Preparation method of ferroferric oxide chitosan microspheres and protein immobilization method

Abstract

The application discloses a preparation method of ferroferric oxide chitosan microspheres and a protein immobilization method, wherein the preparation method of the ferroferric oxide chitosan microspheres comprises using glutaraldehyde with a specific concentration as a crosslinking agent to crosslink chitosan and ferroferric oxide, and chitosan uniformly and firmly wraps the ferroferric oxide particles, and finally forms round microspheres after incubation for a period of time; the protein immobilization method comprises microsphere modification and fusion protein modification; the microspheres take magnetic ferroferric oxide as the core, can be quickly separated from a reaction system by relying on external magnetic force of the system, and are convenient, fast, simple and efficient; the application establishes a fusion protein production method based on a tag ACP to provide mercapto, and the immobilization process is independent of the target protein, has the generality of protein immobilization, and can be convenient for co-immobilization of various related proteins (enzymes).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and in particular to a preparation method of ferroferric oxide chitosan microspheres and a protein immobilization method. BACKGROUND

[0002] The immobilization technology of protein (enzyme) refers to a kind of technology that water-soluble protein is fixed on solid material by physical or chemical method, and the immobilized protein can still maintain its biological activity. The method of protein immobilization technology has many, and in general, it is divided into two categories of physical method and chemical method, and is specifically divided into embedding method fixation, adsorption method fixation, chemical crosslinking method fixation and covalent binding method fixation.

[0003] Chitosan is also called soluble chitin, soluble chitin or deacyl chitin. Chitosan is a kind of amino polysaccharide extracted from the shell of shrimp, crab and the like, which is rich in source, has the characteristics of biocompatibility, biodegradation, non-toxicity, antibiosis, chelation of heavy metal ions, high affinity to protein and the like, and is a very good immobilization carrier. The surface of chitosan molecule is rich in amine group and hydroxyl group, and the reactivity is relatively high. The chitosan can be modified by functional modification or modification method to improve its solubility and be endowed with various functional properties. The magnetic chitosan obtained by combining chitosan with magnetic substance can improve its stability and mechanical strength, so that it is easy to separate and is widely used in various fields.

[0004] Ferroferric oxide chitosan microsphere is a new type of functional polymer material, which is superfine powder containing magnetic ferroferric oxide in the inside, so as to form a polymer microsphere with magnetic response. The microsphere can introduce various reactive functional groups on the surface of the microsphere by chemical method, and can also combine active substances such as protein, cell and specific antibody through covalent bond. The most important thing is that it can show strong magnetic response by external magnetic field, so as to be quickly separated.

[0005] How to provide a simple and efficient preparation method of ferroferric oxide chitosan microspheres with controllable size is a problem to be solved at present. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide a preparation method of ferroferric oxide chitosan microspheres. The preparation method provided by the present application is simple and efficient, the size of the microspheres can be controlled by the concentration of chitosan acetate, the preparation is convenient, and the amine group content on the surface is rich and uniform.

[0007] To achieve the above purpose, the technical scheme of the present application is as follows:

[0008] A preparation method of ferroferric oxide chitosan microspheres, comprising the following steps:

[0009] 1) Mix Fe3O4 with chitosan acetic acid solution, and treat with ultrasonic oscillation to disperse uniformly;

[0010] 2) Mix the mixture obtained in step 1) with liquid paraffin and Span-80 at room temperature to mix uniformly;

[0011] 3) Add glutaraldehyde to the mixture obtained in step 2) and mix uniformly at 35-45℃;

[0012] 4) Adjust the pH value of the mixture obtained in step 3) to 9.0-10.0, and heat to 60-80℃ to stir and react;

[0013] 5) Cool the mixture obtained in step 4) to room temperature, wash, collect with a magnet, and obtain Fe3O4 chitosan microspheres.

[0014] In the present application, the Fe3O4 chitosan microspheres, i.e. magnetic microspheres, are prepared by inverse suspension crosslinking method. 30%-60% mass concentration of glutaraldehyde, preferably 50% concentration of glutaraldehyde, is used as crosslinking agent to crosslink chitosan and Fe3O4. Chitosan uniformly and firmly wraps Fe3O4 particles, and after a period of incubation, round microspheres are finally formed. In the process, chitosan acetic acid of various concentrations can be prepared in advance, and Fe3O4 chitosan microspheres of different sizes can be prepared by using chitosan acetic acid of different concentrations.

