Multimodal Affinity-Modulated Efficient Separation of Lysozyme with a Hierarchical MXene@MOF Hybrid Framework.

The development of abiotic protein affinity adsorbents remains challenging for the accurate acquisition and analysis of specific protein species. Inspired by bacterial cell walls, a hierarchical hybrid framework is fabricated through the oriented growth of an Fe-based metal organic framework (MOF) on V2C MXene for the efficient separation of lysozyme (Lys). After directed evolution of adsorptive materials, the MXene@MOF composite rich in hydroxyl groups (termed as MX@MOF-DH) is found exerting exceptional affinity for Lys. Benefiting from hydrogen-bonding, coordination, and electrostatic interaction-mediated multimodal and multivalent affinity, MX@MOF-DH reveals rapid adsorption rate (5 min), superb enrichment factor (83.1), and favorable binding capacity (609.7 mg g-1), which outperforms other latest adsorbents. Moreover, femtomolar sensitivity is achieved even in the presence of high-abundant interfering proteins, as confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis. This work not only provides an efficient approach for selective enrichment of lysozyme but also paves an avenue to construct the protein affinity reagents for specific biological medicine and analysis applications.

Towards Enhanced Tunability of Aqueous Biphasic Systems: Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins.

This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.

Rapid aggregation of amyloid-like protein enhanced by mTGase to prepare functional wool fabrics for efficient and sustainable remove heavy metals from wastewater.

To counteract the increasing severity of water pollution and purify water sources, wastewater treatment materials are essential. In particular, it is necessary to improve the bonding strength between the adsorption material and the substrate in a long-term humid environment, and resist the invasion of microorganisms to prolong the service life. In this study, an amyloid-like aggregation method of lysozyme catalyzed by microbial transglutaminase (mTGase). Lysozyme self-assembles into an amyloid-like phase-transited lysozyme (PTL) in the presence of a reducing agent. Simultaneously, mTGase catalyzes acyl transfer reactions within lysozyme molecules or between lysozyme and keratin molecules, and driving PTL assembly on the wool fiber (TG-PTL@wool). This process enhances the grafting amount and fastness of PTL on the wool. Moreover, the tensile strength of wool fabric increased to 523 N. TG-PTL@wool achieves a 97.32 % removal rate of heavy metals, maintaining a removal rate of over 95 % after 5 cycles. TG-PTL@wool has excellent antibacterial property (99 %), and it remains above 90 % after 50 times of circulating washing. This study proved that mTGase can enhance the amyloid aggregation of lysozyme and enhance the bonding strength between PTL coating and substrate. Moreover, TG-PTL@wool provides a sustainable, efficient and cleaner solution for removing heavy metals from water.

Preparation of carboxyl-functionalized silica core-shell microspheres and their applications in weak cation exchange chromatography, heavy metal removal, and lysozyme enrichment.

Chromatography is a technique of separation based on adsorption and/or interaction of target molecules with stationary phases. Herein, we report the design and fabrication of BTDA@SiO2 core-shell microspheres as a new class of stationary phase and demonstrate its impressive performance for chromatographic separations. The silica microspheres of BTDA@SiO2 were synthesized by in situ method with 1,3,5-benzenetricarboxaldehyde and 3,5-diaminobenzoic to separate peptides and proteins on high-performance liquid chromatography. The BTDA@SiO2 core-shell structure has a high specific surface area and retention factor of 4.27 and 8.31 for anionic and cationic peptides, respectively. The separation factor and resolution were high as well. A typical chromatogram illustrated nearly baseline resolution of the two peptides in less than 3 min. The BTDA@SiO2 was also highly stable in the pH range of 1 to 14. Furthermore, the prepared BTDA@SiO2 core-shell material not only be used for chromatographic separation but also as heavy metal removal from water. Using a BTDA@SiO2, we also achieved a lysozyme enrichment with a maximum saturated adsorption capacity reaching 714 mg/g. In summary, BTDA@SiO2 has great application prospects and significance in separation and purification systems.

Taylor Dispersion-Induced Phase Separation for the Efficient Characterisation of Protein Condensate Formation.

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid-liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion-induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL-3 and Ddx4. Uniquely accessible to this method, we find an unexpected re-entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α-synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS-modifying compounds can be screened highly efficiently.

Macromolecular protein crystallisation with biotemplate of live cells.

