A rapid photoresponsive self-assembled reverse micelle for separating and purifying proteins and a preparation method thereof

Abstract

The application discloses a kind of quick light response self-assembly reverse micelles of separation and purification protein and preparation method thereof, belong to functional material technical field;Its preparation method includes the following steps: with furfural and malonic acid ring (sub) isopropyl ester as raw material synthesis DASA intermediate and dihexylamine / dodecylamine reaction preparation light control phase transfer nonionic surfactant DASAs, the DASAs synthesized has good phase transfer capacity and cyclic regeneration, the DASAs are dissolved in organic phase with cetyltrimethylammonium bromide CTAB self-assembly and form quick light response self-assembly reverse micelles, the quick light response self-assembly reverse micelles has good selectivity and back extraction rate to protein.The application is not only made protein keep high activity by self-assembly reverse micelle, and its quick light response makes the extracted protein be released quickly by light, greatly improve the efficiency of protein extraction, greatly promote the industrial application of liquid-liquid extraction high activity, high purity protein.

Description

Technical Field

[0001] This invention belongs to the field of functional materials technology, specifically relating to a rapid photoresponsive self-assembled reverse micelle for separating and purifying proteins and its preparation method. Background Technology

[0002] In recent years, with the development of genetic engineering technology, the development and demand for protein drugs have been increasing daily, becoming an important component of pharmaceutical products. Protein drugs are characterized by high activity, strong specificity, low toxicity, and well-defined functions. Because these biological products have extremely stringent requirements for activity and purity, new demands have been placed on separation and purification technologies. On the one hand, these technologies must be highly efficient; more importantly, they must ensure the activity of the protein.

[0003] Currently, the main method for separating and purifying proteins while ensuring their activity is reverse micelle extraction. While reverse micelle extraction can preserve protein activity during the separation and purification process, it is time-consuming. Some research groups have used reverse micelle extraction to separate proteins, but the extraction time can reach 48 to 72 hours, which greatly limits the application of reverse micelle extraction for proteins.

[0004] Therefore, there is an urgent need to design a separation and purification method that can ensure protein activity, improve protein extraction efficiency, and promote the industrial application of high-purity proteins. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of low efficiency in the existing protein separation and purification methods, and to provide a rapid photoresponsive self-assembled reverse micelle for protein separation and purification and its preparation method. This method not only maintains the high activity of the protein, but also allows the extracted protein to be released rapidly by light irradiation due to its rapid photoresponsiveness, which greatly improves the efficiency of protein extraction and greatly promotes the industrial application of liquid-liquid extraction of high-activity and high-purity proteins.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for preparing rapid photoresponsive self-assembled reverse micelles for protein separation and purification, comprising the following steps:

[0007] S1. Preparation of photoresponsive phase-transfer surfactants (DASAs)

[0008] DASA intermediates, alkylamines and tetrahydrofuran TFH were added to a flask, with the molar ratio of DASA intermediates to alkylamines being 1:1. The reaction was carried out at room temperature in a water bath, and the resulting mixture was evaporated to dryness to obtain a light purple DASA solid. The solid was ground into powder and repeatedly washed and purified with n-hexane to finally obtain the photoresponsive phase transfer surfactant DASAs.

[0009] The synthetic route for DASAs is as follows:

[0010]

[0011] S2. Preparation of fast photoresponsive self-assembled reverse micelles

[0012] Photoresponsive phase transfer surfactants (DASAs) with a concentration of 0.05 g / L to 0.5 g / L were dissolved in a mixed solution of toluene and n-propanol. Then, CTAB with a concentration of 6 g / L to 15 g / L was added for ultrasonic dissolution. Finally, an equal volume of distilled water was added and stirred in a vortex manner to form photoresponsive self-assembled reverse micelles in the upper organic phase.

[0013] In step S1, the DASAs intermediate is prepared by the following method:

[0014] Furfural and cyclo(isopropyl)malonic acid were placed in a flask with distilled water at a molar ratio of 1:1 and reacted in a water bath at 75°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, dissolved, purified, and evaporated to dryness to obtain a pale yellow solid DASA intermediate.

