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

文档序号:1551191 发布日期:2020-01-21 浏览:44次 中文

阅读说明:本技术 一种适应于鱼类原肌球蛋白纯化的琼脂糖硼酸亲和材料的制备方法 (Preparation method of agarose boric acid affinity material suitable for purifying fish tropomyosin ) 是由 曹立民 殷佳珞 郑洪伟 林洪 隋建新 王博成 于 2019-07-08 设计创作,主要内容包括:本发明涉及分离技术领域,公开了一种适应于鱼类原肌球蛋白纯化的琼脂糖硼酸亲和材料的制备方法,本发明以3,5-二氟-4-甲酰基苯硼酸为功能单体,三(2-氨基乙基)胺为多支化配体,琼脂糖微球为基质材料,制备了一种新的琼脂糖硼酸亲和材料,并将其首次用于鱼类原肌球蛋白的分离纯化中,并用不同的鱼类样品确定了最佳使用条件。结果显示,当琼脂糖浓度为3%,上样平衡液pH为7.4,HAc洗脱液浓度为100mM时,所得原肌球蛋白纯度在90%以上,柱容量约为1.85mg/mL以上。与传统方法相比,此技术可以显著缩短纯化时间(从数天缩减至3-4h),且不使用有机溶剂,产物纯度与传统方法相当。(The invention relates to the technical field of separation, and discloses a preparation method of an agarose boric acid affinity material suitable for fish tropomyosin purification. As a result, it was found that, when the agarose concentration was 3%, the pH of the equilibrium loading solution was 7.4, and the concentration of the HAc eluent was 100mM, the purity of the tropomyosin obtained was 90% or more, and the column capacity was about 1.85mg/mL or more. Compared with the traditional method, the technology can remarkably shorten the purification time (from a plurality of days to 3-4h), does not use organic solvents, and has the product purity equivalent to that of the traditional method.)

1. A preparation method of an agarose boric acid affinity material suitable for purifying fish tropomyosin is characterized by comprising the following steps:

1) preparation of agarose microspheres: heating agarose aqueous solution with the concentration of 1.5-4wt% to be completely dissolved as water phase; adding span 80 into liquid paraffin according to the proportion of 14-18 g/400mL, and uniformly stirring in a constant-temperature water bath at 75-85 ℃ to obtain an oil phase; adding the water phase into the oil phase according to the volume ratio of 1:6-6.5 under the condition of continuous stirring, then cooling to room temperature, and continuously stirring to form microspheres; respectively using petroleum ether, isopropanol and ultrapure water to perform vacuum pumping washing on the obtained microspheres in a Buchner funnel to obtain agarose microspheres, and placing the agarose microspheres in an ethanol solution for storage for later use;

2) epoxidation of agarose microspheres: weighing 5g of agarose microspheres in terms of 5g of agarose microspheres, putting the agarose microspheres in a container, and adding 45-55mL of 0.8-1.0mol/L NaOH; 0.4-0.6g/L NaBH4Performing oscillation activation on 45-55mL and 35-45mL of epoxy chloropropane at 35-45 ℃ for 3-5h, then placing the activated epoxy chloropropane in a Buchner funnel, performing vacuum filtration washing by using deionized water until the activated epoxy chloropropane is neutral, and adding sodium thiosulfate and phenolphthalein into the washing liquid until the washing liquid does not develop color to prepare the epoxidized agarose microspheres;

3) amination of epoxidized agarose microspheres: taking 5g of the epoxidized agarose microspheres, fully mixing 5g of the epoxidized agarose microspheres with 1.8-2.2mL of (tri (2-aminoethyl) amine and 18-22mL of acetonitrile, and stirring at 55-65 ℃ to carry out amination reaction for 10-14h to obtain aminated agarose microspheres;

4) preparation of agarose boronic acid affinity material: washing 3g of aminated agarose microspheres obtained in the step 3) with methanol and acetonitrile respectively based on 3g of aminated agarose microspheres; and then, mixing 18-22mL of methanol with the aminated agarose microspheres, sequentially adding 290-310mg of 3, 5-difluoro-4-formylphenylboronic acid and 580-620mg of sodium cyanoborohydride after ultrasonic treatment, stirring and reacting for 2.5-3.5d at normal temperature, and washing the obtained product with anhydrous methanol, 4-6wt% of sodium bicarbonate, 4-6wt% of sodium chloride and deionized water in a suction filtration device.