Macromolecular protein crystallisation was one of the potential tools to accelerate the biomanufacturing of biopharmaceuticals. In this work, it was the first time to investigate the roles of biotemplates, Saccharomyces cerevisiae live cells, in the crystallisation processes of lysozyme, with different concentrations from 20 to 2.5 mg/mL lysozyme and different concentrations from 0 to 5.0 × 107 (cfu/mL) Saccharomyces cerevisiae cells, during a period of 96 h. During the crystallisation period, the nucleation possibility in droplets, crystal numbers, and cell growth and cell density were observed and analysed. The results indicated the strong interaction between the lysozyme molecules and the cell wall of the S. cerevisiae, proved by the crystallization of lysozyme with fluorescent labels. The biotemplates demonstrated positive influence or negative influence on the nucleation, i.e. shorter or longer induction time, dependent on the concentrations of the lysozyme and the S. cerevisiae cells, and ratios between them. In the biomanufacturing process, target proteins were various cells were commonly mixed with various cells, and this work provides novel insights of new design and application of live cells as biotemplates for purification of macromolecules.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1.

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

General characteristics of the influence of surfactants on the bacteriolytic activity of lysozyme based on the example of enzymatic lysis of Lactobacillus plantarum cells in the presence of Tween 21 and SDS.

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme.

Cationic Covalent Organic Framework as an Ion Exchange Material for Efficient Adsorptive Separation of Biomolecules.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy2+-COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy2+-COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Online preconcentration of lysozyme in hen egg white using responsive polymer coating in CE.

A mixed polymer brushes material based on poly (2-methyl-2-oxazoline)- and poly (acrylic acid)-coated capillary with switchable protein adsorption/desorption properties was applied for online preconcentration of lysozyme in hen egg white during capillary electrophoresis performance. First, lysozyme in simulated egg white was successfully online preconcentrated and the detection signal of lysozyme was amplified. Ovalbumin, ovomucoid, and conalbumin in egg white were verified show negligible interference on the online preconcentration of lysozyme according to the study on electroosmotic flow mobility. Second, a series validation procedure was carried out to evaluate the proposed method performance. There was a good linearity behavior range from 0.1 to 5.0 ng/mL, limit of detection was 20 pg/mL, and limit of quantity was 50 pg/mL, the accuracy and robustness of this method were also excellent. Last, the proposed method has been successfully used to detect and analyze lysozyme in hen egg white, the determined amounts of lysozyme in hen egg white were consistent with reported normal levels and recoveries were in the range of 96.0-99.2%. After 75 consecutive runs, this prepared capillary was still stable for online preconcentration and determination of lysozyme in hen egg white without being affected by complex matrix.

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.

Adsorption and purification performance of lysozyme from chicken egg white using ion exchange nanofiber membrane modified by ethylene diamine and bromoacetic acid.

A high-performance polyacid ion exchange (IEX) nanofiber membrane was used in membrane chromatography for the recovery of lysozyme from chicken egg white (CEW). The polyacid IEX nanofiber membrane (P-BrA) was prepared by the functionalization of polyacrylonitrile (PAN) nanofiber membrane with ethylene diamine (EDA) and bromoacetic acid (BrA). The adsorption performance of P-BrA was evaluated under various operating conditions using Pall filter holder. The results showed that optimal conditions of IEX membrane chromatography for lysozyme adsorption were 10% (w/v) of CEW, pH 9 and 0.1 mL/min. The purification factor and yield of lysozyme were 402 and 91%, respectively. The adsorption process was further scaled up to a larger loading volume, and the purification performance was found to be consistent. Furthermore, the regeneration of IEX nanofiber membrane was achieved under mild conditions. The adsorption process was repeated for five times and the adsorption capacity of adsorber was found to be unaffected.

Taurine suppresses liquid-liquid phase separation of lysozyme protein.

Taurine is a compatible osmolyte that confers stability to proteins. Recent studies have revealed that liquid-liquid phase separation (LLPS) of proteins underlie the formation of membraneless organelles in cells. In the present study, we evaluated the role of taurine on LLPS of hen egg lysozyme. We demonstrated that taurine decreases the turbidity of the polyethylene glycol-induced crowding solution of lysozyme. We also demonstrated that taurine attenuates LLPS-dependent cloudiness of lysozyme solution with 0.5 or 1 M NaCl at a critical temperature. Moreover, we observed that taurine inhibits LLPS formation of a heteroprotein mix solution of lysozyme and ovalbumin. These data indicate that taurine can modulate the formation of LLPS of proteins.

