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)

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the purification of natural and recombinant therapeutic products and is beginning to find industrial applications. The design, optimization, and scale-up of a chromatographic process using IMAC demands a thorough understanding to be developed regarding the fundamental factors governing the various interactions between immobilized metal ions and proteins. Consequently, there is an immediate need to find out a theory that is able to account for these interactions most efficiently in a qualitative as well as a quantitative manner. In view of this requirement, the interactions of several model proteins (lysozyme, ovalbumin, bovine serum albumin, conalbumin, and wheat germ agglutinin) with metal (Cu(II), Ni(II))-chelated IDA (iminodiacetate) and tris(2-aminoethyl)amine were investigated. The adsorption data were analyzed using four isotherm models, viz., the general affinity interaction theory/Langmuir model, the Freundlich model, the Temkin model, and the Langmuir-Freundlich model, and the sorption parameters were computed. Although the first three models were applicable to some protein-IMA-M(II) systems, the Langmuir-Freundlich model appeared to be the most efficient model for explaining the interactions of proteins with IMA-M(II) gels. Also, this model was able to explain cooperativity and binding heterogeneity in quantitative terms. It is envisaged that this analysis would be useful in developing an improved understanding of protein-immobilized metal ion interactions and providing guidelines for designing preparative-scale separations using IMAC.
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PMID:Interactions of proteins with immobilized metal ions: a comparative analysis using various isotherm models. 1115 83

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).
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PMID:Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography. 1121 18

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.
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PMID:Physiological function of exopolysaccharides produced by Lactococcus lactis. 1125 13

This study was to determine the median lethal concentration (LC50) of copper to Javenese carp, Puntius gonionotus (Bleeker), and the immune response after the fish were exposed to sublethal levels of copper and challenged with formalin killed Aeromonas hydrophila. The LC50 of copper on P. gonionotus at 24, 48, 72, 96 and 120 h were estimated as 2.17, 0.91, 0.57, 0.53 and 0.42 mg l(-1), respectively. To determine the effect of copper on the immune system, fish were exposed for 66 days to 0.05, 0.10 and 0.15 mg Cu l(-1). After 56 days of initial exposure to copper, fish were challenged with 0.1 ml of 4.5 x 10(5) cfu ml(-1) formalin killed A. hydrophila and maintained in the same concentration of copper. After the challenge, the immune response was monitored for 2 weeks using haematological and serological assays. During the initial phase of exposure to copper, significant changes were noted in the white blood cell, lysozyme, potential killing activity, total plasma protein, total immunoglobulin and haematocrit levels between the control and treated fish. One week after challenge with A. hydrophila, there was a significant increase in the values of white blood cells, total protein and total immunoglobulin compared to the values before the challenge. However, these values were not significantly different (P>0.05) between the control and the treated fish. In contrast, NBT and lysozyme assays exhibited a significant difference (P<0.05) in fish exposed to 0.10 mg Cu l(-1) (0.525 +/- 0.17; 24.42 +/- 3.35 x 10(2) micromg ml(-1)) and 0.15 mg Cu 1(-1) (0.536 +/- 0.19; 21.78 +/- 1.29 x 10(2) micromg ml(-1)) compared to the control (0.746 +/- 0.31; 30.73 +/- 5.42 x 10(2) micromg ml(-1)) after the bacterial challenge (day 61). There was however no significant difference (P>0.05) in NBT and lysozyme levels in fish exposed to lower level of copper (0.05 mg Cu l(-1)), suggesting the absence of immunosuppressive effects at lower level of exposure.
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PMID:Immunological parameters of Javanese carp Puntius gonionotus (Bleeker) exposed to copper and challenged with Aeromonas hydrophila. 1141 16

Continuous rod of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) was prepared by a free radical polymerization within the confines of a stainless-steel column. The epoxide groups of the rod were modified by a reaction with iminodiacetic acid (IDA) that affords the active site to form metal IDA chelates used for immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of bovine serum albumin on the Cu2+-IDA continuous rod column was investigated in the range from 5.0 to 9.0. Purification of lysozyme from egg white and human serum albumin (HSA) on the commercially available HSA solution were performed on the naked IDA and Cu2+-IDA continuous rod columns, respectively; and the purity of the obtained fractions was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.
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PMID:Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). 1155 31

