improve lysozyme adsorption capacity, and glycerol monomethacrylate serves as spacer to further optimize the layers structure and ligands density of the grafted tentacles for satisfactory adsorption capacity. The maximum static and dynamic adsorption capacity (10% breakthrough) of BP-PCM reach to 169.6 and 102.6 mg mL−1, respectively. Moreover, BP-PCM displays weak nonspecific adsorption and is able to successfully enrich lysozyme from diluted chicken egg white, indicating the excellent selectivity. The results demonstrated that BP-PCM is promising for use as high-capacity protein chromatography.

Purification of lysozyme from chicken egg white by high-density cation exchange adsorbents in stirred fluidized bed adsorption system

Lysozyme from crude chicken egg white (CEW) feedstock was successfully purified using a stirred fluidized bed adsorption system ion exchange chromatography where STREAMLINE SP and SP-XL high density adsorbents were selected as the adsorption carrier. The thermodynamic and kinetic studies were carried out to understand the characteristics of lysozyme adsorption by adsorbents under various conditions, including adsorption pH, temperature, lysozyme concentration and salt concentrations. Results showed that SP and SP-XL adsorbents achieved optimum lysozyme adsorption at pH 9 with capacity of ~139.77 and ~251.26 mg/mL, respectively. The optimal conditions obtained from batch studies were directly employed to operate in SFBA process. For SP-XL adsorbent, the recovery yield and purification factor of lysozyme were 93.78% and ~40 folds, respectively. For SP adsorbent, lysozyme can be eluted ~100% with purification factor of ~26 folds. These two adsorbents are highly suitable for use in direct recovery of lysozyme from crude CEW.

Strong and weak cation-exchange groups generated cryogels films for adsorption and purification of lysozyme from chicken egg white

Here, the macroporous poly(hydroxylmethyl methacrylate/glycidyl methacrylate [p(HEMA-GMA)] cryogels with large porous surface were prepared, and then the epoxy groups of the p(HEMA-GMA) cryogels were systematically modified into strong and weak cationic groups. The effects of initial protein concentrations, adsorption time, pH, salt concentrations and temperatures on adsorption efficiency of cation exchange cryogels for lysozyme were determined. The maximum lysozyme adsorption capacities of strong and weak cation exchange cryogels were found to be 188.3 and 79.7 mg/g cryogel at 25 °C, respectively. The performance of the strong cationic cryogel was evaluated by purification of lysozyme from egg white. The activity of the isolated lysozyme was found to be 21,347 U/mg. The cationic cryogel maintained its expected high adsorption capacity and efficiency of the purification levels during repeated adsorption desorption processes. Finally, the purpose of this work is the design a cation exchange system for purification of lysozyme from egg-white.

Highly efficient dye removal and lysozyme purification using strong and weak cation-exchange nanofiber membranes

Electrospinning technology was applied for the preparation of polyacrylonitrile (PAN) nanofiber membrane in this work. After hot pressing, alkaline hydrolysis and neutralization treatment, a weak acid cation exchange membrane (P-COOH) was prepared. By the covalent coupling reaction between the acidic membrane and aminomethane sulfonic acid (AMSA), a strong acidic nanofiber membrane (P-SO3H) was obtained. The surface morphology, chemical structure, and thermal stability of the prepared ion exchange membranes were analyzed via SEM, FTIR and TGA. Analytical results showed that the membranes were prepared successfully and thermally stable. The ion exchange membrane (IEX) was conducted with the newly designed membrane reactor, and different operating conditions affecting the adsorption efficiency of Toluidine Blue dye (TBO) were investigated by dynamic flow process. The results showed that dynamic binding capacity (DBC) of weak and strong IEX membranes for TBO dye was ~170 mg/g in a dynamic flow process. Simultaneously, the ion exchange membranes were also used for purifying lysozyme from chicken egg white (CEW). Results illustrated that the recovery yield and purification factor of lysozyme were 93.43% and 29.23 times (P-COOH); 90.72% and 36.22 times (P-SO3H), respectively. It was revealed that two type ion exchange membranes were very suitable as an adsorber for use in dye waste treatment and lysozyme purification process. P-SO3H strong ion-exchange membrane was more effective either removal of TBO dye or purification of lysozyme. The ion exchange membranes not only effectively purified lysozyme from CEW solution, but also effectively removed dye from wastewater.

