Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To develop a polymeric matrix for efficiently loading cationic biomolecules, polyelectrolyte hydrogels carrying pendant phosphate groups were synthesized by copolymerizing 2-methacryloyloxyethyl dihydrogen phosphate with N-isopropylacrylamide and N, N1-methylene-bis-acrylamide. The phosphate-carrying monomer yielded anionic hydrogels, which formed ionic complexes with the cationic protein, lysozyme. It was shown that the amount of complexed lysozyme reached 2.1 g g-1 dry gel, corresponding to 1.3 x 10(-3) mol phosphate group per gram lysozyme, when 40 mol% of phosphate-carrying monomer was incorporated in a hydrogel. When the hydrogel complexed with lysozyme was placed in deionized water and various KCl solutions, of varying concentrations of up to 0.5 M KCl, no lysozyme was released in deionized water, while increasing amounts of lysozyme were released as the KCl concentration increased. This confirmed that lysozyme was loaded in the hydrogel through electrostatic interactions. It was further found that the complexed lysozyme retained its enzymatic activity after being released from the hydrogel. These results suggest the use of this system for the controlled release of cationic protein drugs.
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PMID:Lysozyme loading and release from hydrogels carrying pendant phosphate groups. 950 2

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C14) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K = 0.8 to K = 5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.
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PMID:Aqueous polymer two-phase systems formed by new thermoseparating polymers. 1089 44

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.
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PMID:Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers. 1127 34

Affinity precipitation, especially secondary effect affinity precipitation, has repeatedly been suggested as a valuable technique for the biotechnical downstream process. The present lack of applications is related to the scarcity of predictable affinity macroligands and to the fact that rather high affinity constants are required in affinity precipitation (K(D) < 10(-10)). The latter are rarely found in nature, at least in the case of small affinity ligands (affinity tags), and are usually difficult to handle (complex dissociation) once one has found them. In this article we describe a new type of thermoresponsive affinity macroligand. The base polymer (poly-N-isopropylacrylamide, or PNIPAAm) is produced by chain transfer polymerization. As a consequence, the structure, as well as the solubility behavior, is very homogeneous (polydispersity < 1.2), whereas the average molecular mass is small (<5000 g/mol). In pure water, the base polymer shows sharp thermoprecipitation at 32.2 degrees C. Each oligomer carries a single amino end group, which allows easy and defined coupling of the affinity ligand, while preserving the ligand's activity to the highest possible degree. Herein, the oligomer was coupled to iminobiotin. The ensuing affinity macroligand has a high affinity to avidin (and avidin-tagged molecules) at elevated pH (<10), but releases the avidin easily at lower pH (approximately 4). The affinity macroligands were used to purify avidin from solutions containing large amounts of lysozyme as well as from cell culture supernatants containing 5% fetal calf serum. In both cases, pure avidin was recovered (residual protein contamination below the detection limit), with yields of >90%.
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PMID:Use of the avidin (imino)biotin system as a general approach to affinity precipitation. 1129 Oct 32

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.
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PMID:Affinity separation using an Fv antibody fragment-"smart" polymer conjugate. 1211 15

Poly(N-isopropylacrylamide) (PNIPA) microgels may offer several advantages over PNIPA-modified surfaces when used as sorbents in temperature-sensitive chromatography. Yet, a full exploitation of these advantages requires a better understanding of the mechanisms controlling the separation process. As a model system, we have studied the binding of three proteins (bovine serum albumin (BSA), ovalbumin, and lysozyme) to PNIPA microgels. Binding experiments were conducted both below (25 degrees C) and above (37 degrees C) the volume phase transition temperature of the gel, T(c). The analysis of the binding isotherms has shown that although an average gel particle contained a larger amount of protein below the phase transition temperature, the concentration of the protein within the particle was higher above this temperature. These findings were attributed to changes in the binding loci due to temperature swings around T(c): whereas a sorption mechanism is dominant below this temperature, surface-adsorption was more important above it. A comparison between the three studied proteins has shown that below T(c) the binding increases with a decrease in the molecular weight. On the other hand, no significant difference in the bound protein amounts was observed above the phase transition temperature. Our results imply that, despite the increase in the gel's hydrophobicity above the phase transition temperature, the resolution in bioseparations based on PNIPA gels is not necessarily better above T(c).
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PMID:Mechanisms controlling the temperature-dependent binding of proteins to poly(N-isopropylacrylamide) microgels. 1465 48

