Separation of lysozyme by adopting zymoprotein affine magnetic nano particles

A magnetic nanoparticle and enzyme protein technology, applied in the direction of enzymes, hydrolases, glycosylases, etc., can solve the problems of low separation efficiency at low temperature, enzyme denaturation and inactivation, etc., and achieve large-capacity adsorption, little impact on activity, and enhanced separation speed effect

Inactive Publication Date: 2014-07-09
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention aims to solve the shortcomings of low temperature separation efficiency and high temperature that can easily lead to enzyme denaturation and inactivation in the conventional lysozyme extraction process, and proposes to use Fe 3 o 4 Nanoparticles as magnetic core layer, amorphous SiO 2 Coating magnetic nanoparticles, modifying the surface of nanoparticles with core-shell structure, introducing protein affinity ligands, and preparing a new type of enzyme-protein affinity magnetic nanoparticles, the structural formula is shown in formula 1

Method used

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  • Separation of lysozyme by adopting zymoprotein affine magnetic nano particles
  • Separation of lysozyme by adopting zymoprotein affine magnetic nano particles
  • Separation of lysozyme by adopting zymoprotein affine magnetic nano particles

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Effect test

Embodiment 1

[0024] 5.5g FeCl 3 .6H 2 O and 2.8 g FeSO 4 .7H 2 O was dissolved in 50 mL of deionized water, at room temperature, under the protection of nitrogen, 2 mol / L sodium hydroxide solution was slowly added dropwise to it until the pH value of the solution was 11, and the reaction was stopped after stirring for 60 min. The solid particles obtained by magnetic separation are mixed with alcohol water solution and deionized water several times until the surface of the particles is neutral, and the magnetic Fe 3 o 4 Nanoparticles. 0.5g magnetic Fe 3 o 4Put the nanoparticles into a mixed solution of 150 mL of ethanol and water (volume ratio 4:1), adjust the pH value to 10 with 2 mol / L sodium hydroxide solution, and add 30 mL of ethyl silicate alcohol dropwise under nitrogen protection. Water mixed solution (containing 1.0 mL of ethyl silicate) was reacted at room temperature for 5 h. After magnetic separation, wash with alcohol-water mixed solution, ion water, and absolute ethano...

Embodiment 2

[0028] 5.5g FeCl 3 .6H 2 O and 2.8 g FeSO 4 .7H 2 O was dissolved in 50 mL of deionized water, at room temperature, under the protection of nitrogen, 2 mol / L sodium hydroxide solution was slowly added dropwise to it until the pH value of the solution was 11, and the reaction was stopped after stirring for 4 h. The solid particles obtained by magnetic separation are mixed with alcohol water solution and deionized water several times until the surface of the particles is neutral, and the magnetic Fe 3 o 4 Nanoparticles. 0.5g magnetic Fe 3 o 4 Put the nanoparticles into a mixed solution of 150 mL of ethanol and water (volume ratio 4:1), adjust the pH value to 10 with 2 mol / L sodium hydroxide solution, and add 30 mL of ethyl silicate alcohol dropwise under nitrogen protection. Mix the solution with water (containing 1.0 mL of ethyl silicate), and finish the reaction at room temperature for 5 h. After magnetic separation, wash with alcohol-water mixed solution, ion water, a...

Embodiment 3

[0032] 5.5g FeCl 3 .6H 2 O and 2.8 g FeSO 4 .7H 2 O was dissolved in 50 mL of deionized water, and under nitrogen protection, 2 mol / L sodium hydroxide solution was slowly added dropwise at 50 °C until the pH of the solution was 11, and the reaction was stopped after stirring for 6 h. The solid particles obtained by magnetic separation are mixed with alcohol water solution and deionized water several times until the surface of the particles is neutral, and the magnetic Fe 3 o 4 Nanoparticles. 0.5g magnetic Fe 3 o 4 Put the nanoparticles into a mixed solution of 150 mL of ethanol and water (volume ratio 4:1), adjust the pH value to 10 with 2 mol / L sodium hydroxide solution, and add 30 mL of ethyl silicate alcohol dropwise under nitrogen protection. Water mixed solution (containing 1.5 mL of ethyl silicate) was reacted at room temperature for 5 h. After magnetic separation, wash with alcohol-water mixed solution, ion water, and absolute ethanol three times until the surfa...

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Abstract

The invention discloses a method for separating lysozyme by adopting zymoprotein affine magnetic nano particles and in particular relates to a method for separating lysozyme from egg white by adopting a magnetic nano adsorption material. Fe3O4 nano particles are adopted as a magnetic nuclear layer, magnetic nano particles are coated by amorphous form SiO2, surface modification is carried out on nano particles in a core-shell structure, and protein affine dye ligand is introduced, so that a novel zymoprotein affine magnetic nano particle is obtained. The novel zymoprotein affine magnetic nano particles are added into egg white and are saturated after adsorption is carried out for 24-72 hours at the temperature of 20 DEG C, zymoprotein affine magnetic nano particles saturated through adsorption are added into a phosphate buffer liquid (pH is equal to 10.0), and desorption is carried out for 24 hours at the temperature of 37 DEG C; magnetic separation is carried out, so as to obtain a liquid, a desorbed liquid stays overnight when being placed in a refrigerator at the temperature of 4 DEG C, and centrifugal separation is carried out, so that solid enzyme powder is obtained. The separation method has the advantages that extraction and separation of lysozyme in the egg white are simple, conditions are mild, influence on activity of lysozyme is small and recovery rate is high; the zymoprotein affine magnetic nano particles saturated through adsorption can be directly applied to the next adsorption separation process after being eluted for separating lysozyme, and lysozyme separation efficiency is hardly influenced after the zymoprotein affine magnetic nano particles are recycled for multiple times.

Description

technical field [0001] The invention relates to a method for separating lysozyme, in particular to a method for separating lysozyme from egg white by using a magnetic nanometer adsorption material, and belongs to the technical field of fine chemicals. Background technique [0002] Lysozyme is a hydrolase that specifically acts on the cell wall of microorganisms, also known as muramidase or globulin G, N-acetyl murein hydrolase. The earliest report on lysozyme was that Nicohe first published the existence of the lytic factor of Bacillus citrus in 1907; in 1922, Flemmning found that there were strong bacteriolytic active ingredients in human nasal mucus, saliva and tears, and named the factor with lytic activity as Lysozyme (lysozyme): In 1967, the British Phillips Group first published the x-ray diffraction of the substrate complex of egg white lysozyme, and its research results have a pivotal position in the history of modern enzyme chemistry. [0003] Lysozyme widely exist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36B01J20/22B01J20/28B01J20/30
CPCC12N9/2462C12Y302/01017
Inventor 李美凤
Owner QINGDAO UNIV
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