[0015] Further, in step 1), the ultrasonic time is 15-30 min, and the ultrasonic intensity is 30-60 KW; in step 2), the stirring time is 15-20 min, and the stirring speed is 200-400 r / min; in step 3), the stirring time is 30-60 min, and the stirring speed is 200-400 r / min; in step 4), the stirring time is 100-120 min, and the stirring speed is 200-400 r / min.

[0016] Further, in step 1), the concentration of chitosan acetic acid solution is 0.01-0.03 g / ml, and the solid-liquid ratio of Fe3O4 to chitosan acetic acid solution is 1 g:100-300 ml; preferably 1 g:150-250 ml;

[0017] In step 5), the washing is specifically washing with petroleum ether, acetone, alcohol and distilled water in sequence.

[0018] Further, the solid-liquid ratio of Fe3O4 to liquid paraffin is 1 g:250-400 ml; the solid-liquid ratio of Fe3O4 to Span-80 is 1 g:10-20 ml; and the solid-liquid ratio of Fe3O4 to glutaraldehyde is 1 g:20-40 ml.

[0019] Further, in step 4), the pH value of the mixed solution is adjusted to 9.0-10.0 by using 0.8-1.5 mol / L NaOH, preferably 1 mol / L.

[0020] The second object of the present application is to provide the ferroferric oxide chitosan microspheres prepared by any of the above preparation methods.

[0021] The third object of the present application is to provide a method for fixing proteins, comprising the following steps:

[0022] (1) Modification of the fusion protein

[0023] The thiol groups on the surface of the fusion protein with ACP tag are blocked, and then free thiol groups are introduced, and the modified fusion protein is obtained after salting out;

[0024] (2) Modification of the magnetic microspheres

[0025] The ferroferric oxide chitosan microspheres obtained by the above preparation method are reacted with a crosslinking agent to obtain modified magnetic microspheres;

[0026] (3) The modified fusion protein and the modified magnetic microspheres are mixed, the pH value is adjusted to 6.5-7.5, and the reaction is fixed, then the microspheres are collected and washed.

[0027] The method for fixing proteins provided by the present application, the method for obtaining the fusion protein comprises: transforming the constructed plasmids pET28b-ACP-GFP, pET28b-ACP-mCherry and pET28b-ACP-TEV into BL21(DE3) strains, picking single colonies for seed liquid expansion, adding inducer IPTG when OD600 reaches 0.6, and expressing at 25℃ for 24h. Then centrifuge at 12000rpm to obtain the bacterial body for crushing, and finally purify to obtain ACP-GFP, ACP-mCherry and ACP-TEV; that is, the fusion protein with ACP tag: ACP-GFP, ACP-mCherry and ACP-TEV is obtained.

[0028] Further, in step (1), the thiol blocking is specifically dialyzing the purified ACP-tagged fusion protein sample into a thiol blocking buffer and adding a 3,4-dibromomaleimide solution with a final concentration of 8-12 mM, and placing it in a 20-30 °C constant temperature incubator for 3-5 h; wherein the ACP-tagged fusion protein sample is one or more of ACP-GFP, ACP-mCherry and ACP-TEV obtained by the above method; preferably, a 3,4-dibromomaleimide solution with a final concentration of 10 mM is added, and it is placed in a 25 °C constant temperature incubator for 4 h; wherein the thiol blocking buffer is a 100 mM PBS buffer with pH 8.0.

[0029] Further, in step (1), introducing free thiol groups is specifically dialyzing the sample obtained after thiol blocking into a CoA modification buffer, adding Sfp enzyme according to a ratio of 1:8-12 (preferably a ratio of 1:10) of Sfp to substrate, and the final concentration of CoA is 1-10 mM, and placing it in a 35-40 °C constant temperature incubator for 3-5 h; preferably, Sfp enzyme and CoA with a final concentration of 10 mM are added, and it is placed in a 37 °C constant temperature incubator for 4 h; wherein the CoA buffer: 10 mM MgCl2, 50 mM Hepes, pH 7.5 adjusted with HCl.