2. The method of claim 1, wherein: in step 1), the concentration of the agarose aqueous solution is 3 wt%.

3. The method of claim 1 or 2, wherein: in the step 1), the rotation speed of the first continuous stirring is 800-; the rotation speed of the second continuous stirring is 600-.

4. The method of claim 1 or 2, wherein: in the step 1), the dosage ratio of the petroleum ether, the isopropanol and the ultrapure water is (2-4) to (3-5); the concentration of the ethanol solution is 15-25wt%, and the preservation temperature is 1-5 ℃.

5. The method of claim 1 or 2, wherein: in step 2), the NaBH4The concentration is 0.5g/L, the NaOH concentration is 0.9mol/L, the addition amount of the epichlorohydrin is 40mL, and the oscillation activation time is 4 h.

6. The method of claim 1, wherein: in the step 4), washing the obtained 3g of aminated agarose microspheres with methanol and acetonitrile respectively; and then, mixing 18-22mL of methanol with the aminated agarose microspheres, sequentially adding 310mg of 3, 5-difluoro-4-formylphenylboronic acid 290 and 620mg of sodium cyanoborohydride 580 after ultrasonic treatment, stirring and reacting for 2.5-3.5d at normal temperature, then adding 1.5-2.5mL of formaldehyde solution and 550mg of sodium cyanoborohydride 450, continuously stirring and reacting for 46-50h at normal temperature, and washing the obtained product with anhydrous methanol, 4-6wt% of sodium bicarbonate, 4-6wt% of sodium chloride and deionized water in a suction filtration device.

7. The method of claim 1 or 6, wherein: in the step 4), the dosage ratio of the methanol to the acetonitrile is 35-45 mL; the dosage of the anhydrous methanol, the sodium bicarbonate, the sodium chloride and the deionized water is 35-45mL, 18-22mL and 27-33 mL.

8. A method for purifying fish tropomyosin, characterized by comprising the steps of:

A) pretreatment of a fish meat sample: weighing 1.8-2.2g of minced fish meat sample, adding 6-10mL of phosphate buffer solution with pH =6.5-9, homogenizing, and standing for 1.5-2.5 h; heating the obtained mixture in boiling water bath for 13-17min, cooling to room temperature, and centrifuging to obtain supernatant as fish coarse extract;

B) preparation of agarose boronic acid affinity column: measuring 0.8-1.2mL of the agarose boric acid affinity material of any one of claims 1-7, filling the agarose boric acid affinity material into an SPE column, placing gaskets at the upper end and the lower end of the filler, and connecting the SPE column filled with the filler to a protein purification instrument;

C) purifying fish tropomyosin: firstly, pre-washing the affinity column with phosphate buffer solution with pH =6.5-9 at the flow rate of 0.4-0.6mL/min for 25-35 min; after the signal of the protein purifier is stable, passing 2.8-3.2mL of fish meat crude extract through a column for purification at the flow rate of 0.3-0.5 mL/min; then, non-specifically adsorbed impurities that were not bound to the affinity column were washed off with a phosphate buffer of pH =6.5-9 at a flow rate of 0.3-0.5 mL/min; finally, eluting with 25-150mM HAc at a flow rate of 0.3-0.5mL/min, collecting eluate, ultrasonic washing, dialyzing, lyophilizing, and storing.

9. The purification process of claim 8, wherein: in step A) and step C), the pH of the phosphate buffer solution is 7.4; and in step C), the concentration of the HAc is 100 nM.

10. The purification process according to claim 8 or 9, characterized in that: in the step A), the centrifugation conditions are as follows: 4000-5000rpm for 8-12 min.

Technical Field

The invention relates to the technical field of separation, in particular to a preparation method of an agarose boric acid affinity material suitable for purifying fish tropomyosin.

