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Preparation method of agarose boric acid affinity material suitable for fish tropomyosin purification

A tropomyosin and agarose technology, applied in the field of separation, can solve the problems of unable to obtain tropomyosin, unable to obtain ideal effects and the like, and achieve the effects of shortening purification time, simplifying purification steps and broad application prospects

Active Publication Date: 2020-01-21
OCEAN UNIV OF CHINA
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the affinity material prepared by this method is only suitable for the separation of bacteria, and it cannot be used for the purification of fish tropomyosin to obtain ideal results.
The reason is that the material selects polyethyleneimine as a hyperbranched ligand in order to increase the modification rate of phenylboronic acid and improve the enrichment of bacteria, but compared with the (tri(2-aminoethyl)amine used in the method of the present invention , there will be more exposed amino groups on the surface of the material after modification, and its application in the purification of tropomyosin from complex fish samples will cause a large amount of non-specific adsorption, and higher purity tropomyosin cannot be obtained

Method used

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  • Preparation method of agarose boric acid affinity material suitable for fish tropomyosin purification
  • Preparation method of agarose boric acid affinity material suitable for fish tropomyosin purification
  • Preparation method of agarose boric acid affinity material suitable for fish tropomyosin purification

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preparation example Construction

[0040] A kind of preparation method that is adapted to the agarose boronic acid affinity material of fish tropomyosin purification, comprises the following steps (as figure 1 shown):

[0041] 1) Preparation of agarose microspheres: Heat the agarose aqueous solution with a concentration of 1.5-4wt% until it is completely dissolved as the water phase; add Span 80 to liquid paraffin at a ratio of 14-18 g / 400mL, and dissolve it at 75-85 Stir well in a constant temperature water bath at ℃ and use it as the oil phase; under continuous stirring (800-1200rpm), add the water phase to the oil phase (volume ratio oil phase:water phase=6-6.5:1), then cool to room temperature, and continue Stir (600-1000rpm, 3-7min) to make microsphere molding; Gained microspheres are used sherwood oil, isopropanol and ultrapure water respectively (the consumption ratio of sherwood oil, isopropanol and ultrapure water is (2-4 ):(2-4):(3-5)) were vacuum washed in a Buchner funnel to obtain agarose microsp...

Embodiment

[0051] Materials and Instruments

[0052] Agarose dry powder (Shanghai Macklin Biochemical Technology Co., Ltd.), epichlorohydrin (Sinopharm Chemical Reagent Co., Ltd.), tris(2-aminoethyl)amine (Shanghai Macklin Biochemical Technology Co., Ltd.), 3,5-di Fluoro-4-formylphenylboronic acid (Shanghai Yuanye Biotechnology Co., Ltd.), SDS-PAGE kit (Soleibao Company), SPE empty column (Shanghai Anpu Experimental Technology Co., Ltd.), all chemical reagents are analytical Grade, flounder, bluefin tuna, monkfish (purchased from Qingdao Liqun Supermarket), tilapia (purchased from Guangzhou farmers market)

[0053] Fourier transform near-infrared (FT-IR) system (Thermo Fisher Scientific Corporation), TEM transmission electron microscope system, constant temperature stirring water bath, protein purification instrument (Shanghai Qingpu Huxi Instrument Factory), DY12Y-electrophoresis instrument (Beijing Liuyi Instrument Factory).

[0054] Preparation of agarose boronic acid affinity mater...

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Abstract

The invention relates to the technical field of separation. The invention discloses a preparation method of an agarose boric acid affinity material suitable for fish tropomyosin purification. The novel agarose boric acid affinity material is prepared by taking 3,5-difluoro-4-formylphenylboronic acid as a functional monomer, tri (2-aminoethyl)amine as a multi-branched ligand and agarose microspheres as a matrix material, and is used for separation and purification of fish tropomyosin for the first time, wherein optimal use conditions are determined by using different fish samples. Results showthat when the agarose concentration is 3%, the pH value of the loading equilibrium liquid is 7.4 and the concentration of the HAc eluent is 100 mM, the purity of the obtained tropomyosin reaches 90% or above, and the column capacity reaches 1.85 mg / mL or above. Compared with a traditional method, the method can remarkably shorten the purification time (shortened from several days to 3-4 hours), noorganic solvent is used, and the product purity is equivalent to that of the traditional method.

Description

technical field [0001] The invention relates to the field of separation technology, in particular to a method for preparing an agarose boronic acid affinity material suitable for the purification of fish tropomyosin. Background technique [0002] Food allergy is one of the main problems of public health security at present. According to the statistics of WHO / IUIS allergen database, tropomyosin is the main allergen in many invertebrates (such as: shrimp, crab, shellfish). However, in recent years, tropomyosin in some fish (such as: tilapia, monkfish, bluefin tuna, albacore tuna, zebrafish, etc.) potential allergens, which has aroused widespread concern. Therefore, an in-depth understanding of the sensitization mechanism of tropomyosin in fish and other aquatic products, and reasonable monitoring and labeling of tropomyosin in different foods are extremely important for effectively controlling the allergic safety problems caused by tropomyosin. important. [0003] Efficient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/285B01D15/38C07K14/46C07K1/22
CPCB01D15/3804B01J20/285C07K14/461
Inventor 曹立民殷佳珞郑洪伟林洪隋建新王博成
Owner OCEAN UNIV OF CHINA
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