Sulfonated sodium alginate grafted agarose gel chromatographic medium as well as preparation method and application

A technology of agarose gel and sodium alginate is applied in the field of sulfonated alginate grafted agarose gel chromatography medium and preparation, and in the field of protein chromatography separation, and can solve the problems of time-consuming, low charge density and salt tolerance of biological products. low problems, to achieve the effect of easy regeneration, high adsorption capacity and low cost

Inactive Publication Date: 2018-07-06
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, sodium alginate grafted agarose gel media can only improve the adsorption capacity and mass transfer rate of proteins in the low salt concentration range due to the low charge density on the grafted polymer.
In addition, due to the low salt tolerance of some traditional media, samples containing high salt need to be diluted before injection, which is very time-consuming and expensive in the separation process of biological products (Biotechnology Journal, 2015, 10(12): 1929-1934)

Method used

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  • Sulfonated sodium alginate grafted agarose gel chromatographic medium as well as preparation method and application
  • Sulfonated sodium alginate grafted agarose gel chromatographic medium as well as preparation method and application
  • Sulfonated sodium alginate grafted agarose gel chromatographic medium as well as preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0031]1) Add 5.0mL of NaOH (1.0mol / L), 3.0mL of DMSO and 3.0mL of allyl bromide to 5g of sodium alginate-grafted agarose gel medium, place it in a water bath shaker at 30°C, 120rpm reaction 24h. The reaction product is sequentially washed with 0.1mol / L NaOH, 25% v / v ethanol, 0.5mol / L NaCl and a large amount of deionized water until the cleaning solution is detected by potassium permanganate solution and does not change color;

[0032] 2) Suspend 5 g of the medium obtained from the reaction in step 1) in 5 mL of sodium metabisulfite aqueous solution (200 mg / mL), titrate to pH 6.3 with 1 mol / L NaOH to produce sodium sulfonate, and place in a water bath shaker at 20 ° C , 200rpm reaction 12h. The reaction product was washed repeatedly with deionized water to obtain a sulfonated sodium alginate grafted agarose gel chromatographic medium with an ion exchange capacity of 276 mmol / L as shown in the structural formula.

Embodiment 2

[0034] 1) Add 2.5mL of NaOH (0.6mol / L), 1.5mL of DMSO and 1.5mL of allyl bromide to 5g of sodium alginate grafted agarose gel medium, place in a water bath shaker at 20°C, 200rpm reaction for 12h. The reaction product is sequentially washed with 0.1mol / L NaOH, 25% v / v ethanol, 0.5mol / L NaCl and a large amount of deionized water until the cleaning solution is detected by potassium permanganate solution and does not change color;

[0035] 2) Suspend 5 g of the medium obtained from the reaction in step 1) in 50 mL of sodium metabisulfite aqueous solution (20 mg / mL), titrate with 1 mol / L NaOH to pH 6.0 to produce sodium sulfonate, place in a water bath shaker at 25 ° C, 150rpm reaction for 18h. The reaction product was washed repeatedly with deionized water to obtain a sulfonated sodium alginate grafted agarose gel chromatographic medium with an ion exchange capacity of 256 mmol / L as shown in the structural formula.

Embodiment 3

[0037] 1) Add 1.5mL of NaOH (4.0mol / L), 0.9mL of DMSO and 0.9mL of allyl bromide to 3g of sodium alginate grafted agarose gel medium, place in a water bath shaker at 25°C, 120rpm reaction 48h. The reaction product is sequentially washed with 0.1mol / L NaOH, 25% v / v ethanol, 0.5mol / L NaCl and a large amount of deionized water until the cleaning solution is detected by potassium permanganate solution and does not change color;

[0038] 2) Suspend 3 g of the medium obtained from the reaction in step 1) in 6 mL of sodium metabisulfite aqueous solution (100 mg / mL), titrate with 1 mol / L NaOH to pH 6.5 to produce sodium sulfonate, place in a water bath shaker at 30 ° C, 120rpm reaction 48h. The reaction product was washed repeatedly with deionized water to obtain a sulfonated sodium alginate grafted agarose gel chromatographic medium with an ion exchange capacity of 378 mmol / L as shown in the structural formula.

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Abstract

The invention relates to a sulfonated sodium alginate grafted agarose gel chromatographic medium as well as a preparation method and application. The preparation method comprises the following steps:firstly, selecting the sodium alginate grafted agarose gel chromatographic medium with a suitable ion exchange capacity as a basic medium for carrying out sulfonic group modification; secondly, controlling the concentration of sodium hydroxide in an allyl bromide activation process; finally, determining the concentration of sodium pyrosulfite and determining the pH (Potential of Hydrogen) value ofthe solution and sulfonation time. According to the sulfonated sodium alginate grafted agarose gel chromatographic medium provided by the invention, the static saturated adsorption capability on lysozyme can reach 358mg / mL and the lowest dynamic binding capacity under a 10 percent penetration condition is also 100mg / mL or more. The medium has the advantages of simple preparation method and low cost, and has a wide application prospect in efficient separation and purification of protein.

Description

technical field [0001] The invention relates to a sulfonated sodium alginate grafted agarose gel chromatographic medium, a preparation method and an application, and belongs to the protein chromatographic separation technology in the field of biotechnology. Background technique [0002] As an efficient separation technique, ion exchange chromatography is widely used in the separation and purification of proteins. In recent years, grafted ion-exchange chromatography can improve the adsorption capacity and mass transfer rate of proteins by grafting polymers on the base medium, which has become a research hotspot. [0003] Sodium alginate is a kind of natural linear polysaccharide extracted from brown algae, which is composed of β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit) through 1,4-glycoside It is a random block copolymer composed of different GG, GM or MM segments in a certain proportion (Journal of Controlled Release, 2006, 114(1): 1-14). At present, sodi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/291B01J20/30C07K1/22
CPCB01J20/291C07K1/22
Inventor 孙彦李宪秀余林玲
Owner TIANJIN UNIV
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