Background

The problem of food allergy is one of the major problems of public health safety at present, and according to the statistics of WHO/IUIS allergen database, tropomyosin is the main allergen in many invertebrates (such as shrimps, crabs and shellfish). However, in recent years, tropomyosin has been reported to have sensitization in some fish species (e.g., tilapia, angry, bluefin tuna, long fin tuna, zebrafish, etc.), and is considered to be a potential allergen, thus attracting much attention. Therefore, the method has the advantages of deeply degrading the sensitization mechanism of tropomyosin in fish and other aquatic products, reasonably monitoring and labeling the tropomyosin in different foods, and being extremely important for effectively controlling the allergic safety problem caused by the tropomyosin.

Efficient separation and purification are of great importance for understanding the physical, chemical and biological properties of tropomyosin, which is also a key process for establishing analytical techniques such as various immunoassays. The method for extracting and purifying tropomyosin commonly used at present mainly comprises the following steps: firstly, extracting by using an organic reagent to obtain a crude protein extract, then using the crude protein extract as a raw material, obtaining a crude tropomyosin by adopting an ammonium sulfate salting-out method or an isoelectric point precipitation method, and finally further purifying the tropomyosin by generally using a chromatography technology. The traditional method has high sample processing amount, but the whole process takes long time (usually 2-3 days), the steps are more, the purification effect is often unstable due to multi-step operation, and the use of organic reagents has the risk of protein denaturation. Therefore, the development of a method for extracting and purifying tropomyosin, which is efficient and convenient, has a very important meaning.

Boron Affinity (BA) technology is an effective means for selectively separating and enriching cis-dihydroxy compounds. Based on the reversible covalent binding effect between the boron affinity material and the cis-dihydroxy compound, the main principle is that boric acid and ortho-dihydroxy in the cis-dihydroxy biomolecule generate covalent reaction under a higher pH environment (usually the pH is more than or equal to 8.5) to generate a five-membered or six-membered cyclic compound; when the environment is switched to acidic (usually pH < 3.0), the tetrahedral boron atom is converted to a planar boron atom, and the cyclic compound formed by the reaction is dissociated to release the cis-dihydroxy biomolecule.

The selection of the material matrix is more critical in the preparation of the boron affinity material, and the material matrix is generally divided into a rigid inorganic medium and a polysaccharide soft gel. Rigid inorganic media such as silica gel, metal oxides, porous glass and the like have the advantages of ideal mechanical strength, high column efficiency and the like, and the ancient Shuqing and the like have reported that main allergens in peanuts, milk, soybeans and nuts in chocolate are purified by using an integral silica gel column and a phenylboronic acid group. However, the non-specific adsorption of the matrix is strong, the application pH range is narrow, the biocompatibility is poor, and the boron affinity material used in the report has a high loading pH value (pH is 11), and protein denaturation can be caused in the pH environment, so that the application range of the material in a biological sample is greatly limited. The soft polysaccharide glue includes agarose, cellulose, dextran, etc., and agarose is one kind of widely used separating medium owing to its physical and chemical stability, easy modification, low toxicity and biocompatibility.

The prior patent CN201811039827.8 of the applicant discloses a preparation method and application of a polyethyleneimine hyperbranched agarose base boron affinity material, wherein the preparation method comprises the following steps: 1) preparing agarose microspheres; 2) preparing epoxy agarose; 3) Performing amination modification on the epoxidized agarose; 4) preparation of phenyl boronic acid agarose. According to the agarose-based boron affinity material with high column capacity and high biocompatibility, agarose is selected as a base frame, polyethyleneimine is used as a hyperbranched ligand to be modified, and 3, 5-difluoro-4-formylphenylboronic acid is used as an affinity group. The invention overcomes a series of technical problems and successfully applies the boron affinity material to the recognition and separation of cell level. However, the affinity material prepared by the method is only suitable for separating bacteria, and the ideal effect cannot be obtained when the affinity material is used for purifying fish tropomyosin. The reason is that the material selects polyethyleneimine as hyperbranched ligand to increase the modification rate of phenylboronic acid and improve the enrichment amount of bacteria, but compared with the (tri (2-aminoethyl) amine used in the method, the surface of the modified material has more naked amino groups, and the application of the modified material in the purification of the tropomyosin of the complex fish sample can cause a large amount of nonspecific adsorption and can not obtain the tropomyosin with higher purity.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of an agarose boric acid affinity material suitable for purifying fish tropomyosin. The method adopts tri (2-aminoethyl) amine as a multi-branched ligand, 3, 5-difluoro-4-formylphenylboronic acid with strong boron affinity and low pKa value (6.5) as an affinity group, obtains the agarose boric acid affinity material with high column efficiency and good adsorption and separation performance by optimizing the conditions of agarose microsphere preparation, loading pH environment, elution pH environment and the like, and applies the agarose boric acid affinity material to the separation and purification of the tropomyosin of paralichthys olivaceus, tilapia mossambica, anglerfish and tuna bluefin. Compared with the traditional method, the purification time (from several days to 3-4h) can be obviously shortened by adopting the boron affinity material to purify the fish tropomyosin, and the purity of the product is equivalent to that of the traditional method without using an organic solvent.