[0030] Further, in step (1), the salting-out is specifically adding saturated ammonium sulfate solution to the sample after introducing free thiol groups, so that the final concentration of ammonium sulfate reaches 70-80%, and then ice-bathing for 3-5 h, then centrifuging to remove the supernatant, and dissolving the precipitated protein with 10 mM PBS buffer; preferably, saturated ammonium sulfate solution is added so that the final concentration of ammonium sulfate reaches 75%, and then ice-bathing for 4 h, then centrifuging at a speed of 10000-15000 rpm at low temperature (0-5 °C) for 20 min to remove the supernatant, and dissolving the precipitated protein with the immobilization buffer.

[0031] Further, step (2) specifically includes:

[0032] The above obtained ferroferric oxide chitosan microspheres are swelled in 80-120 mM PBS buffer with pH 7-9, then the microspheres are collected from the solution by a magnet and transferred to a solution of 8-12 mM Sulfo-SMCC, the pH is adjusted to 7-8, then the reaction is completed by slow shaking, to obtain modified magnetic microspheres. Preferably, the concentration of the PBS buffer is 100 mM, and the pH is 7; the concentration of Sulfo-SMCC is 10 mM; the shaking reaction time is 3-5 h, preferably 4 h; wherein the solid-liquid ratio of ferroferric oxide chitosan microspheres to Sulfo-SMCC is 2-6 mg: 1 mL, preferably 4 mg: 1 mL.

[0033] Further, in step (3), 3,4-dibromomaleimide is added, i.e., 3,4-dibromomaleimide, the modified fusion protein and the modified microspheres are mixed, the pH is adjusted to 6.5-7.5, and slow shaking is performed for 3-5 h; after completion of the fixation, the microspheres are collected by a magnet, the surface of the microspheres is washed with PBS, and then the microspheres are soaked in 80-120 mM PBS buffer solution and stored at 0-6°C; preferably, the shaking time is 4 h, and the soaking is performed in 100 mM PBS buffer solution at 4°C.

[0034] In the present application, the ACP tag is used to mediate protein fixation on the surface of the magnetic microspheres, which mainly includes two steps: 1) modification of the ACP-X (X represents GFP, mCherry or TEV) protein, i.e., the thiol group on the surface of the fusion protein is blocked by 3,4-dibromomaleimide, and then the ACP is modified by Sfp enzyme to obtain a free thiol group; and 2) modification of the magnetic microspheres, i.e., the large number of amine groups on the surface of the magnetic microspheres are reacted with the crosslinking agent Sulfo-SMCC, and then reacted with the thiol group containing the ACP tag to mediate protein immobilization. Sulfo-SMCC is a bifunctional crosslinking agent that can specifically react with thiol and amine groups, and contains a maleimide group and an N-hydroxysuccinimide active ester at both ends of the molecule. The maleimide group can covalently react with the thiol group, and the N-hydroxysuccinimide active ester specifically binds with the amine group.

[0035] The present application has at least the following beneficial effects:

[0036] (1) The microspheres have a magnetic ferroferric oxide core, and can be quickly separated from the reaction system by relying on the external magnetic force of the system, which is convenient, fast, simple and efficient.

[0037] (2) The present application uses chitosan, a biocompatible polymer, and the size of the magnetic microspheres can be controlled by the concentration of chitosan acetate, which is convenient to prepare, and the amine group on the surface is abundant and uniformly distributed.

[0038] (3) The crosslinking agent makes full use of the amine group on the surface of chitosan, has low price, high reactivity, is uniformly distributed on the surface of chitosan, and is convenient to operate.

[0039] (4) The present application uses Sulfo-SMCC modified chitosan to specifically react with thiol, establishes a fusion protein production method based on the ACP tag providing thiol, and the immobilization process does not depend on the target protein, has universality for protein fixation, and can facilitate the co-immobilization of various related proteins (enzymes). BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1(a), (b), (c) and (d) are optical microscope images of microspheres prepared using different concentrations of chitosan acetate; wherein the scale bar is 10 μm, a is microspheres prepared using 0.010 g / ml chitosan acetate; b is microspheres prepared using 0.015 g / ml chitosan acetate; c is microspheres prepared using 0.020 g / ml chitosan acetate; d is microspheres prepared using 0.025 g / ml chitosan acetate.

[0041] Figure 2 is a comparison of the average diameters of 100 randomly selected microspheres prepared using four different concentrations of chitosan acetate.

[0042] Figure 3 is the suspended light transmittance of microspheres prepared using four different concentrations of chitosan acetate.

[0043] Figure 4 is a dynamic analysis chart of the protein immobilization process.

