CN102068965B - Method for preparing chitosan separation medium suitable for protein purification - Google Patents

Abstract

The invention relates to a method for preparing a chitosan separation medium suitable for protein purification, which comprises the following steps of: (1) dispersing acetic acid solution of chitosan into liquid paraffin with stirring to form chitosan particles in the presence of cyclohexane serving as a porogen and a small amount of span80, and further cross-linking the chitosan particles to form a chitosan skeleton under the action of glutaraldehyde serving as a cross-linking agent; (2) swelling the chitosan skeleton, and reacting a hydroxyl group on the chitosan skeleton with epoxy chloropropane in dimethyl sulfoxide (DMSO)/NaOH mixed solution to introduce an epoxy group into the chitosan skeleton so as to obtain a grafted chitosan skeleton; and (3) adding iminodiacetic acid (IDA)/NaOH mixed solution into the grafted chitosan skeleton, reacting at the temperature of between 20 and 80 DEG C for 1 to 10 hours, and performing suction-filtration to obtain the chitosan separation medium. The chitosan separation medium solves the leakage problem of metal ions to a large extent, has the advantages of process stability, high repeatability and the like, and is suitable for mass production.

Description

A kind of preparation method of chitosan separation medium suitable for protein purification

1. Technical field

The invention belongs to the technical field of natural polymer materials, and in particular relates to a preparation method of a chitosan separation medium suitable for protein purification.

2. Background technology

Although there are many methods for protein separation at present, metal chelation chromatography (IMAC) has the advantages of simple and convenient preparation of chelation medium, large exchange capacity, mild separation conditions, strong versatility, and easy to scale up, especially in the purification process of proteins. Among them, its mild elution conditions can better maintain the biological activity of proteins and other advantages, making its application more and more attention, and will become the most potential chromatography method in protein separation and purification.

However, most of the conventional metal chelate chromatography (IMAC) chromatography columns use soft substrates such as dextran or agarose as carriers. On the one hand, these media are mostly imported and expensive, and their preparation techniques have been adopted by some foreign biological products. On the other hand, metal ions are easy to leak during the use of these media, resulting in a decrease in the purity of protein separation. Therefore, scholars at home and abroad have been looking for IMAC carriers with low price, good hydrophilicity and strong rigidity. Chitosan is the only natural alkaline polysaccharide found so far. Its structure and performance are similar to agarose and dextran. It is rich in resources and has good biocompatibility. Its molecules contain active amine groups (-NH ₂ ) and hydroxyl groups (-OH), easy to derivatize to obtain porous materials with various adsorption properties, which can be directly or modified as carriers for various chromatography and protein separation and purification.

Chitosan in natural state is mostly in powder form and has small specific surface area. As an adsorption carrier, it is difficult to recover both the carrier and the adsorbate, thus limiting its application. Therefore, chitosan is prepared into monodisperse and narrowly distributed polymer microspheres, so that the functions of chitosan and polymer microspheres are consistent, so that it can be used in biomedicine and other fields.

In recent years, the research on porous chitosan films and microspheres has been reported in the literature. The research on chitosan has developed rapidly, but most of the research has focused on the use of chitosan to modify drug delivery systems such as liposomes, microspheres, and microcapsules. There are not many reports about it as a separation medium, and the conditions of the prepared chitosan separation medium are not stable, and the adsorption capacity is not high.

3. Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of chitosan separation medium suitable for protein purification.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

A kind of preparation method of the chitosan separation medium suitable for protein purification, comprises the steps:

(1) Preparation of chitosan skeleton: under stirring, the acetic acid solution of chitosan is dispersed in liquid paraffin, and in the presence of pore-forming agent cyclohexane and a little span 80, chitosan particles are formed; Under the action of the linking agent glutaraldehyde, the chitosan particles are further cross-linked to form a chitosan skeleton;

(2) Chitosan grafting: the chitosan skeleton is first swelled; then in the DMSO/NaOH mixture, the hydroxyl groups on the chitosan skeleton react with epichlorohydrin to introduce epoxy on the chitosan skeleton. base, obtain the chitosan skeleton after grafting;

(3) Preparation of chitosan separation medium: add the IDA/NaOH mixed solution to the grafted chitosan skeleton, react at a temperature of 20 to 80°C for 1 to 10 hours, and suction filter to obtain the chitosan separation medium ; In the IDA/NaOH mixed solution, the concentration of IDA is 0.5-3.0 mol/L, and the concentration of NaOH is 0.5-3.0 mol/L. Among them, IDA is iminodiacetic acid.