The specific technical scheme of the invention is as follows: a preparation method of an agarose boric acid affinity material suitable for purifying fish tropomyosin comprises the following steps:

1) preparation of agarose microspheres: heating agarose aqueous solution with the concentration of 1.5-4wt% to be completely dissolved as water phase; adding span 80 into liquid paraffin according to the proportion of 14-18 g/400mL, and uniformly stirring in a constant-temperature water bath at 75-85 ℃ to obtain an oil phase; adding the water phase into the oil phase according to the volume ratio of 1:6-6.5 under the condition of continuous stirring, then cooling to room temperature, and continuously stirring to form microspheres; and (3) respectively using petroleum ether, isopropanol and ultrapure water to perform vacuum pumping washing on the obtained microspheres in a Buchner funnel to obtain agarose microspheres, and placing the agarose microspheres in an ethanol solution for storage for later use.

2) Epoxidation of agarose microspheres: weighing 5g of agarose microspheres in terms of 5g of agarose microspheres, putting the agarose microspheres in a container, and adding 45-55mL of 0.8-1.0 mol/LNaOH; 0.4-0.6g/LNaBH4Performing shaking activation on 45-55mL and 35-45mL of epoxy chloropropane at 35-45 ℃ for 3-5h, then placing the materials in a Buchner funnel, performing vacuum filtration and washing by using deionized water until the materials are neutral, and adding sodium thiosulfate and phenolphthalein into the washing liquid until the materials do not develop color; and obtaining the epoxy agarose microspheres.

3) Amination of epoxidized agarose microspheres: and (3) fully mixing 5 times of the epoxidized agarose microspheres with 1.8-2.2mL of (tri (2-aminoethyl) amine and 18-22mL of acetonitrile, and stirring at 55-65 ℃ to carry out amination reaction for 10-14h to obtain the aminated agarose microspheres.

4) Preparation of agarose boronic acid affinity material: washing 3g of aminated agarose microspheres obtained in the step 3) with methanol and acetonitrile respectively; then 18-22mL of methanol is mixed with the aminated agarose microspheres, 290 mg of 3, 5-difluoro-4-formylphenylboronic acid and 580 mg of sodium cyanoborohydride are sequentially added after ultrasonic treatment, the mixture is stirred and reacted for 2.5-3.5d at normal temperature, and the obtained product is washed by anhydrous methanol, 4-6wt% of sodium bicarbonate, 4-6wt% of sodium chloride and deionized water in a suction filtration device.

Preferably, in step 1), the concentration of the agarose aqueous solution is 3 wt%.

