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New method for separating out and purifying high-purity lysozyme from egg white

A high-efficiency, chromatographic packing technology, applied in the field of high-performance liquid chromatography and active protein preparation, can solve the problems of protein irreversible adsorption and strong hydrophobic interaction, etc., and achieve simple and mature preparation process, easy control and amplification, good selectivity and The effect of load

Inactive Publication Date: 2015-12-16
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The composition of its stationary phase is to modify some hydrophobic groups on the surface of the matrix. However, the traditional HIC mode has the defect that the hydrophobic interaction is too strong and it is easy to cause irreversible protein adsorption.

Method used

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  • New method for separating out and purifying high-purity lysozyme from egg white
  • New method for separating out and purifying high-purity lysozyme from egg white
  • New method for separating out and purifying high-purity lysozyme from egg white

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Effect test

Embodiment 1

[0031] (1) Preparation of high-efficiency hydrophobic interaction chromatography packings regulated by electrostatic groups

[0032]Use high-purity spherical silica gel as a blank matrix, firstly carry out acid treatment, reflux the silica gel with 1:1 hydrochloric acid at 120 degrees for 3-4 hours, cool, then wash repeatedly with distilled water until the solution is neutral, and dry it in vacuum overnight to make the silica gel surface The silanol is maintained at an appropriate level. Then activate the acid-treated silica gel with γ-glycidoxypropyltrimethoxysilane (TM-560), that is, slowly add an appropriate amount of TM-560 dropwise in a 50mmol / L sodium acetate solution at 90°C, React with silica gel for 4h, then wash with water, methanol and water successively, and dry in vacuum. The reaction between tryptophan and epoxidized silica gel is carried out in a dimethyl sulfoxide solution dried with sodium wire. After adding the reactant, it is ultrasonicated for 10 minutes, ...

Embodiment 2

[0036] (1) Preparation of high-efficiency hydrophobic interaction chromatography packings regulated by electrostatic groups

[0037] Use high-purity spherical silica gel as a blank matrix, firstly carry out acid treatment, reflux the silica gel with 1:1 hydrochloric acid at 120°C for 3-4 hours, cool, then wash repeatedly with distilled water until the solution is neutral, and dry it in vacuum overnight to make the silica gel surface The silanol is maintained at an appropriate level. Then activate the acid-treated silica gel with γ-glycidoxypropyltrimethoxysilane (TM-560), that is, slowly add an appropriate amount of TM-560 dropwise in a 50mmol / L sodium acetate solution at 90°C, React with silica gel for 4h, then wash with water, methanol and water successively, and dry in vacuum. The reaction between tryptophan and epoxidized silica gel is carried out in a dimethyl sulfoxide solution dried with sodium wire. After adding the reactant, it is ultrasonicated for 10 minutes, react...

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Abstract

The invention discloses a new method for separating out and purifying high-purity lysozyme from egg white. The method includes the steps that firstly, the surface of a silica gel matrix is decorated with tryptophan molecules serving as ligand of filler, and novel high performance hydrophobic interaction chromatography (HPHIC) filler regulated and controlled by static groups is synthesized; secondly, a chromatographic column which the HPHIC filler is filled with has the reserved characteristics of mixed mode chromatography, and has high selectivity and load capacity for natural protein separation; thirdly, the filler is applied to separation of an egg white sample, and high-purity and high-activity protein lysozyme with the purity of 99% and the quality recycling rate of 97.8% can be obtained. A salt-water elution system is adopted for the separation and purification process, and the new method is safe, environmentally friendly and beneficial for preparing lysozyme on a widened scale.

Description

field of invention [0001] The invention relates to a preparation of high-efficiency hydrophobic interaction chromatographic packing with tryptophan as ligand and electrostatic group regulation and its application to the new method of separating and purifying lysozyme from egg white, which belongs to active protein preparation technology and high-performance liquid chromatography Cross-cutting fields of technology. Background technique [0002] Egg white is an important natural protein source sample, rich in functional and active proteins. The four main components in egg albumin are 54% ovalbumin (OVA), 13% ovotransferrin (ovo), 3.5% lysozyme (Lys) and 3.5% ovomucin (ovomucin). , ovm) [Li-Chan E., Nakai S. Critical reviews in poultry biology, 1989, 2(1): 21-58]. Among them, Lys is a bactericide, antiviral agent, and analgesic. In order to obtain lysozyme with high purity and quality recovery from egg white, the main preparation methods are traditional crystallization, preci...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30B01D15/32B01D15/36C12N9/36
Inventor 王骊丽杨毅聪高栋谭福春李维敏
Owner NORTHWEST UNIV
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