Egg white lysozyme purification by a stirred cell contactor equipped with a weak ion-exchange nanofiber membrane: Process development and scale-up

A weak ion-exchange membrane (P-COOH) was synthesized by alkaline hydrolysis of a polyacrylonitrile nanofiber membrane prepared by electrospinning process. The P-COOH membrane was characterized for its physical properties and its application for purification of lysozyme from chicken egg white was investigated. The lysozyme adsorption efficiency of the P-COOH membrane operating in a stirred cell contactor (Millipore, Model 8010) was evaluated. The effects of key parameters such as the feed concentration, the rotating speed, the flow rate of feed and the operating pressure were studied. The results showed successful purification of lysozyme with a high recovery yield of 98% and a purification factor of 63 in a single step. The purification strategy was scaled-up to the higher feedstock loading volume of 32.7 and 70 mL using stirred cell contactors of Model 8050 and 8200, respectively. The scale-up processes achieved similar purification results, proving linear scalability of the purification technique adopted.

Preparation of supermacroporous cryogels with improved mechanical strength for efficient purification of lysozyme from chicken egg white.

A novel, facile, and robust strategy was proposed to increase the pore size and mechanical strength of cryogels. By mixing the monomers of acrylamide and 2-hydroxyethyl methacrylate as the precursor, a monolithic copolymer cryogel with large interconnected pores and thick pore walls was prepared. Hydrogen bonding between the two monomers contributed to the entanglement and aggregation of the copolymers, thickening the pore walls and resulting in larger pore sizes. Analysis via mercury porosimetry demonstrated that the interconnected pore diameter of the copolymer cryogel ranged from 10-350 µm, which was far larger than that of the cryogels from one monomer (10-50 µm). Additionally, the thicker pore walls of the copolymer cryogel improved its mechanical strength. Affinity cryogels were prepared through covalent immobilization using Tris(hydroxymethyl)aminomethane as a coupling agent, and the affinity binding of lysozymes on Tris-cryogel was evaluated by the Langmuir isothermal adsorption with the maximum adsorption capacity of 360mg/g. Compared with that of the Tris-cryogels produced from one monomer, the copolymer Tris-cryogel exhibited higher adsorption capacity and lysozyme purity, when the chicken egg white solution flowed solely driven by gravity. This work provides a new avenue for designing and developing supermacroporous cryogels for bioseparation.

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography.

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9mL/min background buffer of 20mM pH 5.5 Tris-Acetic acid, 350V, and 14°C as well as 2mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Eco-friendly polylactic acid/rice husk ash mixed matrix membrane for efficient purification of lysozyme from chicken egg white

An eco-friendly and inexpensive ion-exchange membrane was developed for efficient purification of lysozyme from chicken egg white. Agricultural waste particles (rice husk ash, RHA) were incorporated into biodegradable polymeric matrix (polylatic acid, PLA) to fabricate porous PLA/RHA mixed matrix membrane (MMM) via water-vapour-induced phase inversion. The resulted membrane had an ion-exchange capacity (IEC) of 844 μmol/g membrane. In batch operation at pH 8, the protein adsorption equilibrium was attained within 2 h of contact time and the maximum lysozyme adsorption capacity was 4.49 μmol/g membrane. The adsorptivity of PLA/RHA MMM was essentially contributed from the filled RHA particles. In dynamic operation using one piece of PLA/RHA MMM at 122 L/h/m2 for a feed lysozyme concentration identical to that in mucin-free egg white (0.5 mg/mL), the breakthrough volume was about 50 MMM volume. The lysozyme recovery was 68–70% with a near 100% purity and a purification fold of 35 after an adsorption/washing/elution cycle treating mucin-free egg white mixture. The MMM performance was only deteriorated at a minor percentage during three successive cycles. These results demonstrate that the PLA/RHA MMM process is a green and efficient approach for isolating lysozyme from chicken egg white.

Effective purification of lysozyme from chicken egg white by tris(hydroxymethyl)aminomethane affinity nanofiber membrane

Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.