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)

A poly(vinyl alcohol) (PVA) coated particulate perfluoropolymer (FEP) support has been functionalised with ion-exchange groups for use in ion-exchange chromatography of proteins. Anion-exchange (DEAE and Q) and cation-exchange (SP) groups were introduced to PVA-FEP which had previously been activated using cyanuric chloride. The equilibrium adsorption capacities of SP-PVA-FEP were 31.8 and 25.2 mg ml-1 for lysozyme and IgG respectively while for DEAE-PVA-FEP, the equilibrium adsorption capacities were 14.9 and 9.7 mg ml-1 for beta-lactoglobulin and HSA respectively. The equilibrium adsorption capacities for Q-PVA-FEP were determined to be 17.2 and 13.5 mg ml-1 for beta-lactoglobulin and HSA respectively. Experiments carried out to investigate the resolving power of materials showed that both SP and Q-PVA-FEP were able to resolve proteins with only small differences in their isoelectric points and that this resolution could be maintained at a flow-rate of 1500 cm h-1. SP-PVA-FEP was used to purify lysozyme from egg whites where a 50-fold purification, to homogeneity, was achieved in 98% yield. The anion exchanger, Q-PVA-FEP could be used to purify G6PDH from a clarified homogenate of bakers' yeast 14.3-fold in 81% yield.
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PMID:Preparation and use of ion-exchange chromatographic supports based on perfluoropolymers. 922 92

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K(m) = 0.70 microM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.
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PMID:Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli. 968 61

The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose. Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.
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PMID:Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose. 975 52

Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221-236 of human cytoskeletal alpha-actinin. An immunoaffinity purified rabbit polyclonal anti-chicken alpha-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of alpha-actinin produced by thermolysin digestion of chicken gizzard alpha-actinin, but not intact alpha-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrate-resistant acid phosphatase staining. We conclude that an extracellular alpha-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.
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PMID:A fragment of alpha-actinin promotes monocyte/macrophage maturation in vitro. 1002 73

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.
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PMID:Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes. 1086 95

Acetobacter strains able to produce a thick pellicle at 37 degrees C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37 degrees C or 40 degrees C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30 degrees C than at 37 degrees C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.
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PMID:Purification and characterization of a novel polysaccharide involved in the pellicle produced by a thermotolerant Acetobacter strain. 1203 50

Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which otherwise bound strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.
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PMID:Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption. 1215 16

Pseudomonas aeruginosa PAO1 was used in this study. Isolation of outer membrane was accomplished by treating the cell envelope with EDTA and lysozyme, followed by centrifugation. The outer membrane (10 mg of protein) was mixed with 34 mmol/L octyl beta-glucoside-5 mmol/L EDTA-10 mmol/L Tris-HCl (pH 8.0) and subjected to supersonic oscillation for 2 min. The centrifuged supernatant (100 kgf for 30 min at 20 degrees C) was applied onto a DEAE ion-exchange high performance liquid chromatographic column (TSK gel-DEAE-5PW column, 0.75 cm x 7.5 cm i.d.) that was equilibrated with a solution of 10 mmol/L Tris-HCl buffer (pH 8.0) containing 2.5 mmol/L beta-C12E8 and 1 mmol/L EDTA. The column was washed with the same solution and eluted with a linear gradient of 0-0.5 mol/L NaCl in the same solution and fractions A, B, C were collected. Proteins in these fractions were analyzed by SDS-polyacrylamide gel electrophoresis and quantified by the method of Lowry et al. Protein E(Mr 43,000), G(Mr 25,000) and H (Mr 19,000) flowed through the column without adsorption in fraction A. Protein C(Mr 70,000), D(Mr 46,000) and a small amount of F (Mr 34,000) were eluted in fraction B. Fraction A was concentrated with ultrafiltration and applied again onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L beta-C12E8 and 1 mmol/L MgCl2. Fraction B was subjected to DEAE ion-exchange HPLC column in the presence of EDTA. This fraction was then applied onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L octylglucoside and 1 mmol/L EDTA. By these procedures protein C, D and E were purified to apparent homogeneity as judged by SDS-PAGE. In this work, we purified the outer membrane proteins of Pseudomonas aeruginosa, and used a new technique selectively solubilizing the cytoplasmic membrane with sodium lauryl sarcosinate for isolating the outer membrane proteins of Pseudomonas aeruginosa because of its relative simplicity.
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PMID:[Purification of outer membrane proteins in Pseudomonas aeruginosa by high performance ion-exchange liquid chromatography]. 1254 27

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Streptomyces sp. M-20 and purified by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration. No exochitinase activity was found in the culture filtrate. The molecular mass of the purified chitinase was 20 kDa, estimated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed by activity staining with Calcofluor White M2R. Chitinase was optimally active at pH of 5.0 and at 30 degrees C. The enzyme was stable from pH 4 to 8, and up to 40 degrees C. Among the metals and inhibitors that were tested, the Hg(+), Hg(2+), and p-chloromercuribenzoic acid completely inhibited the enzyme activity. The chitinase activity was high on colloidal chitin, chitotriose, and chitooligosaccharide. The purified chitinase showed antifungal activity against Botrytis cinerea, and lysozyme activity against the cell wall of Botrytis cinerea.
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PMID:Purification and characterization of chitinase from Streptomyces sp. M-20. 1268 17

1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described.
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PMID:THE CELL-BOUND ALPHA-AMYLASES OF STREPTOCOCCUS BOVIS. 1434 85


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