The research of the invention team finds that the concentration of agarose has obvious influence on the internal structure of the agarose microspheres. The influence of the concentration of agarose on the purification effect of tropomyosin is examined by taking the crude extract of the fish flesh of paralichthys olivaceus as a sample, and the experimental results show that when the concentration of agarose is 1.5%, 2.5%, 3.0% and 4.0%, the purity of tropomyosin in the eluate is about 95%, 93%, 92% and 86%, respectively, and the column capacities of affinity columns are about 1.56mg/ml, 1.87mg/ml, 1.92mg/ml and 1.77mg/ml, respectively. The purity of tropomyosin in the eluent is slightly reduced along with the increase of the concentration of agarose, and the analysis shows that when the concentration of agarose is lower, the prepared agarose microspheres have smaller particle size, larger specific surface area and increased modification amount of boron groups, so that the specific adsorption capacity of the affinity column on the tropomyosin is improved. When the concentration of agarose is increased, the viscosity of the agarose solution is increased, so that the dispersibility of liquid drops in an oil phase is reduced when the agarose microspheres are prepared, the particle size is increased, the uniformity is also reduced, the molecular sieve function of the agarose microspheres is possibly adversely affected, and the purification effect of the affinity column is reduced. However, in terms of column capacity, when the concentration of the agarose solution is less than 3%, a phenomenon of high column pressure and poor flow-through occurs during the experiment, which results in incomplete elution of tropomyosin bound to the column, thereby decreasing the column capacity of the affinity column. Therefore, combining both tropomyosin purity and column capacity, an agarose concentration of 3% is the optimum condition.

Preferably, in the step 1), the rotation speed of the first continuous stirring is 800-; the rotation speed of the second continuous stirring is 600-.

Preferably, in the step 1), the usage ratio of the petroleum ether, the isopropanol and the ultrapure water is (2-4) to (3-5); the concentration of the ethanol solution is 15-25wt%, and the preservation temperature is 1-5 ℃.

Preferably, in step 2), the NaBH is4The concentration is 0.5g/L, the NaOH concentration is 0.9mol/L, the addition amount of the epichlorohydrin is 40mL, and the oscillation activation time is 4 h.

The basis of the modification of the agarose boric acid affinity material is the modification of an epoxy group, and the team of the invention finds that the density of the epoxy group is closely related to the modification of the boron affinity group in researchThe increase of the density of the epoxy group is beneficial to the increase of the boron affinity group, thereby improving the column capacity and the purification capacity of the affinity column. This study investigated the concentration of NaOH, NaBH4The effect of the concentration of (A), the amount of epichlorohydrin added and the reaction time on the epoxy group density, and the experimental results show that the reaction time is not affected by NaBH4The concentration is 0.5g/L, the NaOH concentration is 0.9mol/L, the addition amount of the epichlorohydrin is 40%, and the epoxy group density on the surface of the agarose microsphere can reach 151.74 mu mol/g when the reaction time is 4 hours. The shape of the modified agarose boron affinity material is characterized by a Scanning Electron Microscope (SEM), the characterization result shows that the surface of the obtained material is smooth, no obvious crack or fragment is attached, the particle size of the material is uniform, 200 microspheres are randomly selected from SEM images for measurement, and the size distribution of the microspheres is between 40 and 160 mu m.

Preferably, in step 4), the obtained 3g of aminated agarose microspheres are washed with methanol and acetonitrile, respectively; and then, mixing 18-22mL of methanol with the aminated agarose microspheres, sequentially adding 310mg of 3, 5-difluoro-4-formylphenylboronic acid 290 and 620mg of sodium cyanoborohydride 580 after ultrasonic treatment, stirring and reacting for 2.5-3.5d at normal temperature, then adding 1.5-2.5mL of formaldehyde solution and 550mg of sodium cyanoborohydride 450, continuously stirring and reacting for 46-50h at normal temperature, and washing the obtained product with anhydrous methanol, 4-6wt% of sodium bicarbonate, 4-6wt% of sodium chloride and deionized water in a suction filtration device.

The team of the invention finds that the effect of the step 4) according to the scheme further improves the space. Therefore, after a large number of experiments, the modification of formaldehyde and sodium cyanoborohydride is added on the original basis, and the purpose is to seal the exposed amino group of the unmodified 3, 5-difluoro-4-formylphenylboronic acid, so that the nonspecific adsorption caused by residual amino groups is reduced, and the purity of the obtained tropomyosin is improved.

Preferably, in the step 4), the using ratio of the methanol to the acetonitrile is 35-45 mL; the dosage of the anhydrous methanol, the sodium bicarbonate, the sodium chloride and the deionized water is 35-45mL, 18-22mL and 27-33 mL.