[0015] The synthetic route for DASA intermediates is as follows:

[0016]

[0017] In step S1, the alkylamine used is dihexylamine or dodecylamine, and the reaction time at room temperature is 30 min.

[0018] In step S2, the concentration ratio of toluene to n-propanol is 4:1 to 3, where toluene is the solvent and n-propanol is the co-solvent.

[0019] Preferably, the concentration ratio of toluene to n-propanol is 4:1, the concentration of DASAs is 0.2 g / L, and the concentration of CTAB is 7 g / L.

[0020] In step S2, the ultrasonic dissolution time is 30 minutes, and the vortex stirring time is 10 minutes.

[0021] The present invention also discloses a fast photoresponsive self-assembled reverse micelle for separating and purifying proteins obtained by the above preparation method.

[0022] The beneficial effects of this invention are: the fast photoresponsive self-assembled reverse micelles prepared by this invention can not only maintain the high activity of proteins, but also enable the extracted proteins to be released rapidly by light irradiation due to their fast photoresponsiveness, which greatly improves the efficiency of protein extraction and greatly promotes the industrial application of liquid-liquid extraction of high-activity and high-purity proteins. Attached Figure Description

[0023] Figure 1 DASA intermediates prepared for this invention 1 H NMR spectrum;

[0024] Figure 2 The DASAs prepared for this invention 1 H NMR spectrum;

[0025] Figure 3 A flowchart illustrating the phase transfer capability test of the photoresponsive phase transfer surfactants DASAs prepared in this invention;

[0026] Figure 4 Transmission electron microscope (TEM) image at 50 nm after extraction and back-extraction of proteins by the fast photoresponsive self-assembled reverse micelles prepared according to the present invention.

[0027] Figure 5 The curve of the pre-extraction rate of Hb extracted by the rapid photoresponsive self-assembled reverse micelles prepared in this invention as a function of CTAB concentration is shown.

[0028] Figure 6 The curve showing the pre-extraction rate of Hb extracted by the rapid photoresponsive self-assembled reverse micelles prepared in this invention as a function of DASA concentration is shown.

[0029] Figure 7 This is a bar chart comparing the extraction rates of Hb after 10 min under light and no light conditions using the fast photoresponsive self-assembled reverse micelles prepared in this invention.

[0030] Figure 8 This is a curve showing the extraction rate of the fast photoresponsive self-assembled reverse micelles used in this invention for extracting mixed proteins. Detailed Implementation

[0031] The present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments.

[0032] In the following embodiments, the intermediates of DASAs and the preparation methods of DASAs are as follows:

[0033] The preparation of S0-1 and DASA intermediates includes the following steps:

[0034] 1 g of furfural, 1.44 g of cyclo(isopropyl)malonate and 25 ml of distilled water were placed in a flask and reacted in a water bath at 75 °C for 2 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, dissolved in 30 ml of dichloromethane, and purified by extraction with 30 ml of saturated NaHSO3, 30 ml of distilled water, 30 ml of saturated NaHCO3, and 30 ml of saturated NaCl. The dichloromethane was removed by rotary evaporation to obtain a pale yellow solid.

[0035] like Figure 1 The image shows the prepared DASA intermediate. 1 H NMR spectrum. 1 H NMR (400MHz, CDCl3) δ8.40(d,J=3.8Hz,1H),8.28(s,1H),7.81(d,J=1.6Hz,1H),6.72-

[0036] 6.67 (m, 1H), 1.71 (s, 6H).

[0037] S0-2. The preparation of photoresponsive phase-transfer surfactants (DASAs) includes the following steps:

[0038] DASA intermediate, dihexylamine, and 40 ml of tetrahydrofuran (TFH) were added to a flask. The amount of DASA intermediate added was 2.22 g (0.01 M), and the amount of dihexylamine added was 1.9 g (0.01 M). The mixture was reacted in a water bath at room temperature for 15 min. The solvent was removed by rotary evaporation to obtain a light purple crude DASA solid. The solid was ground into powder and purified by repeated washing with n-hexane to obtain the DASA solid.