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.
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PMID:Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study. 1182 Feb 89

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.
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PMID:Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5. 1207 47

Different adsorbents have been reported in the literature for protein purification. The authors have developed a novel and new approach to obtain high protein adsorption capacity utilizing a 2-methacrylamidoalanine-containing membrane. Amino acid ligand 2-methacrylamidoalanine (MAAL) monomer was synthesized using methacryloyl chloride and alanine. Poly(2-hydroxyethylmethacrylate-co-2-methacrylamidoalanine) [p(HEMA-co-MAAL)] membranes were then prepared by UV-initiated photopolymerization of HEMA and MAAL in the presence of an initiator (azobisisobutyronitrile, AIBN). The synthesized MAAL monomer was characterized by NMR. p(HEMA-co-MAAL) membranes were characterized by swelling studies, porosimeter, SEM, FTIR, and elemental analysis. These membranes have macropores in the size range of 5-10 microm. Cu(II) ions (25.9 mmol/m2) were chelated on these membranes. p(HEMA-co-MAAL) membranes were used to study the adsorption of lysozyme from aqueous media containing different amounts of lysozyme (0.1-3.0 mg/l) and at different pH values (4.0-8.0). The non-specific adsorption of lysozyme on the pHEMA membranes was negligible (0.9 microg/cm2). Incorporation of MAAL increased the lysozyme adsorption significantly up to 2.96 mg/cm2. The lysozyme adsorption capacity of the Cu(II) incorporated membranes (9.98 mg/cm2) was greater than that of the p(HEMA-co-MAAL) membranes. More than 90% of the adsorbed lysozyme was desorbed in 1 h in the desorption medium containing 1.0 M NaCl and 0.025 M EDTA. The metal-chelate affinity membranes are suitable for repeated use for more than ten cycles without a noticeable loss of capacity.
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PMID:Poly(hydroxyethyl methacrylate-co-methacrylamidoalanine) membranes and their utilization as metal-chelate affinity adsorbents for lysozyme adsorption. 1218 59

To obtain the SDS-PAGE-pure human lysozyme, the crude enzyme of engineered bacterium E. coli was purified by chromatography on cation ion exchange of Express-Ion S. The optimum reaction temperature and pH of this lysozyme were 45 degrees C and 6.5, respectively. The isoelectric point is pH 8.91, and Km of the enzyme for Micrococcus lysodeikticus is 0.0311 mg/mL. The thermal stability of the engineered enzyme is more sensible than hen egg white lysozyme and human milk lysozyme. The sequence of 5 amino acids in N-end is same as designed, except an Met at the first. The affects of some metal ion on this enzyme were shown. Cu2+ of 0.01 mmol/L could completely inactivate the enzyme.
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PMID:[Purification and properties of human lysozyme in engineered bacterium E. coli]. 1255 2

Immobilized metal affinity membranes (IMAMs) were prepared by immobilizing copper ions on microporous regenerated cellulose membranes through different types of chelating agents (dentate and triazine dye). The resulting chelator utilization percentages were 95% for iminodiacetic acid, 56% for N,N,N-tris(carboxymethyl)ethylenediamine, 52% for Cibacron blue 3GA, and 140% for Cibacron red 3BA. On the other hand, triazine dyes were slightly superior to dentate chelators on metal ion utilization for protein adsorption. In batch single-protein adsorptions, the protein adsorption capacity decreased with increasing molecular size and number of accessible surface histidine residues [lysozyme>bovine serum albumin(BSA)>gamma-globulin], while the binding strength order was the opposite (gamma-globulin>BSA>lysozyme). Moreover, the proportions of specific and nonspecific bindings were evaluated by varying pH and salt concentration conditions. A large fraction of the adsorption capacity was found to come from the nonspecific interactions for the prepared IMAMs. Lastly, batch three-protein adsorptions were performed and weak adsorption competition was observed.
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PMID:Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. 1283 Sep 8


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