A method for purifying fish tropomyosin, comprising the steps of:

A) pretreatment of a fish meat sample: weighing 1.8-2.2g of minced fish meat sample, adding 6-10mL of phosphate buffer solution with pH of 6.5-9, homogenizing, and standing for 1.5-2.5 h; heating the obtained mixture in boiling water bath for 13-17min, cooling to room temperature, and centrifuging to obtain supernatant as fish coarse extract.

B) Preparation of agarose boronic acid affinity column: measuring 0.8-1.2mL of the agarose boronic acid affinity material of any one of claims 1-5, filling the SPE column, placing gaskets on the upper and lower ends of the filler, and connecting the SPE column filled with the filler to a protein purification instrument.

C) Purifying fish tropomyosin: firstly, pre-washing the affinity column with phosphate buffer solution with pH value of 6.5-9 at the flow rate of 0.4-0.6mL/min for 25-35 min; after the signal of the protein purifier is stable, passing 2.8-3.2mL of fish meat crude extract through a column for purification at the flow rate of 0.3-0.5 mL/min; then, non-specifically adsorbed impurities which are not bound to the affinity column are washed away with a phosphate buffer solution having a pH of 6.5 to 9 at a flow rate of 0.3 to 0.5 mL/min; finally, eluting with 25-150mM HAc at a flow rate of 0.3-0.5mL/min, collecting eluate, ultrasonic washing, dialyzing, lyophilizing, and storing.

Preferably, in step a) and step C), the pH of the phosphate buffer is 7.4; and in step C), the concentration of the HAc is 100 nM.

The present team has found in research that, in most cases, the reversible covalent reaction binding force between boronic acid affinity ligands and cis-dihydroxy compounds is dependent on the pH environment of the load. In general, boronic acid affinity ligands will only exhibit their affinity when the pH of the environment is above the pKa of the boronic acid. In the experiment of the present invention, when the pH of the equilibration fluid (phosphate buffer) was in the range of 6.5 to 9.0, the tropomyosin purity in the eluate was about 81%, 82%, 89%, 66% and 54%, respectively, and the column capacities were about 1.70mg/mL, 1.76mg/mL, 1.88mg/mL, 0.57mg/mL and 0.54mg/mL, respectively. In summary, pH7.4 is the optimal condition for loading the equilibration fluid. The experimental results show that the separation and purification capacity of the agarose boronic acid affinity column increases gradually as the pH increases gradually (from 6.5 to 7.4), which can be attributed to the basic affinity properties of the boron affinity ligand. However, when the pH is higher than 8.0, the separating and purifying ability of the affinity column rather tends to decrease, which may be caused by a complex interaction among various factors such as pKa of tropomyosin, affinity of boronic acid ligand, and ionic strength. For example, increasing the pH of the loading environment may promote specific affinity for the boronic acid ligand, but on the other hand, as the pH increases, the ionic forces in the environment increase, and the presence of some negative charge makes the affinity column somewhat repulsive to tropomyosin.

The present group also found that elution conditions also play a crucial role in the application of agarose boronic acid affinity columns. When the HAc concentration ranged from 25mM to 150mM, tropomyosin was approximately 89%, 88%, 93%, 86% and 83% pure in the eluate, and the corresponding column capacities were approximately 1.01mg/mL, 1.31mg/mL, 1.94mg/mL, 1.87mg/mL and 1.91mg/mL, respectively. At HAc concentrations less than 100mM, tropomyosin does not appear to elute completely from the column, whereas at concentrations above 100mM some non-specifically adsorbed proteins are eluted as well. The optimal concentration of HAc eluate was 100mM, taking into account the purity and column capacity of tropomyosin.

Preferably, in step a), the centrifugation conditions are: 4000-5000rpm for 8-12 min.

