muramidase and glycidyl-methacrylate

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

Topics: Adsorption; Cellulose; Chemistry Techniques, Analytical; Chromatography; Diphosphonates; Epoxy Compounds; Ligands; Methacrylates; Muramidase; Polymerization; Porosity

Catheter-associated infections still represent a challenging thread because of the likelihood of biofilm formation. The aim of this work was the surface modification of catheters to immobilize lysozyme and acylase under mild conditions while preserving antimicrobial and anti-quorum sensing performances. Glycidyl methacrylate (GMA) was grafted onto poly(vinyl chloride) (PVC) catheters by a pre-irradiation method. The effects of monomer concentration, pre-irradiation dose, reaction time, monomer concentration and reaction temperature were investigated. The grafting process was monitored using FTIR-ATR spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and swelling data. Lysozyme was directly immobilized onto PVC-g-GMA maintaining the hydrolytic activity, which hindered Staphylococcus aureus adhesion. For acylase immobilization, the PVC-g-GMA catheters were reacted with ethylenediamine and glutaraldehyde in order to facilitate acylase covalent binding. Free acylase in solution demonstrated notably capability to act as quorum sensing inhibitor, as observed using Chromobacterium violaceum as biosensor, by degrading a wide variety of acylated homoserine lactones (AHLs), including those produced by Pseudomonas aeruginosa and Acinetobacter baumannii. Acylase-immobilized PVC-g-GMA catheters were challenged against degradation of AHLs and the activity monitored using both the biosensor and HPLC-MS. Relevantly, the functionalized catheters completely degraded all tested AHL signals, opening new ways of preventing biofilm formation on medical devices.

Topics: Amidohydrolases; Bacterial Adhesion; Catheters; Enzymes, Immobilized; Epoxy Compounds; Lactones; Methacrylates; Muramidase; Polyvinyl Chloride; Quorum Sensing; Staphylococcus aureus

Designing biomaterials capable of functioning in harsh environments is vital for a range of applications. Using molecular dynamics simulations, we show that conjugating lysozymes with a copolymer [poly(GMA- stat-OEGMA)] comprising glycidyl methacrylate (GMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) results in a dramatic increase of stability of these enzymes at high temperatures provided that the concentration of the copolymer in the close vicinity of the enzyme exceeds a critical value. In our simulations, we use triads containing the same ratio of GMA to OEGMA units as in our recent experiments (N. S. Yadavalli et al., ACS Catalysis, 2017, 7, 8675). We focus on the dynamics of the conjugate at high temperatures and on its structural stability as a function of the copolymer/water content in the vicinity of the enzyme. We show that the dynamics of phase separation in the water-copolymer mixture surrounding the enzyme is critical for the structural stability of the enzyme. Specifically, restricting water access promotes the structural stability of the lysozyme at high temperatures. We identified critical water concentration below which we observe a robust stabilization; the phase separation is no longer observed at this low fraction of water so that the water domains promoting unfolding are no longer formed in the vicinity of the enzyme. This understanding provides a basis for future studies on designing a range of enzyme-copolymer conjugates with improved stability.

Topics: Catalysis; Enzyme Stability; Epoxy Compounds; Hot Temperature; Methacrylates; Molecular Dynamics Simulation; Muramidase; Polymerization; Polymers; Protein Conformation; Water

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24% GMA, 16% EDMA, 20% cyclohexanol, and 40% 1-dodecanol (all% as w/w) containing 1% of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 μL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 μL s(-1)) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Topics: Animals; Cattle; Chickens; Chromatography, Ion Exchange; Cytochromes c; Epoxy Compounds; Horses; Methacrylates; Muramidase; Myoglobin; Polymers; Ribonuclease, Pancreatic

