Lysozyme preparation

Lysozyme, also known as muramidase (muramidase) or N-acetyl murein polysaccharide hydrolase (N-acetylmuramide glycanohydrlase), is a kind of bacteria in the hydrolysis of the enzyme alkaline mucopolysaccharides. Primarily through the destruction of the cell wall of N-acetyl muramic acid and N-acetyl glucosamine between the β-1, 4 glycosidic bond, so that the insoluble cell wall mucopolysaccharides into soluble glycopeptides contents escape rupture cell walls leaving bacteria dissolved. Lysozyme can be negatively charged with direct binding of viral proteins, and DNA, RNA, apoprotein form complex salts, the virus inactivation. Thus, the enzyme has antibacterial, anti-inflammatory, anti-viral effects.
Lysozyme is a clone of biological engineering technology, extraction and preparation, it is a natural enzyme, safety green additive, no resistance.
The enzyme exists in a variety of human tissues, birds and poultry egg white, mammal tears, saliva, plasma, urine, milk and other body fluids and micro-organisms also contain this enzyme, of which the most abundant egg white. Extracted from the isolated egg lysozyme is 18 kinds of amino acid residues 129 a single polypeptide chain. It is rich in basic amino acids, 4 bonds maintain the enzyme conformation, is a basic protein, the N-terminal lysine, C terminal leucine. Decomposable Micrococcus lysodeikticus, Bacillus megaterium, yellow Sarcina other Gram-positive bacteria.
Lysozyme less than the isoelectric point wide pH range, with a positive charge molecules can be adsorbed on the weakly acidic cation exchange resin, eluted salting, lysozyme can be obtained precipitate was then purified product can be obtained. Adsorption resin egg wash ↓ 724 ↓ ↓ eluted with a buffer solution of ammonium sulfate salting-dialysis ↓ NaOH to basic protein precipitate freeze dried powder was dried lysozyme Lysozyme 5 ~ 10 ℃ under 540kg fresh egg white added to the processed Good 80kg 724 resin and stirred adsorbed 6h, allowed to stand at 0 ~ 5 ℃ overnight. Decanted upper egg white, centrifugal drying resin, washed repeatedly with distilled water adhesion egg protein, and the resin into the column with a 0? 15mol / L, pH = 6? 5 phosphate buffer solution, about 150L resin was washed, then Approximately 10% ammonium sulfate solution 600L eluate. Eluate was added ammonium sulfate to give a final volume of 40% with ammonium sulfate, a white precipitate formed, cool place overnight. Siphon supernatant drained precipitate filtered off with suction, dissolved in distilled water and then with 1 X precipitate was thin paste, and then into the dialysis bags, about 5 ℃ conditions, dialyzed against distilled water for about 24h, the intermediate changing the water 2-3 times . Precipitate was removed by centrifugation, and then a small amount of precipitate was washed once by centrifugation was combined with the washings. Beyond dialysis supernatant was slowly added 1mol / L sodium hydroxide solution, while constantly stirring, the pH increased to 8? 0 ~ 9? 0, if the white precipitate is removed by centrifugation. Then 3mol / L hydrochloric acid to adjust pH = 5? 0, freeze-drying, white sheet of lysozyme. Can also be used centrifugate 3mol / L hydrochloric acid pH = 3? 5, was added slowly with stirring a 5% solid sodium chloride, at a temperature of about 5 ℃ stand 48h, centrifuged, washed with acetone precipitate 0 ℃, drying, lysozyme.


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From: Biochemical Reagent