[0044] Figure 5 is a live observation of ACP-GFP and ACP-mCherry immobilized on microspheres under an inverted fluorescence microscope; wherein (a) is a comparison of the observation results under a fluorescence microscope and an optical microscope after ACP-GFP is immobilized on microspheres, and (b) is a comparison of the observation results under a fluorescence microscope and an optical microscope after ACP-mCherry is immobilized on microspheres.

[0045] Figure 6 is SDS-PAGE electrophoresis detection of the enzyme cleavage experiment of the target protein ACP-TEV immobilized on microspheres; wherein a1 and b1 are fluorescence observations after only ACP-GFP is immobilized; a2 and b2 are fluorescence observations after ACP-GFP and ACP-mCherry are mixed at a ratio of 2:1 and then immobilized; a3 and b3 are fluorescence observations after ACP-GFP and ACP-mCherry are mixed at a ratio of 1:1 and then immobilized; a4 and b4 are fluorescence observations after ACP-GFP and ACP-mCherry are mixed at a ratio of 1:2 and then immobilized; a5 and b5 are fluorescence observations after only ACP-mCherry is immobilized; the photographs are taken under the same field of view and exposure time.

[0046] Figure 7For inverted fluorescence microscope observation of ACP-GFP and ACP-mCherry co-immobilized proteins; wherein, the substrate is fusion protein YebF-Metch, and YebF and Metch contain a specific protease TEV recognition site; M in the figure is protein Marker; lane S1 is the fusion protein YebF-Metch without enzyme digestion; lane S2 is the result after treatment with recombinant expressed ACP-TEV in the reaction system; lane S3 is the result after treatment with ACP-TEV immobilized microspheres.

[0047] Figure 8 For the immobilization process chart. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0049] In addition, the technical solutions of various embodiments of the present application can be combined with each other, but it must be based on the fact that a person skilled in the art can realize it. When the combination of technical solutions appears to be contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the scope of protection required by the present application.

[0050] The method proposed by the present application will be specifically described below through specific embodiments:

[0051] Example 1 Preparation of ferroferric oxide magnetic microspheres using different concentrations of chitosan acetate

[0052] (1) Preparation of chitosan acetate

[0053] Prepare chitosan acetate solutions with different concentrations: 0.01 g / ml, 0.015 g / ml, 0.020 g / ml and 0.025 g / ml of chitosan acetate solution, as follows: 0.010 g / mL chitosan acetate: weigh 0.2 g of chitosan powder, use 19.8 mL of ddH2O and 200 μL of acetic acid solution; 0.015 g / mL chitosan acetate: weigh 0.3 g of chitosan powder, use 19.8 mL of ddH2O and 200 μL of acetic acid solution; 0.020 g / mL chitosan acetate: weigh 0.4 g of chitosan powder, use 19.8 mL of ddH2O and 200 μL of acetic acid solution; 0.025 g / mL chitosan acetate: weigh 0.5 g of chitosan powder, use 19.8 mL of ddH2O and 200 μL of acetic acid solution.

[0054] (2) Preparation of magnetic microspheres using inverse suspension crosslinking method

[0055] a. 0.25 g Fe3O4 was mixed with 50 mL chitosan-acetic acid solution (0.01 g / mL, 0.015 g / mL, 0.020 g / mL or 0.025 g / mL) respectively, and ultrasonic oscillation was performed for 20 min to make Fe3O4 fully dispersed in chitosan-acetic acid.

[0056] b. Slowly added to 80 mL liquid paraffin and 4 mL Span-80, and stirred at room temperature for 20 min.

[0057] c. 8 mL glutaraldehyde was added to the solution, and heated to 40°C for 1 h.

[0058] d. 1 mol / L NaOH was added to the solution to adjust the pH value to 9.0-10.0, and then reacted in a 70°C water bath for 120 min.

[0059] e. After stopping heating, the solution temperature was allowed to drop to room temperature, and the product was collected by a magnet after being fully washed with petroleum ether, acetone, alcohol and distilled water.

[0060] f. The diameter of the microspheres and the sedimentation after suspension are shown in Figure 1 、 Figure 2 and Figure 3 .