Motivated by the demand for more economical capture and polishing steps in downstream processing of protein therapeutics, a novel strong cation-exchange chromatography stationary phase based on polyethylene terephthalate (PET) high surface area short-cut fibers is presented. The fiber surface is modified by grafting glycidyl methacrylate (GMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and a subsequent derivatization leading to sulfonic acid groups. The obtained cation-exchange fibers have been characterized and compared to commercially available resin and membrane based adsorbers. High volumetric static binding capacities for lysozyme (90mg/mL) and polyclonal human IgG (hIgG, 92mg/mL) were found, suggesting an efficient multi-layer binding within the grafted hydrogel layer. A packed bed of randomly orientated fibers has been tested for packing efficiency, permeability and chromatographic performance. High dynamic binding capacities for lysozyme (50mg/mL) and hIgG (54mg/mL) were found nearly independent of the bed-residence time, revealing a fast mass-transport mechanism. Height equivalent to a theoretical plate (HETP) values in the order of 0.1 cm and a peak asymmetry factor (AF) of 1.8 have been determined by tracer experiments. Additionally inverse size-exclusion chromatography (iSEC) revealed a bimodal structure within the fiber bed, consisting of larger transport channels, formed by the voidage between the fibers, and a hydrogel layer with porous properties.

Topics: Adsorption; Cations; Chromatography, Ion Exchange; Epoxy Compounds; Humans; Hydrogels; Immunoglobulin G; Methacrylates; Muramidase; Permeability; Polyethylene Terephthalates; Polymerization; Sulfonic Acids

In this report, protein repelling silicone hydrogels with improved hydrophilicity were prepared by photo-polymerization of silicone-containing monomer and glycidyl methacrylate followed by grafting zwitterionic amino acids. The grafted silicone hydrogels possessed excellent hydrophilic surfaces due to the enrichment of amino acids, which was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, contact angle, and equilibrium water content measurements. Remarkable resistance to bovine serum albumin and lysozyme fouling was observed for the silicone hydrogels immobilized with neutrally charged amino acids because of the formation of zwitterionic surfaces with pairs of protonated secondary ammonium cations and deprotonated carboxyl anions. Meanwhile, the silicone hydrogels grafted with positively or negatively charged amino acids were able to repulse same charged protein with reduced deposition and attract oppositely charged protein with increased adsorption. Preliminary cytotoxicity test indicated that the zwitterionic silicone hydrogels were non-cytotoxic. Similarly, three types of natural amino acids, including serine, aspartic acid and histidine, modified silicone hydrogel contact lenses exhibited excellent hydrophilicity and non-damage to the rabbit's eyes, but only serine modified zwitterionic contact lens showed superior protein fouling resistance compared with the current commercial hydrogel contact lens, which may have great potential application in ophthalmology.

Topics: Adsorption; Amino Acids; Animals; Cattle; Cell Line; Cell Survival; Contact Lenses, Hydrophilic; Epoxy Compounds; Hydrogels; Hydrophobic and Hydrophilic Interactions; Light; Methacrylates; Mice; Muramidase; Polymerization; Rabbits; Serum Albumin, Bovine; Silicones; Surface Properties; Water; Wettability

In this study, poly (methyl methacrylate-glycidyl methacrylate) [poly(MMA-GMA)] cryogels were prepared by radical cryocopolymerization of MMA with GMA as a functional comonomer. Reactive Green 19 dye was then attached to the cryogel by nucleophilic substitution reaction, and this dye-attached cryogel column was used for lysozyme adsorption. Characterization of the cryogel was performed by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, Brunauer-Emmett-Teller, and energy dispersive X-ray analysis. Pore size of the cryogels was 15-30 μm and pores were interconnected structure. Attached amount of Reactive Green 19 to cryogel support was calculated as 106.25 μmol/g cryogel. Lysozyme adsorption studies were carried out by using a continuous system. It was found that the maximum amount of lysozyme adsorption (32 mg/g cryogel) obtained from experimental results was found to be approximately same with the calculated Langmuir adsorption capacity (33 mg/g cryogel). Desorption of adsorbed lysozyme was carried out by using 1.5 M NaCl in pH 4.5 acetate buffer, and desorption yield was found to be 97.4%. Cryogels were very stable, and it was found that there was no remarkable reduction in the adsorption capacity at the end of ten adsorption-desorption cycles. As a result, Reactive Green 19-attached cryogels have great advantages such as easy preparation, rapid adsorption, and desorption, being economic and allowing the direct separation of proteins.