Use of magnetic Fe3O4 nanoparticles coated with bovine serum albumin for the separation of lysozyme from chicken egg white.

Fe3O4 magnetic nanoparticles modified with tetraethyl orthosilicate and bovine serum albumin (Fe3O4@TEOS@BSA) were synthesized and efficiently used to separate lysozyme from egg white. Glutaraldehyde was used to crosslink the bovine serum albumine molecules around the nanoparticles. The surface modifications were attested by transmission electron microscopy, infrared spectroscopy, thermogravimetry analysis, and zeta potential. The material was thermally stable, and its surface charge was pH-dependent. The best lysozyme adsorption and desorption were obtained at pHs 10.0 and 5.0, respectively. The pseudo-second-order model fitted well into the lysozyme adsorption kinetic data and the time for the equilibrium was 15 min. The adsorption equilibrium results were best described by the Freundlich model. Fe3O4@TEOS@BSA particles were very efficient to extract lysozyme from chicken egg, according to the SDS-PAGE analyses. The extracted molecules maintained their enzymatic activity in about 90%. Fe3O4@TEOS@BSA particles were easily recycled, with their reuse being possible 5 times with the same performance.

Isolation of a broad spectrum antimicrobial producing thermophilic Bacillus and characterization of its antimicrobial protein.

The hot spring water of Atri in India was believed to have disease curing property. An antibacterial producing organism was isolated and identified as Bacillus paralicheniformis by morphology, microscopy, and 16S-rRNA. Its secretion inhibited bacteria, yeast, and fungus in well-diffusion-method. The secreted antimicrobial was a 16.74 kDa protein homologous of chicken-lysozyme-C. The novel lysozyme's activities were recorded under different parameters. It was active from pH 5-9 and endured up to 60 °C for 120 min. Complete cell wall lysis of S. flexneri and P. aeruginosa was observed under a microscope at 4500× with a minimum inhibitory concentration of 7.8 µg/ml, while others required a higher dose, i.e., 13 µg/ml, and 20 µg/ml for E.coli and S. typhimurium, respectively. The discovered lysozyme has the extraordinary potential to lyse Gram-positive bacteria, yeast, fungus, and more efficiently lyse chick-lysozyme-C resistant lipopolysaccharide rich Gram-negative bacteria's outer cell wall.

New possibilities for egg white lysozyme: heat-denatured lysozyme partially inactivates select foot-and-mouth disease virus strains.

Foot-and-mouth disease (FMD) is one of the most contagious diseases of cloven-hoofed animals. Disinfectants are used to inactivate FMD virus (FMDV) in Japan. Reports that heat-denatured lysozyme inactivates bacteria as well as viruses, such as norovirus and hepatitis A virus, led us to determine its effects on FMDV. We show here that heat-denatured lysozyme partially inhibited the infectivity of FMDV O/JPN/2010-1/14C but of FMDVs A/TAI/46-1/2015 and Asia1/Shamir (ISR/3/89). Further, heat-denatured lysozyme variably reduced RNA loads of FMDVs O/JPN/2010-1/14C, O/MOG/2/Ca/BU/2017, O/Taiwan/1997, Asia1/Shamir (ISR/3/89), Asia1/TUR/49/2011, SAT1/KEN/117/2009, SAT2/SAU/6/2000 and SAT3/ZIM/3/83 but could not those of O/JPN/2000, A/TAI/46-1/2015, A22/IRQ/24/64, A15/TAI/1/60 and C/PHI/7/84. These findings indicate that heat-denatured lysozyme may serve as a new disinfectant against FMDV.

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

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.

Selective Enrichment of Low-abundance Compounds in a Mixture by Capillary Electrophoresis.

Recently, we developed a new approach for the selective enrichment of low-abundance compounds in biological samples by capillary electrophoresis. As a model test, the low-abundance compound lysozyme was successfully fractionated from a mixture containing high-abundance compound BSA (1:4500) using a custom-made apparatus. The feasibility of this approach for real complex biological samples was verified by rat serum, wherein three low-abundance proteins with high charge/mass ratios were detected.

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.