This work was focused on the investigation of temperature and pH-responsive polymeric composite membranes and their permeability to proteins and peptides in response to environmental stimuli. The composite membranes were prepared from nanoparticles of poly(N-isopropylacrylamide-co-methacrylic acid) of various NIPAAm:MAA ratios dispersed in a matrix of a hydrophobic polymer. N-Benzoyl-L-tyrosine ethyl ester HCl, momany peptide, Leuprolide, vitamin B(12), insulin, and lysozyme were used as model solutes. The morphology of the membranes was examined with SEM and permeation of the solutes was measured using side-by-side diffusion cells at varied temperatures and pH. Permeability of the solutes across the membranes increased with increasing temperature or particle concentration, while decreased with increasing pH and molecular size of the solutes. Membranes containing nanoparticles of more NIPAAm units exhibited higher thermal sensitivity, and those with higher MAA content showed more pH responsiveness, which was in line with the temperature and pH-responsive volume change of the nanoparticles. The change in permeability was quickly detected following the application of the stimuli. These results and partition study using vitamin B(12) supported the proposed gel-pore mechanism of solute permeation through these composite membranes.
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PMID:Temperature and pH-responsive polymeric composite membranes for controlled delivery of proteins and peptides. 1511 Apr 79

In order to prepare a polymer matrix capable of loading protein at high density, anionic hydrogels were synthesized by copolymerizing a monomer carrying a pendant phosphate group, methacryloyl-polyoxyethyl phosphate, with N-isopropylacrylamide and N,N'-methylene-bis-acrylamide, and the stumuli-sensitivity of hydrogels was characterized. The number of repeating ethylene glycol units in the phosphate carrying monomer was 1, 2, 5 or 8. Lysozyme bearing a positive net charge was immobilized in the hydrogel through formation of polyelectrolyte complex. It was shown that the amount of complexed lysozyme reached to 1.7 g/g dry gel, when high content of a phosphate-carrying monomer with 5 ethylene glycol units was incorporated into a hydrogel. It was further found that lysozyme complexed with phosphate-carrying network could be released by immersion of the lysozyme/hydrogel composite in a phosphate buffer solution of pH 7.4 owing to the pH-sensitivity of the hydorgel but no lysozyme was released at pH 1.4. The initial rate of lysozyme release was varied depending on the length of the ethylene glycol chains possessed by a network polymer and the content of the phosphate-carrying monomer unit. Lysozyme released from the phosphate-carrying hydrogel was proved to retain enzymatic activity at a level similar to the activity of lysozyme, which had been kept in buffer solution.
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PMID:Synthesis and characterization of stimuli-sensitive hydrogels having a different length of ethylene glycol chains carrying phosphate groups: loading and release of lysozyme. 1564 73

Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by muLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by (1)H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed.
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PMID:In situ preparation of protein-"smart" polymer conjugates with retention of bioactivity. 1631 41

A controlled-release device that responds to a specific molecular signal is an ideal goal in drug delivery and tissue engineering. A molecular recognition ion gating membrane, in which a copolymer of N-isopropylacrylamide and benzo[18]-crown-6-acrylamide was grafted onto the surface of the porous polyethylene film, was used to control the permeability of vitamin B12 and lysozyme in response to a specific ion signal. The observed response depended on the amount of grafted copolymer. When the grafting ratio was below 15%, the membrane pores opened by Ca2+ and closed by Ba2+. The permeability of model drugs became higher by opening of the pores. On the other hand, when the grafting ratio was above 15%, the properties of the membrane changed. The permeability of model drugs became lower by Ca2+ due to dehydration of the grafted copolymer. The opposite responses were observed at different grafting ratios.
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PMID:Controlled release of model drugs through a molecular recognition ion gating membrane in response to a specific ion signal. 1658 80


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