Compared with the prior art, the invention has the beneficial effects that: the invention takes 3, 5-difluoro-4-formyl phenylboronic acid as a functional monomer, tri (2-aminoethyl) amine as a multi-branched ligand and agarose microspheres as a matrix material to prepare a novel agarose boric acid affinity column, and the affinity column is firstly used for separating and purifying fish tropomyosin, and different fish samples are used for verifying the optimal use condition of the affinity column, thus establishing a novel purification system. The results of the experiment show that when the agarose concentration is 3%, the pH of the equilibrium loading solution is 7.4, and the concentration of the HAc eluent is 100mM, the purity of the tropomyosin obtained is more than 90%, and the column capacity is more than about 1.85 mg/mL. Compared with the traditional method, the technology can obviously shorten the purification time, simplify the purification steps and have wide application prospect in the aspect of separating tropomyosin and other allergens.

Drawings

FIG. 1 is a diagram showing the preparation process of the agarose boron affinity material of the invention;

FIG. 2 is an image of epoxy modification conditions of agarose microspheres; wherein, a.NaBH4The influence of concentration on the density of epoxy groups on the surface of the microsphere, the influence of NaOH concentration on the density of the epoxy groups on the surface of the microsphere, the influence of the addition amount of epichlorohydrin on the density of the epoxy groups on the surface of the microsphere, and the influence of reaction time on the density of the epoxy groups on the surface of the microsphere;

FIG. 3 is a representation of agarose boronic acid affinity material; the method comprises the following steps of a, representing an agarose boric acid affinity material by a scanning electron microscope, b, representing the content of an element of the agarose boric acid affinity material, and c, representing the infrared spectrum of the agarose boric acid affinity material: (a) unmodified agarose microspheres, (b) aminated modified agarose material, (c) agarose boronic acid affinity material;

FIG. 4 is a graph of the effect of different agarose concentrations on tropomyosin purification; wherein, a.1.5% agarose concentration b.2.5% agarose concentration c.3% agarose concentration d.4% agarose concentration; m: protein molecular weight standards, lane 1: fish meal crude extract, lane 2: load flow-through, lanes 3-5: leacheate, lane 6: eluting the solution;

FIG. 5 is a graph showing the effect of pH on tropomyosin purification on various loading equilibrating solutions; wherein, a.pH 6.5 sample loading balance liquid b.pH 7.0 sample loading balance liquid c.pH 7.4 sample loading balance liquid d.pH 8.0 sample loading balance liquid e.pH 9.0 sample loading balance liquid; m: protein molecular weight standards, lane 1: fish meal crude extract, lane 2: dilute 2-fold fish meal crude extract, lane 3: loading effluent, lane 4-5: leacheate, lane 6: eluting the solution;

FIG. 6 is a graph showing the effect of different concentrations of HAc eluate on tropomyosin purification; wherein, a.25mM HAc eluate, b.75mM HAc eluate, c.100mM HAc eluate, d.125mM HAc eluate, e.150mM HAc eluate; m: protein molecular weight standards, lane 1: fish meal crude extract, lane 2: load flow-through, lanes 3-4: eluate, lane 5: eluting the solution;

FIG. 7 is an application of agarose boric acid affinity column to purification of tropomyosin in four fishes (Paralichthys olivaceus, tuna bluefin, tilapia, anglerfish); wherein, M: protein molecular weight standards, lane 1: crude extract of flounder meat, lane 2: flounder fish eluate, lane 3: crude extract from bluefin tuna, lane 4: bluefin tuna eluate, lane 5: crude extract of tilapia, lane 6: tilapia eluate, lane 7: crude anglerfish extract, lane 8: ankang fish eluent.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

A method for preparing agarose boric acid affinity material suitable for purifying fish tropomyosin comprises the following steps (as shown in figure 1):

1) preparation of agarose microspheres: heating agarose aqueous solution with the concentration of 1.5-4wt% to be completely dissolved as water phase; adding span 80 into liquid paraffin according to the proportion of 14-18 g/400mL, and uniformly stirring in a constant-temperature water bath at 75-85 ℃ to obtain an oil phase; adding the water phase into the oil phase (the volume ratio of the oil phase to the water phase is 6-6.5: 1) under the condition of continuous stirring (800-; and (3) respectively using petroleum ether, isopropanol and ultrapure water (the dosage ratio of the petroleum ether to the isopropanol to the ultrapure water is (2-4) to (3-5)) to carry out vacuum pumping and washing on the obtained microspheres in a Buchner funnel to obtain agarose microspheres, and storing the agarose microspheres in 15-25wt% ethanol solution at 1-5 ℃ for later use.