[0061] The relationship between the concentration of chitosan-acetic acid and the particle size of the prepared microspheres is shown in Table 1 below:

[0062] Table 1: Relationship between the concentration of chitosan-acetic acid and the particle size of the prepared microspheres

[0063]

[0064] From Figure 1 、 Figure 2 and Table 1 above, it can be seen that as the concentration of chitosan-acetic acid increases, the size of the microspheres gradually increases; when the concentration of chitosan-acetic acid is 0.010 g / mL, the variance of the microsphere diameter is small, and the size is relatively uniform; as the concentration of chitosan increases, the size of the microspheres changes, and the non-uniformity of the diameter increases. Among the four concentrations of chitosan-acetic acid, the microspheres prepared at a concentration of 0.010 have the smallest volume, the largest specific surface area, and the size is relatively uniform.

[0065] From Figure 3The results show that the light transmittance of the four kinds of microspheres changed greatly within two hours after standing, then the change of light transmittance slowed down, and the microspheres in the suspension tended to be stable. The light transmittance of the microspheres prepared from 0.025 g / ml chitosan-acetic acid changed the most, from 28% at the beginning to 62% and then tended to be stable. The most stable among the four kinds of microspheres was the microspheres prepared from 0.010 g / ml chitosan-acetic acid, with light transmittance only from 8% to 21%. Because the greater the light transmittance, the clearer the solution, and the more microspheres that settle down, the microspheres prepared from 0.010 g / ml chitosan-acetic acid concentration have the best suspension, the longest suspension time, and the least settled microspheres among the four kinds of microspheres.

[0066] Example 2 Surface of magnetic microspheres using ACP tag to mediate immobilization of target protein

[0067] Binding Figure 8 As shown, comprising the following steps:

[0068] (1) Modification of fusion protein

[0069] a. Thiol blocking reaction: dialyze the purified fusion protein sample into thiol blocking buffer, and add 3,4-dibromomaleimide solution with a final concentration of 10 mM, and place in a 25°C constant temperature incubator for reaction for 4 h.

[0070] b. Introduction of thiol group: dialyze the sample modified by 3,4-dibromomaleimide into CoA modification buffer, add CoA with a final concentration of 10 mM and appropriate amount of Sfp enzyme (added according to the ratio of Sfp enzyme to substrate 1:10), and place in a 37°C constant temperature incubator for reaction for 4 h.

[0071] c. Ammonium sulfate salting-out: add saturated ammonium sulfate solution to the protein solution, so that the final concentration of ammonium sulfate reaches 75%, ice bath for 4 h, then remove the supernatant after high-speed low-temperature centrifugation for 20 min, and dissolve the precipitated protein with immobilization buffer.

[0072] (2) Surface immobilization of magnetic microspheres

[0073] a. Microsphere pretreatment: take 20 mg of ferroferric oxide chitosan microspheres, swell in 100 mM PBS buffer with pH 7.0, then collect the microspheres from the solution with a magnet, transfer to a 5 mL solution of Sulfo-SMCC with a concentration of 10 mM, adjust the pH to 7-8, then slowly oscillate for 4 h to covalently bind SMCC to the surface of the microspheres.

[0074] b.Fixation of target protein: mix the protein modified by 3,4-dibromomaleimide and Sfp in turn with the magnetic microspheres, adjust the pH to 6.5-7.5, slowly shake for 4 h, collect the microspheres by magnet after the fixation is completed, wash the surface of the microspheres with PBS, and then immerse in 100 mM PBS buffer solution at 4°C. The fixation result is shown in Figure 4 and Table 2.

[0075] Table 2: Change of protein concentration in solution during fixation

[0076]

[0077] Example 3 Co-fixation of GFP and mCherry on the surface of magnetic microspheres

[0078] GFP and mCherry were used as model proteins to explore the co-fixation method on the surface of the microspheres. The ACP-GFP and ACP-mCherry samples were modified by 3,4-dibromomaleimide and Sfp in turn according to the method determined previously, and then the modified ACP-GFP and ACP-mCherry were mixed in different proportions, with the total protein concentration controlled at about 4 mg / ml. The fixation results are shown in Table 2 and Figure 5 and Table 3. Figure 6 These results show that the proportion of co-fixed proteins can be controlled by the protein proportion in the mixed solution before fixation, and is independent of the properties of the fixed proteins.