Topics: Adsorption; Chromatography, Gel; Coloring Agents; Cryogels; Epoxy Compounds; Hydrogen-Ion Concentration; Methacrylates; Microscopy, Electron, Scanning; Muramidase; Polymethyl Methacrylate; Porosity; Spectroscopy, Fourier Transform Infrared

The immobilization of enzymes is of paramount importance to maintain their activity and stability. In this study, surface-initiated atom-transfer radical polymerization was applied to prepare poly(2-hydroxyethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) brushes on glass slides. The polymerization kinetics was followed by using a quartz crystal microbalance with dissipation monitoring and ellipsometry in terms of mass and thickness growth, respectively. The surface chemical compositions of the obtained polymer brushes were characterized by X-ray photoelectron spectroscopy. Their mass, thickness, and enzyme-immobilization ability could be easily tuned by the initiator reaction time, monomer ratio, and polymerization time. The antibacterial activity and stability of the immobilized lysozymes were studied by fluorescent staining and bacteria lysis assay, which revealed that the lysozymes on the copolymer brushes had good stability during storage at 4 °C for up to 30 days.

Topics: Bacteria; Enzymes, Immobilized; Epoxy Compounds; Methacrylates; Muramidase; Photoelectron Spectroscopy; Polymers

Based on the needs of new packing materials for rapid and efficient separation, purification and analysis of biomacromolecules, a novel sulfonic acid-type strong cation exchange resin (SP-G-PGMA SCX resin) was prepared. The porous poly(glycidyl methacrylate) microspheres (PGMA) were selected as the matrix and glucose was used as the hydrophilic modifier to block the hydrophobic domains of PGMA beads. Glucose modification on PGMA beads improved the biocompatibility and reduced the non-specific adsorption so as to increase the recoveries of protein. The PGMA beads possess the porous structure and the relatively high specific surface area, which make the PGMA-based resins good permeability and high loading capacity. The application of such SP-G-PGMA SCX resin for the chromatographic separation of biomacromolecules was explored. Four basic proteins were baseline separated within 6 min with the column size of 100 mm x 4.6 mm. The adsorption capacity of lysozyme on SP-G-PGMA SCX resin was determined as 39.5 g/L. The results make the material promising for the separation and purification of biomacromolecules.

Topics: Adsorption; Cation Exchange Resins; Cations; Epoxy Compounds; Hydrophobic and Hydrophilic Interactions; Methacrylates; Microspheres; Muramidase; Porosity; Proteins

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).

Topics: Absorption; Animals; Chickens; Egg White; Epoxy Compounds; Hydrogen-Ion Concentration; Magnetics; Methacrylates; Microspheres; Muramidase; Polymers; Reproducibility of Results; Temperature

In order to achieve a simple covalent hydrophilic polymer coating on poly(dimethylsiloxane) (PDMS) microfluidic chip, epoxy modified hydrophilic polymers were synthesized in aqueous solution with a persulfate radical initiation system, and crosslinked onto PDMS pretreated by oxygen plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). Glycidyl methacrylate (GMA) was copolymerized with acrylamide (poly(AAM-co-GMA)) or dimethylacrylamide (poly(DAM-co-GMA)), and graft polymerized with polyvinylpyrrolidone (PVP-g-GMA) or polyvinylalcohol (PVA-g-GMA). The epoxy groups in the polymers were determined by UV spectra after derivation with benzylamine. Reflection absorption infrared spectroscopy (RAIRS) confirmed covalent grafting of GMA-modified polymers onto PDMS surface. Electroosmotic flow (EOF) in the polymer grafted microchannel was strongly suppressed within the range pH 3-11. Surface adsorption of lysozyme and bovine serum albumin (BSA) was reduced to less than 10% relative to that on the native PDMS surface. On the GMA-modified polymer coated PDMS microchip, basic proteins, peptides, and sodium dodecyl sulfate (SDS) denatured proteins were separated successfully.