The step (1) of the present invention utilizes a reverse-phase suspension polymerization method to prepare chitosan skeletons with gel pores and bead structures. Further, the present invention specifically recommends that the step (1) be carried out as follows: dissolving chitosan in an acetic acid solution with a mass fraction of 1.0 to 3.0% to prepare a chitosan solution with a concentration of 0.01 to 0.05 g/mL; Add liquid paraffin (oil phase), cyclohexane and a little Span 80 in turn to the reaction vessel, stir for 30-60 minutes, then add the prepared chitosan solution (water phase), heat to 20-80°C and stir for 0.5-1.5 hours Then add glutaraldehyde, adjust the pH to 8-12, then raise the temperature to 30-80°C, continue the reaction for 2-6h, suction filter while hot, wash and dry to obtain the chitosan skeleton; the chitosan solution and liquid The feeding volume ratio of paraffin wax is 1: 0.5～5, preferably 1: 1～3; the molar ratio of the crosslinking agent glutaraldehyde and chitosan amino is 1: 0.1～10, preferably 1: 0.5～3 ; The volume ratio of the pore-forming agent cyclohexane to liquid paraffin is 5-50:100, preferably 10-30:100. A small amount of span 80 is enough, for example, based on 100mL of liquid paraffin, just add 5-10 drops of span 80.

Further, in step (1), it is preferred to heat to 50-60°C and stir for 0.5-1.0h; then add glutaraldehyde, adjust the pH to 9-10, then raise the temperature to 60-70°C, and continue the reaction for 2.5-4h.

The step (2) of the present invention activates the chitosan skeleton prepared in the step (1), and introduces an epoxy group on the chitosan skeleton. Further, the present invention specifically recommends that the described step (2) is carried out as follows: take the chitosan skeleton, and after fully swelling with water, wash it successively with dimethyl sulfoxide solution of 10%～100% gradient concentration; Add DMSO/NaOH solution and epichlorohydrin to the final product, shake and react at 20-80°C for 1-10 hours, wash with distilled water after the reaction, until no epoxy group is detected in the cleaning solution; the DMSO/NaOH mixed The solution is obtained by preparing DMSO and 0.1-1.0mol/L NaOH aqueous solution according to the volume ratio of 1:0.1-10.0, preferably according to the volume ratio of 1:0.5-2.0; the feeding of the epichlorohydrin and DMSO/NaOH mixed solution The volume ratio is 1-20:100, preferably 5-15:100.

Furthermore, in step (2), it is preferable to shake the reaction at 40-50° C., and the reaction time is preferably 3-5 hours.

In the above method, the epoxy group density on the chitosan skeleton can be measured by the sodium thiosulfate method.

In step (3) of the present invention, in the IDA/NaOH mixed solution, the IDA concentration is preferably 1 mol/L, and the NaOH concentration is preferably 1 mol/L. The reaction temperature in step (3) is preferably 55-65° C., and the reaction time is preferably 3-8 h.

Compared with the prior art, the present invention has the beneficial effects that: the chitosan separation medium preparation method of the present invention is simple to operate, and the particle size uniformity is obviously improved, has very strong acid and alkali resistance, and has more Amino group has a stronger chelating ability for metal ions, the density of epoxy group modification can reach 0.1mmol/g, the amount of Cu ²⁺ chelation is significantly increased, and the problem of metal ion leakage is largely solved, and the maximum adsorption of BSA The amount can reach 128mg/g, has the advantages of process stability and good reproducibility, and is suitable for large-scale production.

4. Description of drawings

Fig. 1 is the molecular structure schematic diagram of the chitosan separation medium prepared by the present invention.

Fig. 2 is the scanning electron micrograph of the chitosan separation medium prepared by the present invention.

Fig. 3 is the particle size distribution diagram of the chitosan separation medium prepared by the present invention.

