Method for separating and purifying egg white lysozyme

A technology for separation and purification of egg white lysozyme is applied in the field of separation and preparation of lysozyme to achieve the effects of easy large-scale industrial production, short production cycle, and overcoming low purity and yield.

Inactive Publication Date: 2017-05-24
泰州苏鹏蛋业生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method combines ion exchange method, two-phase extraction method and membrane separation method, which overcomes the defect that it is difficult to obtain higher purity and yield by a single separation and purification method

Method used

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  • Method for separating and purifying egg white lysozyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Collect fresh egg whites, add citric acid-disodium hydrogen phosphate buffer solution with pH 4.6 according to the volume ratio of egg whites and buffer solution at a ratio of 1:2, stir evenly, and let stand overnight in a cold place; centrifuge at 8000rpm to remove the precipitate, and collect the supernatant Solution A, use 0.5M sodium hydroxide solution to adjust the pH to 6; load the CM-agarose ion exchange medium into the column, pretreat it into sodium form, and equilibrate it with citric acid-disodium hydrogen phosphate buffer solution at pH 6; The volume ratio of liquid to medium was 3:1, and the unexchanged protein was washed with deionized water, and finally eluted with 1M sodium chloride solution for 2 column volumes, and the eluate was collected. Use 1M hydrochloric acid to adjust the eluent to pH 5.5, stir evenly for 1 hour, centrifuge at 8000rpm to remove the precipitate, and collect the supernatant B; add polyethylene glycol 2000 to the obtained supernatan...

Embodiment 2

[0026] Collect fresh egg whites, add citric acid-disodium hydrogen phosphate buffer solution with pH 4.6 according to the volume ratio of egg whites and buffer solution at a ratio of 1:2, stir evenly, and let stand overnight in a cold place; centrifuge at 8000rpm to remove the precipitate, and collect the supernatant Solution A, use 0.5M sodium hydroxide solution to adjust the pH to 6; load the CM-agarose ion exchange medium into the column, pretreat it into sodium form, and equilibrate it with citric acid-disodium hydrogen phosphate buffer solution at pH 6; The volume ratio of liquid to medium was 3:1, and the unexchanged protein was washed with deionized water, and finally eluted with 1M sodium chloride solution for 2 column volumes, and the eluate was collected. Use 1M hydrochloric acid to adjust the eluent to pH 5.5, stir evenly for 1 hour, centrifuge at 8000rpm to remove the precipitate, and collect the supernatant B; add polyethylene glycol 2000 to the obtained supernatan...

Embodiment 3

[0028] Collect fresh egg whites, add citric acid-disodium hydrogen phosphate buffer solution with pH 4.6 according to the volume ratio of egg whites and buffer solution at a ratio of 1:2, stir evenly, and let stand overnight in a cold place; centrifuge at 8000rpm to remove the precipitate, and collect the supernatant Solution A, use 0.5M sodium hydroxide solution to adjust the pH to 6; load the CM-agarose ion exchange medium into the column, pretreat it into sodium form, and equilibrate it with citric acid-disodium hydrogen phosphate buffer solution at pH 6; The volume ratio of liquid to medium was 3:1, and the unexchanged protein was washed with deionized water, and finally eluted with 1M sodium chloride solution for 2 column volumes, and the eluate was collected. Use 1M hydrochloric acid to adjust the eluent to pH 5.5, stir evenly for 1 hour, centrifuge at 8000rpm to remove the precipitate, and collect the supernatant B; add polyethylene glycol 2000 to the obtained supernatan...

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Abstract

The invention relates to a method for separating and purifying egg white lysozyme. The method comprises the following main steps: collecting egg white of fresh eggs, adding a buffer solution in proportion and carrying out uniform mixing under stirring; then successively carrying out standing and centrifugation to remove precipitates so as to obtain supernatant A; adjusting the pH value of the supernatant A to 6 and then loading a sample of the supernatant A into a chromatographic column packed with a CM-agarose ion exchange medium; carrying out elution by using a 1M sodium chloride solution and adjusting the pH value of eluate to 5.5; carrying out centrifugation to remove precipitates and adding polyethylene glycol 2000 into obtained supernatant B until the concentration of polyethylene glycol 2000 reaches 20 to 25%; successively carrying out stirring and centrifugation; adding a saturated sodium sulfate solution into supernatant C obtained in the previous step until the concentration of sodium sulfate reaches 15 to 20%; successively carrying out uniform mixing and centrifugation; subjecting supernatant D obtained in the previous step to ultrafiltration; and subjecting a solution obtained after ultrafiltration to vacuum freeze drying so as to obtain lysozyme. The method provided by the invention employs a combination of an ion exchange process, a two-aqueous-phase extraction process and a membrane separation process to overcome the defects of low yield and low purity of a single separation and purification method, and is suitable for large-scale production.

Description

technical field [0001] The invention relates to the technical fields of biology, medicine and food industry, in particular to a method for separating and preparing lysozyme from egg white. Background technique [0002] Lysozyme (Lysozyme) is a glycoside hydrolase, also known as murene or N-acetylmuramic glycan hydrolase, which acts on the β-glucosamine between N-acetylglucosamine and N-acetylmuramic acid. The 1,4 bond can decompose the mucopolysaccharide components in the cell wall of some bacteria, thereby hydrolyzing the bacterial cell wall, and destroying the pathogenic bacteria after phagocytosis in the cell. So it has anti-inflammatory properties, and to protect the body from infection. Lysozyme can not only decompose thick mucin, make purulent wound exudate liquefied and easy to discharge, but also remove necrotic mucosa and accelerate the repair and regeneration of mucosal tissue. The combined use of lysozyme and antibiotics has a good synergistic and synergistic ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36
Inventor 宋锡铭姚永鹏夏海峰杭小伟
Owner 泰州苏鹏蛋业生物科技有限公司
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