[0079] Table 3: Co-fixation of proteins in different proportions

[0080]

[0081] Example 4 Detection of the enzyme activity of ACP-TEV fixed on the surface of magnetic microspheres

[0082] Detection of the activity of the fixed TEV enzyme: mix the fusion protein containing the recognition site of TEV enzyme with the fixed ACP-TEV, and place in a constant temperature of 25°C for 6 h. The fixed ACP-TEV is separated by magnet, and the cleavage of the fusion protein is detected by SDS-PAGE. The enzyme cleavage result is shown in Figure 7 The results show that the protein bands in lanes S2 and S3 are smaller than that in S1, indicating that the fusion protein is successfully cleaved, the ACP-TEV on the fixed microspheres is active, and the enzyme fixation is successful.

[0083] It should be noted that, in this text, the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such a process, method, article or device. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0084] The above-mentioned embodiment numbers of the application are only for description, not representing the advantages and disadvantages of the embodiments.

[0085] The above embodiments are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for immobilizing a protein, characterized by, The method comprises the following steps: (1) modification of the fusion protein The thiol groups on the surface of the fusion protein with ACP tag are blocked, free thiol groups are introduced, and the modified fusion protein is obtained after salting-out; (2) modification of the magnetic microspheres The ferroferric oxide chitosan microspheres are reacted with a crosslinking agent to obtain modified magnetic microspheres; The crosslinking agent is Sulfo-SMCC; The preparation of the ferroferric oxide chitosan microspheres comprises: 1) Fe3O4 is mixed with a chitosan acetic acid solution with a chitosan concentration of 0.01 g / ml to 0.03 g / ml at a solid-liquid ratio of 1 g:100 to 300 ml, and ultrasonic oscillation treatment is performed for 15 to 30 min to uniformly disperse; 2) the mixture obtained in step 1) is mixed with liquid paraffin and Span-80 at room temperature for 15 to 20 min to uniformly mix; 3) glutaraldehyde is added to the mixture obtained in step 2), and stirring is uniformly performed at 35 to 45 ℃ for 30 to 60 min; 4) the pH value of the mixture obtained in step 3) is adjusted to 9.0 to 10.0, and the temperature is raised to 60 to 80 ℃ for stirring reaction for 100 to 120 min; 5) the mixture obtained in step 4) is cooled to room temperature, and after washing, the ferroferric oxide chitosan microspheres are collected by a magnet, and the washing is specifically performed by sequentially using petroleum ether, acetone, alcohol and distilled water to obtain the ferroferric oxide chitosan microspheres; (3) the modified fusion protein is mixed with the modified magnetic microspheres, the pH value is adjusted to 6.5 to 7.5, reaction and fixation are performed, after complete fixation, the microspheres are collected, and washing is performed.

2. The method of immobilizing a protein according to claim 1, wherein In step (1), the thiol group blocking is specifically that the purified fusion protein sample with ACP tag is dialyzed into a thiol group blocking buffer, and a 3,4-dibromomaleimide solution with a final concentration of 8 to 12 mM is added, and the sample is placed in a 20 to 30 ℃ constant temperature incubator for reaction for 3 to 5 h.

3. The method of immobilizing a protein according to claim 1, wherein In step (1), the introduction of free thiol groups is specifically that the sample after thiol group blocking is dialyzed into a CoA modification buffer, Sfp enzyme and CoA with a final concentration of 1 to 10 mM are added, and the sample is placed in a 35 to 40 ℃ constant temperature incubator for reaction for 3 to 5 h.

4. The method of immobilizing a protein according to claim 1, wherein In step (1), the salting-out is specifically that saturated ammonium sulfate solution is added to the sample after introduction of free thiol groups, the final concentration of ammonium sulfate is adjusted to 70 to 80 %, the sample is placed in an ice bath for 3 to 5 h, and then centrifugation is performed to remove the supernatant, and the precipitated protein is dissolved in a fixation buffer.

5. The method of immobilizing a protein according to claim 1, wherein Step (2) specifically comprises the following steps: the ferroferric oxide chitosan microspheres are placed in a PBS buffer with a concentration of 80 to 120 mM and a pH of 7 to 9 to swell, then the microspheres are collected from the solution by a magnet, transferred to a Sulfo-SMCC solution with a concentration of 8 to 12 mM, the pH is adjusted to 7 to 8, then slow oscillation is performed until the reaction is complete, and the modified magnetic microspheres are obtained.

6. The method of immobilizing a protein according to claim 1, wherein In step (3), the reaction time is 3 to 4 h, the washing agent is a PBS buffer, and after washing is completed, the obtained product is soaked in a PBS buffer solution with a concentration of 80 to 120 mM and stored at 0 to 6 ℃.

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