5. Specific implementation

The technical scheme of the present invention will be further described below with specific examples, but protection scope of the present invention is not limited to this:

The preparation of embodiment 1 chitosan skeleton

3.0 g of chitosan (molecular weight 30000 Da, deacetylation degree ≥ 95%) was dissolved in 100 mL of acetic acid solution with a mass fraction of 2.0%, and left standing overnight at room temperature for later use. In a 500mL three-necked bottle equipped with a mechanical stirrer and a thermometer, add 100mL of liquid paraffin, 15mL of cyclohexane and 6 drops of Span 80 in sequence, after stirring for 0.5h, add the above chitosan solution, and heat the system to 55°C with a water bath. ℃, stirred for 1 h, added 3 mL of glutaraldehyde with a mass fraction of 25%; adjusted the pH value to 10 with 10% NaOH solution, then raised the temperature to 65 °C, continued the reaction for 3 h, and filtered the micro The ball is filtered out, washed repeatedly with distilled water, then washed with petroleum ether and absolute ethanol, and vacuum-dried to constant weight to obtain a chitosan skeleton, 90% of which has a particle size distribution of 125-200 μm.

Embodiment 2 chitosan grafting

Take by weighing the product that 0.5g embodiment 1 obtains, after water fully swells, wash with the DMSO aqueous solution of 20%, 50%, 70% successively; Add 27mL DMSO/NaOH solution (DMSO volume fraction is 0.4 , the concentration of NaOH aqueous solution is 0.4mol/L) and epichlorohydrin, so that the volume fraction of epichlorohydrin is 10%, the shaking reaction is 4h, and the reaction temperature is 50°C. After the reaction, rinse with a large amount of distilled water until no epoxy group is detected in the cleaning solution. Chitosan epoxy group modification density was measured by sodium thiosulfate titration method, and the epoxy group modification density reached 0.1mmol/g.

Embodiment 3 chitosan grafting

Take by weighing the product that 0.5g embodiment 1 obtains, after water fully swells, wash with the DMSO aqueous solution of 20%, 50%, 70% successively; Add 27mL DMSO/NaOH solution (DMSO volume fraction is 0.5 , the concentration of NaOH aqueous solution is 0.4mol/L) and epichlorohydrin, so that the volume fraction of epichlorohydrin is 6%, the shaking reaction is 3h, and the reaction temperature is 40°C. After the reaction, rinse with a large amount of distilled water until no epoxy group is detected in the cleaning solution. Chitosan epoxy-modified density was measured by sodium thiosulfate titration method, and the epoxy-modified density reached 0.089mmol/g.

The preparation of embodiment 4 chitosan separation medium

Add 50mL IDA/NaOH mixed solution (the concentrations of IDA and NaOH are 1.0mol/L respectively) to the grafted microspheres prepared in Example 2, react at 60°C for 3h, and filter the obtained product with suction.

Determination of Cu ²⁺ chelation amount: Accurately weigh a certain amount of prepared chitosan separation medium, add CuSO ₄ solution with the same volume and concentration (0.1mol/L) to chelate Cu ²⁺ , after vacuum filtration, The amount of Cu ²⁺ chelation can be known by measuring the amount of residual Cu ²⁺ in the filtrate. Spectrophotometric method (wavelength is 660nm) measures copper ion content in the solution, and Cu ²⁺ chelation amount reaches 162.0mg/g.

Determination of bovine serum albumin (BSA) adsorption capacity: Take 1 mL of the prepared chitosan separation medium, put it on the column with 5 mL, 10 mg/mL BSA, flow rate 20 mL/h, and use 0.05 mol/L, pH7.5 Tris -Elute with HCl buffer, monitor and collect the eluate online, measure the protein in the eluate, and obtain the BSA adsorption capacity. BSA concentration was determined by the Coomassie brilliant blue method (595nm). The maximum adsorption capacity of BSA reached 128.0mg/g.

The preparation of embodiment 5 chitosan separation medium

Add 50 mL of IDA/NaOH mixed solution (the concentrations of IDA and NaOH are 1.0 mol/L respectively) to the grafted microspheres prepared in Example 3, react at 60° C. for 3 h, and filter the obtained product with suction.

The assay method of Cu ²⁺ chelation amount and BSA adsorption amount is the same as embodiment 4, and the result shows: adopt spectrophotometry (wavelength is 660nm) to measure Cu ²⁺ chelation amount, and its chelation amount reaches 103.8mg/g; The BSA content was determined by the Mas Brilliant Blue method (595nm), and the maximum adsorption amount reached 89.2mg/g.