[0039] like Figure 2 The image shows the prepared DASAs. 1 H NMR spectrum

[0040] 1 H NMR (600MHz, CDCl3) δ11.41(d,J=1.4Hz,1H),7.19(d,J=12.3Hz,1H),7.07(s,1H),6.03(t,J=12.3Hz,1H),3.38(td,J=7. 8,5.3Hz,4H),2.17(s,1H),1.77(s,3H),1.67(dq,J=15.4,7.5Hz,12H),1.32(dd,J=6.3,3.1Hz,14H),0.93-0.88(m,7H).

[0041] Test Example 1: Phase transfer capability test of photoresponsive phase transfer surfactants (DASAs), including the following steps:

[0042] 1. Place the toluene solution containing DASAs and an equal volume of distilled water into three transparent glass bottles a, b, and c. Stir under light for 10 minutes, and then let the mixture stand under light for 30 minutes to separate into layers.

[0043] 2. Take the upper toluene phase from glass bottle a and place it in a transparent glass bottle in the dark for 15 minutes, then measure the ultraviolet absorbance at 545 nm.

[0044] 3. Protect glass bottles b and c from light for 15 minutes and measure the ultraviolet absorption at 545 nm of the upper toluene phase. Then, take the lower aqueous phase of glass bottle b and add an equal volume of dichloromethane to a transparent glass bottle. Stir at 65°C for 10 minutes and measure the ultraviolet absorbance of the lower dichloromethane phase.

[0045] 4. Stir the remaining equal volumes of aqueous phase and toluene phase in glass bottle b at 65°C in the dark for 10 minutes, let stand, and measure the ultraviolet absorbance of the upper toluene phase.

[0046] 5. Add an equal volume of dichloromethane to the remaining aqueous phase in glass bottle b and to a transparent glass bottle. Stir at room temperature for 10 minutes, let stand, and measure the UV absorbance of the dichloromethane phase.

[0047] 6. Take the aqueous phase from glass bottle c into another glass bottle, add an equal volume of dichloromethane, stir at room temperature for 10 minutes, and measure the UV absorbance of the dichloromethane phase.

[0048] 7. Substitute the measured UV absorbance of the toluene phase into the standard curve to calculate the toluene phase concentration. Substitute the calculated toluene phase concentration into the dichloromethane standard curve of DASAs to calculate the UV absorbance in the dichloromethane phase. Finally, convert the UV absorbance in dichloromethane to calculate the phase transfer rate.

[0049] from Figure 3 The phase transfer flow chart shows that light irradiation can induce DASAs to enter the aqueous phase, with a transfer rate of 77% from the toluene phase. Light avoidance promotes the transfer of DASAs from the aqueous phase to the toluene phase, with a transfer rate of 65.2%. Heating also promotes the transfer of DASAs from the aqueous phase to the toluene phase; under heating and light-avoidance conditions, the transfer rate from the aqueous phase to the toluene phase is 75.6%. At room temperature, the transfer rate from the aqueous phase to the dichloromethane phase is 88.9%. Under heating and stirring conditions at 65°C, DASAs entering the dichloromethane phase from the aqueous phase begin to decompose, leading to a decrease in recovery rate.

[0050] Example 1: A method for separating and purifying proteins using rapid light-responsive self-assembled reverse micelles for extracting Hb from aqueous solutions, comprising the following steps:

[0051] S1. The preparation of photoresponsive phase-transfer surfactants (DASAs) is as described above.

[0052] S2, Pre-extraction:

[0053] Dissolve 0.02 g of DASAs in 80 ml of a mixed solution of toluene and n-propanol (toluene: n-propanol = 4:1), where toluene is the solvent and n-propanol is the co-solvent. Transfer the mixed solution to a 100 ml volumetric flask and bring the volume to 100 ml. Sonicate for 30 min to prepare a 0.2 g / L DASAs solution. Take 10 ml of the DASAs solution into a 50 ml centrifuge tube, add 0.07 g of cetyltrimethylammonium bromide (CTAB), sonicate for 30 min, add an equal volume of phosphate buffer (pH 7.5, 0.1 M KCl) containing 0.5 mg / ml Hb, vortex for 10 min, centrifuge at 6000 rpm for 10 min, and finally form a photoresponsive self-assembled reverse micelle containing protein in the upper organic phase.