2) Epoxidation of agarose microspheres: based on 5g of agarose microspheres, the step 2) is specifically as follows: weighing 5g of agarose microspheres, placing the agarose microspheres in a container, and adding 45-55mL of 0.8-1.0mol/L NaOH; 0.4-0.6g/L NaBH4Performing shaking activation on 45-55mL and 35-45mL of epoxy chloropropane at 35-45 ℃ for 3-5h, then placing the materials in a Buchner funnel, performing vacuum filtration and washing by using deionized water until the materials are neutral, and adding sodium thiosulfate and phenolphthalein into the washing liquid until the materials do not develop color; and obtaining the epoxy agarose microspheres.

3) Amination of epoxidized agarose microspheres: and (3) fully mixing 5g of epoxidized agarose microspheres with 1.8-2.2mL of (tri (2-aminoethyl) amine and 18-22mL of acetonitrile, and stirring at 55-65 ℃ to carry out amination reaction for 10-14h to obtain the aminated agarose microspheres.

4) Preparation of agarose boronic acid affinity material: washing 3g of aminated agarose microspheres obtained in the step 3) with 35-45mL of methanol and 35-45mL of acetonitrile respectively; then 18-22mL of methanol is mixed with the aminated agarose microspheres, 290 mg of 3, 5-difluoro-4-formylphenylboronic acid and 580 mg of sodium cyanoborohydride are sequentially added after ultrasonic treatment, the mixture is stirred and reacted for 2.5-3.5d at normal temperature, and the obtained product is washed by 35-45mL of anhydrous methanol, 18-22mL of 4-6wt% sodium bicarbonate, 18-22mL of 4-6wt% sodium chloride and 27-33mL of deionized water in a suction filtration device.

Optionally, in step 4), washing the obtained 3g of aminated agarose microspheres with methanol and acetonitrile, respectively; and then, mixing 18-22mL of methanol with the aminated agarose microspheres, sequentially adding 310mg of 3, 5-difluoro-4-formylphenylboronic acid 290 and 620mg of sodium cyanoborohydride 580 after ultrasonic treatment, stirring and reacting for 2.5-3.5d at normal temperature, then adding 1.5-2.5mL of formaldehyde solution and 550mg of sodium cyanoborohydride 450, continuously stirring and reacting for 46-50h at normal temperature, and washing the obtained product with anhydrous methanol, 4-6wt% of sodium bicarbonate, 4-6wt% of sodium chloride and deionized water in a suction filtration device.

A method for purifying fish tropomyosin, comprising the steps of:

A) pretreatment of a fish meat sample: weighing 1.8-2.2g of minced fish meat sample, adding 6-10mL of phosphate buffer solution with pH of 6.5-9, homogenizing, and standing for 1.5-2.5 h; heating the obtained mixture in boiling water bath for 13-17min, cooling to room temperature, and centrifuging (under the conditions of 4000-.

B) Preparation of agarose boronic acid affinity column: measuring 0.8-1.2mL of the agarose boronic acid affinity material of any one of claims 1-5, filling the SPE column, placing gaskets on the upper and lower ends of the filler, and connecting the SPE column filled with the filler to a protein purification instrument.

C) Purifying fish tropomyosin: firstly, pre-washing the affinity column with phosphate buffer solution with pH value of 6.5-9 at the flow rate of 0.4-0.6mL/min for 25-35 min; after the signal of the protein purifier is stable, passing 2.8-3.2mL of fish meat crude extract through a column for purification at the flow rate of 0.3-0.5 mL/min; then, non-specifically adsorbed impurities which are not bound to the affinity column are washed away with a phosphate buffer solution having a pH of 6.5 to 9 at a flow rate of 0.3 to 0.5 mL/min; finally, eluting with 25-150mM HAc at a flow rate of 0.3-0.5mL/min, collecting eluate, ultrasonic washing, dialyzing, lyophilizing, and storing.

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