Claims (8)

1. a preparation method who is suitable for the shitosan separating medium of protein purification, comprise the steps:

(1) under agitation, the acetic acid solution of shitosan is scattered in atoleine, under perforating agent cyclohexane and a little span80 exist, forms chitosan particle; Then under the effect of crosslinking agent glutaraldehyde, chitosan particle further is cross-linked to form the shitosan skeleton; The volume ratio that feeds intake of described chitosan solution and atoleine is 1 ︰ 0.5 ~ 5, and the mol ratio of described crosslinking agent glutaraldehyde and amino of chitosan is 1 ︰ 0.1 ~ 10, and described perforating agent cyclohexane and the atoleine volume ratio that feeds intake is 5 ~ 50 ︰ 100;

(2) the advanced row of shitosan skeleton swelling treatment; Then in the DMSO/NaOH mixed liquor, the hydroxyl on the shitosan skeleton and epichlorohydrin reaction are introduced epoxy radicals on the shitosan skeleton, obtain the shitosan skeleton after grafting; Described DMSO/NaOH mixed liquor is obtained according to volume ratio 1 ︰ 0.1～10.0 preparation by the NaOH aqueous solution of DMSO and 0.1～1.0mol/L; The volume ratio that feeds intake of described epoxychloropropane and DMSO/NaOH mixed liquor is 1～20 ︰ 100;

(3) the IDA/NaOH mixed solution is added shitosan skeleton after grafting, react 1 ~ 10h at 20 ~ 80 ℃ of temperature, suction filtration namely gets described shitosan separating medium; In described IDA/NaOH mixed solution, IDA concentration is 0.5 ~ 3mol/L, and NaOH concentration is 0.5 ~ 3.0mol/L.

2. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 1 is characterized in that described step (1) is specific as follows: it is that in 1.0 ~ 3.0% acetum, preparation obtains the chitosan solution that concentration is 0.01 ~ 0.05g/mL that shitosan is dissolved in mass fraction; Add successively atoleine, cyclohexane and a little Span80 in reaction vessel, stir the chitosan solution that adds preparation to obtain after 30 ~ 60min, be heated to 20 ~ 80 ℃ and stir 0.5 ~ 1.5h; Then add glutaraldehyde, regulate pH to 8 ~ 12, then be warming up to 30 ~ 80 ℃, continue reaction 2 ~ 6h, suction filtration, washing, drying obtain the shitosan skeleton while hot; The volume ratio that feeds intake of described chitosan solution and atoleine is 1 ︰ 0.5 ~ 5, and the mol ratio of described crosslinking agent glutaraldehyde and amino of chitosan is 1 ︰ 0.1 ~ 10, and described perforating agent cyclohexane and the atoleine volume ratio that feeds intake is 5 ~ 50 ︰ 100.

3. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 2, is characterized in that in described step (1), and the volume ratio that feeds intake of described chitosan solution and atoleine is 1 ︰ 1 ~ 3; The mol ratio of described crosslinking agent glutaraldehyde and amino of chitosan is 1 ︰ 0.5 ~ 3; The volume ratio that feeds intake of described perforating agent cyclohexane and atoleine is 10 ~ 30 ︰ 100.

4. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 2, is characterized in that in described step (1), first is heated to 50 ~ 60 ℃ after the chitosan solution that adds preparation to obtain and stirs 0.5 ~ 1.0h; Then add glutaraldehyde, regulate pH to 9 ~ 10, then be warming up to 60 ~ 70 ℃, continue reaction 2.5 ~ 4h.

5. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 1, it is characterized in that described step (2) is specific as follows: take the shitosan skeleton, after the abundant swelling of water, the dimethyl sulphoxide solution that is 10%～100% gradient concentration with volume fraction successively cleans; Add DMSO/NaOH solution and epoxychloropropane in product after process, at 20 ~ 80 ℃ of oscillating reactions 1 ~ 10h, clean with distilled water after reaction finishes, until detect without epoxy radicals in cleaning fluid.

6. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 5, it is characterized in that in described step (2), described DMSO/NaOH mixed liquor is obtained according to volume ratio 1 ︰ 0.5～2.0 preparation by the NaOH aqueous solution of DMSO and 0.1～1.0mol/L; The volume ratio that feeds intake of described epoxychloropropane and DMSO/NaOH mixed liquor is 5～15 ︰ 100.

7. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 5, is characterized in that in described step (2), at 40 ~ 50 ℃ of oscillating reactions, reaction time 3 ~ 5h.

8. the preparation method who is suitable for the shitosan separating medium of protein purification as claimed in claim 1, is characterized in that in described step (3), reaction temperature is 55 ~ 65 ℃, and the reaction time is 3 ~ 8h.

Images