[0054] S3, Post-extraction:

[0055] Take 5 ml of the organic phase from step S2 above and add it to a 50 ml centrifuge tube. Add 5 ml of phosphate buffer solution with pH = 7.5 and 0.3 M KBr. Vortex for 10 min under light and room temperature conditions, and measure the UV absorbance of the lower aqueous phase.

[0056] like Figure 4 As shown, where Figure 4 (a) is a 50 nm transmission electron microscope image of protein extracted by the fast photoresponsive self-assembled reverse micelles prepared in this invention. Figure 4 (b) is a 50nm transmission electron microscope image after back-extraction (b); from Figure 4 (a) and Figure 4 (b) It can be seen that uniform reverse micelles are formed in the organic phase. Before back-extraction, the reverse micelles contain particulate Hb protein. After back-extraction, the Hb protein in the reverse micelles disappears because the Hb protein is extracted into the aqueous phase.

[0057] Example 2: The difference from Example 1 is the concentration of surfactants DASAs and CTAB added during the pre-extraction process.

[0058] Dissolve 0.02 g of DASAs in 80 ml of a mixed solution of toluene and n-propanol (toluene: ethanol = 4:1), where toluene is the solvent and n-propanol is the co-solvent. Transfer the mixed solution to a 100 ml volumetric flask and bring the volume to 100 ml. Sonicate for 30 min to prepare a 0.2 g / L DASAs solution. Take 10 ml of the DASAs solution into a transparent glass bottle and adjust the CTAB concentration to 6 g / L, 7 g / L, 8 g / L, 9 g / L, 12 g / L, and 15 g / L respectively. Sonicate for 30 min and add an equal volume of phosphate buffer (pH = 7.5, 0.1 MCl) containing 0.5 mg / ml Hb. Vortex for 10 min. Finally, a photoresponsive self-assembled reverse micelle containing protein is formed in the upper organic phase.

[0059] Post-extraction: The post-extraction process is the same as in Example 1.

[0060] From the appendix Figure 5 It is known that as the concentration of CTAB increases, the pre-extraction rate initially rises and then falls, reaching its maximum at a CTAB concentration of 7 g / L. This is because as the concentration of CTAB increases, the number of reverse micelles formed in the organic phase increases, leading to an increase in the pre-extraction rate. However, as the concentration of CTAB further increases, the number of CTAB molecules that aggregate and precipitate decreases, resulting in a decrease in the pre-extraction rate.

[0061] With the concentration of CTAB determined, the concentrations of DASAs were then sequentially changed to 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, and 0.5 g / L; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Figure 6 It is known that with increasing DASA concentration, pre-extraction initially increases and then decreases, reaching its maximum at a DASA concentration of 0.2 g / L. This is because as the concentration of DASA increases, the repulsive force between cationic surfactants decreases, making it easier to form large, stable reverse micelles, which is beneficial for improving the pre-extraction rate. However, as the proportion of DASA increases, the proportion of cationic surfactants composing the reverse micelles decreases, resulting in a decrease in the charge density within the reverse micelles and a decrease in the interaction force between the reverse micelles and proteins, leading to a decrease in the pre-extraction rate.

[0062] Example 3: Unlike Example 2, the post-extraction process was carried out under light-free conditions.

[0063] Pre-extraction: Same as in Example 2.

[0064] Post-extraction: Take 5 ml of the pre-extraction organic phase and add it to a 50 ml centrifuge tube. Add 5 ml of phosphate buffer (pH 7.5, 0.3 M KBr). Stir in the dark for 10 min at room temperature. Measure the UV absorbance of the lower aqueous phase and calculate the back-extraction rate.

[0065] Depend on Figure 7 It is known that the back-extraction rate under illumination is much higher than that under no-illumination conditions. This is because Hb has a pI of 6.45. Under conditions of low ion concentration and high pH in the back-extraction solution, Hb, being negatively charged, still exhibits electrostatic interaction with CTAB, resulting in greater resistance for Hb to enter the back-extraction phase. Therefore, the extraction time is longer and the extraction rate is lower. Under illumination, the membrane structure of the reverse micelles is disrupted, making it easier for Hb to enter the back-extraction phase.