Topics: Acrylamides; Adsorption; Dimethylpolysiloxanes; Electrophoresis; Epoxy Compounds; Hydrophobic and Hydrophilic Interactions; Methacrylates; Microfluidic Analytical Techniques; Muramidase; Polyvinyl Alcohol; Povidone; Protein Denaturation; Proteins; Serum Albumin, Bovine; Silicones; Surface Properties

Hollow-fibre membranes with different degrees of surface hydrophilicity were obtained by grafting mixtures of glycidyl methacrylate (GMA) and dimethyl acrylamide (DMAA) in various proportions, and Cibacron Blue F3G-A was attached to them through ammonia or glucamine spacers. Membrane hydrophilicity increased with the amount of dimethyl acrylamide in the grafted polymer. As the hydrophilicity increased the permeability decreased from 352 mL/cm2 min MPa for membranes grafted with GMA with ammonia spacer to 12.7 mL/cm2 min MPa for membranes grafted with GMA/DMAA 1/3 with glucamine spacer. Membranes grafted with GMA/DMAA 1/3 with ammonia spacer showed the best performance for BSA and lysozyme adsorption: maximum capacity was 15.3 +/- 2.2 mg BSA/mL membrane and 58.3 +/- 6.6 mg lysozyme/mL membrane while dissociation constants were 0.27 +/- 0.16 and 0.13 +/- 0.12 mg/mL, respectively. Over 80% of adsorbed proteins could be eluted with 2 M NaCl + 20% isopropanol in 20 mM sodium phosphate buffer, pH 7.0.

Topics: Acrylamides; Adsorption; Animals; Cattle; Chromatography, Affinity; Coloring Agents; Epoxy Compounds; Hydrophobic and Hydrophilic Interactions; Membranes, Artificial; Methacrylates; Muramidase; Permeability; Polymers; Proteins; Serum Albumin, Bovine; Surface Properties; Triazines

Non-porous particles having an average diameter of 2.1 microm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S-MMA-GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode. The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S-MMA-GMA) particles were slurry packed into a 1.0 cm x 0.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 microg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level.

Topics: Chromatography, Affinity; Chromatography, High Pressure Liquid; Epoxy Compounds; Methacrylates; Methylmethacrylate; Muramidase; Polymers; Proteins; Serum Albumin, Bovine; Styrene

An affinity monolith with a novel immobilization strategy was developed leading to a tailored pore structure. Hereby the ligand is conjugated to one of the monomers of the polymerization mixture prior to polymerization. After the polymerization, a monolithic structure was obtained either ready to use for affinity chromatography or ready for coupling of additional ligand to further increase the binding capacity. The model ligand, a peptide directed against lysozyme, was conjugated to glycidyl methacrylate prior to the polymerization. With this conjugate, glycidyl methacrylate, and ethylene dimethacrylate, a monolith was formed and tested with lysozyme. A better ligand presentation was achieved indicated by the higher affinity constant compared to a conventional sorbent.

Topics: Chromatography, Affinity; Cross-Linking Reagents; Epoxy Compounds; Ligands; Methacrylates; Muramidase; Peptides; Polymers

A continuous rod of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 300 x 8 mm I.D. chromatographic column. The epoxide groups of the rod have been modified by a reaction with diethylamine that affords ionizable functionalities required for the ion-exchange chromatographic mode. The properties of this rod column have been characterized and the column has been used successfully for the chromatographic separation of proteins. The column exhibits a dynamic capacity that exceeds 300 mg at a flow velocity of 200 cm/min. An excellent selectivity allows the separation of up to 300 mg of a protein mixture in a single run.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Conalbumin; Epoxy Compounds; Methacrylates; Muramidase; Ovalbumin; Proteins; Serum Albumin, Bovine; Trypsin Inhibitors