Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50 degrees C, HM-EOPO, 14 degrees C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance.
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PMID:Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers. 1063 Aug 69

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using (3)H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism, the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains ( approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.
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PMID:Noncovalent adducts of poly(ethylene glycols) with proteins. 1063 81

Protein release from a series of biodegradable poly(ether ester) multiblock copolymers, based on poly(ethylene glycol) (PEG) and poly(butylene terephthalate) (PBT) was investigated. Lysozyme-containing PEG/PBT films and microspheres were prepared using an emulsion technique. Proteins were effectively encapsulated and dense polymer matrices were formed. The swelling in water of PEG/PBT films reached equilibrium within 3 days. The degree of swelling increased with increasing PEG content and with increasing molecular weight of the PEG segment. The release rate of lysozyme from PEG/PBT films could be tailored very precisely by controlling the copolymer composition. Release rates increased with increasing PEG/PBT weight ratio and increasing molecular weight of the PEG segment. For films prepared from block copolymers with PEG blocks of 4000 g/mol, first-order lysozyme release was observed. For matrices prepared from polymers with PEG segments of 1000 and 600 g/mol, the lysozyme release profile followed near zero-order kinetics. A mathematical description of the release mechanism was developed which takes into account the effect of polymer hydrolytic degradation on solute diffusion. The model was found to be consistent with the experimental observations. Finally, determination of the activity of released protein showed that lysozyme was not damaged during the formulation, storage and release periods.
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PMID:Zero-order release of lysozyme from poly(ethylene glycol)/poly(butylene terephthalate) matrices. 1064 Jun 56

Protein-containing films and microspheres, based on poly(ethylene glycol)-poly(butylene terephthalate) (PEG-PBT) multiblock copolymers, were prepared from water-in-oil (w/o) emulsions. The properties of the matrices could be controlled by the water-to-polymer ratio (w/p) in the w/o emulsion. A linear increase in water uptake of the matrices was observed with increasing emulsion w/p. This could be explained by an increase in the number of dispersed water-rich domains in the polymer matrix. At low volume fraction of the dispersed phase (epsilon), lysozyme release was mainly dependent on the permeability of the swollen polymer bulk. Above a critical volume fraction (the percolation threshold epsilon(c)), the dispersed water-rich phase formed an interconnected network, which largely enhanced the permeability of the matrix. Determination of the permeability of PEG-PBT matrices for vitamin B(12) as a function of epsilon confirmed the formation of such an interconnected network. This interconnected network could be used to achieve controlled release of a large protein (bovine serum albumin, BSA) from PEG-PBT films and microspheres. Due to its hydrodynamic diameter, BSA was screened by the polymer network when epsilon was low. However above epsilon(c), the fraction released BSA increased with increasing volume fraction of the dispersed phase. A very rapid BSA release could be obtained, with the majority of the incorporated BSA released within 1 day, as well as a slow and continuous release, lasting for over 150 days. When BSA-containing microspheres were prepared with a volume fraction just below the percolation threshold, a delayed release was observed. This was attributed to the effect of polymer degradation on matrix permeability.
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PMID:Control of protein delivery from amphiphilic poly(ether ester) multiblock copolymers by varying their water content using emulsification techniques. 1074 89

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.
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PMID:Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins. 1074 92

During recombinant E. coli fermentation with high-expression levels inclusion bodies are often formed. Aqueous two-phase systems have been successfully used in the presence of urea for the initial recovery step of inclusion bodies from E. coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we selected T4-lysozyme mutants with different thermal stability as a model. The stabilization of these variants by phase components was investigated measuring the fluorescence emission of tryptophan residues in the protein. Protein structure was stabilized at pH 7 in the order of S0(4)(2-) >> PEG = Dextran > H(2)O. The conformation of proteins was shown to have a strong influence on the partitioning in aqueous two-phase systems. Tryptophan and its homologuous di- and tripeptdides were partitioned in similar phase systems to normalize for contribution from hydrophobic interactions.
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PMID:Aqueous two-phase systems containing urea: influence of protein structure on protein partitioning. 1082 Mar 34

The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent extraction from a water-in-oil (w/o) emulsion in ethanol or methanol did not result in the formation of microspheres, due to massive polymer precipitation caused by rapid solvent extraction in these non-solvents. In a second process, microspheres were first prepared by a water-in-oil-in-water (w/o/w) emulsion system with 4% poly(vinyl alcohol) (PVA) as stabilizer in the external phase, followed by extraction of the remaining solvent. As non-solvents ethanol, methanol and mixtures of methanol and water were employed. However, the use of alcohols in the extraction medium resulted in microspheres which gave an incomplete lysozyme release at a non-constant rate. Complete lysozyme release was obtained from microspheres prepared by an emulsification-solvent evaporation method in PBS containing poly(vinyl pyrrolidone) (PVP) or PVA as stabilizer. PVA was most effective in stabilizing the w/o/w emulsion. Perfectly spherical microspheres were produced, with high protein entrapment efficiencies. These microspheres released lysozyme at an almost constant rate for approximately 28 days. The reproducibility of the w/o/w emulsion process was demonstrated by comparing particle characteristics and release profiles of three batches, prepared under similar conditions.
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PMID:Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. Influence of preparation techniques on particle characteristics and protein delivery. 1082 57

Amphiphilic multiblock copolymers, based on hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks were used as matrix material for protein-loaded microspheres. The efficiency of lysozyme entrapment by a double emulsion method was found to depend on the swelling behavior of the polymers in water and decreased from 100% for polymers with a degree of swelling of less than 1.8 to 11% for PEG-PBT copolymers with a degree of swelling of 3.6. The particle size could be controlled by varying the concentration of the polymer solution used in the microsphere preparation. An increase in the polymer concentration resulted in a proportional increase in the particle size. The in vitro release profiles of the encapsulated model protein lysozyme could be precisely tailored by variation of the copolymer composition and the size of the microspheres. Both a slow continuous release of lysozyme, and a fast release which was completed within a few days could be obtained. The release behavior, attributed to a combination of diffusion and polymer degradation, could be described by a previously developed model.
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PMID:Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 2. Modulation of release rate. 1082 58

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

Amphiphilic poly(ether ester amide) (PEEA) multiblock copolymers were synthesized by polycondensation in the melt from hydrophilic poly(ethylene glycol) (PEG), 1,4-dihydroxybutane and short bisester-bisamide blocks. These amide blocks were prepared by reaction of 1,4-diaminobutane with dimethyl adipate in the melt. A range of multiblock copolymers were prepared, with PEG contents varying from 23-66 wt %. The intrinsic viscosity of the PEEA polymers varied from 0.58-0.78. Differential scanning calorimetry showed melting transitions for the PEG blocks and for the amide-ester blocks, suggesting a phase separated structure. Both the melting temperature and the crystallinity of the hard amide-ester segments decreased with increasing PEG content of the polymers. The equilibrium swelling ratio in phosphate buffered saline (PBS) increased with increasing amount of PEG in the polymers and varied from 1.7 to 3.7, whereas the polymer that contained 66 wt % PEG was soluble in PBS. During incubation of PEEA films in PBS, weight loss and a continuous decrease in the resulting inherent polymer viscosity was observed. The rate of degradation increased with increasing PEG content. The composition of the remaining matrices did not change during degradation. A preliminary investigation of the protein release characteristics of these PEEA copolymers showed that release of the model protein lysozyme was proportional to the square root of time. The release rate was found to increase with increasing degree of swelling of the polymers.
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PMID:Amphiphilic poly(ether ester amide) multiblock copolymers as biodegradable matrices for the controlled release of proteins. 1090 69


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