[0066] Example 4: Applying fast light-responsive self-assembled reverse micelles for protein separation and purification to extract a mixed solution of Hb and BSA, including the following steps:

[0067] 0.02 g of DASAs was dissolved in 80 ml of a mixed solution of toluene and n-propanol (toluene: n-propanol = 4:1), where toluene was the solvent and n-propanol was the co-solvent. The mixed solution was transferred to a 100 ml volumetric flask and brought to a final volume of 100 ml. The solution was sonicated for 30 min to prepare a 0.2 g / L DASAs solution. 10 ml of the DASAs solution was taken into transparent glass bottles, and the CTAB addition was adjusted to 6 g / L, 7 g / L, 8 g / L, 9 g / L, 12 g / L, and 15 g / L, respectively. The solution was sonicated for 30 min, and an equal volume of phosphate buffer (pH 7.5, 0.1 M KCl) containing 0.5 mg / ml Hb and 0.5 mg / ml BSA was added. The mixture was vortexed for 10 min, and finally, a photoresponsive self-assembled reverse micelle containing protein was formed in the upper organic phase.

[0068] like Figure 8 The figure shows the extraction selectivity curves for fast photoresponsive self-assembled reverse micelles when extracting mixed proteins. When the concentrations of Hb and BSA in the mixed protein solution are the same, the selectivity of the reverse micelles for Hb protein ranges from 78.3% to 93.2%, indicating that fast photoresponsive self-assembled reverse micelles have good selectivity.

[0069] The fast photoresponsive self-assembled reverse micelles prepared by this invention not only maintain the high activity of proteins, but also enable the rapid release of extracted proteins through light irradiation due to their fast photoresponsiveness, which greatly improves the efficiency of protein extraction and greatly promotes the industrial application of liquid-liquid extraction of high-activity and high-purity proteins.

[0070] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the spirit and scope of the technical solution of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing rapid photoresponsive self-assembled reverse micelles for protein separation and purification, characterized in that: Includes the following steps: S1. Preparation of photoresponsive phase-transfer surfactants (DASAs) DASA intermediates, alkylamines and tetrahydrofuran were added to a flask, with the molar ratio of DASA intermediates to alkylamines being 1:

1. The reaction was carried out at room temperature in a water bath, and the resulting mixture was evaporated to dryness to obtain a light purple DASA solid. The solid was ground into powder and repeatedly washed and purified with n-hexane to finally obtain the photoresponsive phase transfer surfactant DASAs. The DASA intermediate was prepared by the following method: furfural and isopropyl malonate were placed in a flask with distilled water at a molar ratio of 1:1 and reacted in a water bath at 75°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, dissolved, purified and evaporated to dryness to obtain a pale yellow solid DASA intermediate. The alkylamine used is dihexylamine or dodecylamine, and the reaction time at room temperature is 15 min; S2. Preparation of fast photoresponsive self-assembled reverse micelles The photoresponsive phase transfer surfactants DASAs were dissolved in a mixed solvent of toluene and n-propanol, and then hexadecyltrimethylammonium bromide was added for ultrasonic dissolution. The concentration of DASAs was 0.05 g / L to 0.5 g / L, and the concentration of hexadecyltrimethylammonium bromide was 6 g / L to 15 g / L. Then, an equal volume of distilled water was added and stirred in a vortex manner, and finally, photoresponsive self-assembled reverse micelles were formed in the upper organic phase.

2. The method for preparing rapid photoresponsive self-assembled reverse micelles for separating and purifying proteins according to claim 1, characterized in that: In step S2, toluene is the solvent and n-propanol is the co-solvent in the mixed solvent of toluene and n-propanol.

3. The method for preparing rapid photoresponsive self-assembled reverse micelles for separating and purifying proteins according to claim 2, characterized in that: In step S2, the concentration of DASAs is 0.2 g / L and the concentration of hexadecyltrimethylammonium bromide is 7 g / L.

4. The method for preparing rapid photoresponsive self-assembled reverse micelles for separating and purifying proteins according to claim 1, characterized in that: In step S2, the ultrasonic dissolution time is 30 minutes and the vortex stirring time is 10 minutes.

5. A fast photoresponsive self-assembled reverse micelle for separating and purifying proteins obtained by any of the preparation methods described in claims 1 to 4.

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