Modified PVDF (Polyvinylidene Fluoride) ultrafiltration membrane for separation and purification of ethanol dehydrogenase and preparation method of modified PVDF ultrafiltration membrane

Abstract

The invention belongs to the technical field of medical filter membranes, and particularly discloses a modified PVDF (Polyvinylidene Fluoride) ultrafiltration membrane for separation and purification of ethanol dehydrogenase and a preparation method of the modified PVDF ultrafiltration membrane. According to the membrane, the polypeptide KGDVRDTSK is modified on the surface of the PVDF membrane, so that the hydrophilicity of the PVDF membrane and the rejection rate of ethanol dehydrogenase are remarkably improved, and meanwhile, the activity of the enzyme is maintained. The method solves the limitation of a traditional ultrafiltration membrane in separation and purification of ethanol dehydrogenase, has the characteristics of high efficiency and economy, and is suitable for industrial application.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical filter membranes, and in particular relates to a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase and a preparation method thereof. Background Technique

[0002] In the fields of biopharmaceuticals and bioseparations, alcohol dehydrogenase (ADH), as an important industrial enzyme, its purification process has always been a research hotspot. Traditional separation and purification methods, such as precipitation, ion exchange, gel filtration, etc., although able to achieve a certain degree of purification, have problems such as complex operation, high cost, and large loss of enzyme activity. In recent years, ultrafiltration technology has gradually become an important means in protein separation and purification due to its advantages of high efficiency, energy saving, and simple operation.

[0003] Ultrafiltration membrane technology can effectively remove small molecule impurities in fermentation broth or extract by selecting an appropriate membrane pore size, while concentrating and purifying the target enzyme, significantly improving the enzyme activity and recovery rate. However, traditional ultrafiltration membranes often face problems of membrane fouling and insufficient selectivity when treating protein solutions, resulting in a decrease in membrane flux and separation efficiency. Therefore, developing ultrafiltration membranes with high selectivity and anti-fouling properties has important practical significance for the separation and purification of alcohol dehydrogenase. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase and a preparation method thereof. The obtained ultrafiltration membrane can efficiently separate alcohol dehydrogenase when used for the purification of alcohol dehydrogenase and can efficiently maintain the enzyme activity. The preparation method of the present invention is simple and has practical application prospects.

[0005] On the one hand, the present invention provides a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase. The surface of the ultrafiltration membrane is covalently fixed with the polypeptide KGDVRDTSK, and the polypeptide content is 0.05 - 0.2 mg / cm². This polypeptide modification significantly enhances the specific adsorption ability of the membrane to alcohol dehydrogenase.

[0006] After detection, the water contact angle of the ultrafiltration membrane is less than or equal to 32°, indicating that the membrane surface has good hydrophilicity, which helps to improve the separation efficiency and reduce membrane fouling.

[0007] On the second hand, the present invention provides a preparation method of a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase, including the following steps: (1) Immerse the PVDF ultrafiltration membrane in an alkaline solution for surface activation; (2) Immerse the activated membrane in a polypeptide solution for reaction; (3) Immerse the membrane after the reaction in step b into an ethanol solution of imidazole-2-carboxaldehyde for reaction; (4) Rinse the surface of the membrane with deionized water, and obtain a modified PVDF ultrafiltration membrane after drying.

[0008] In step (1), the alkali solution is an inorganic strong alkali solution, including NaOH, KOH, etc., the concentration of the alkali solution is 0.5 - 1 mol / L, the treatment temperature is 50 - 60 °C, and the treatment time is 2 - 3 h.

[0009] In step (2), the polypeptide is KGDVRDTSK; the concentration of the polypeptide solution is 0.1 - 0.2 mg / mL, the reaction pH value is 7.0 - 7.4, the reaction temperature is 25 - 37 °C, and the reaction time is 4 - 6 h.

[0010] In step (3), the concentration of imidazole-2-carboxaldehyde is 1 - 5 mg / g, and the reaction is carried out at 50 - 70 °C for 6 - 8 h.

[0011] The third aspect of the present invention provides a method for separating and purifying alcohol dehydrogenase, using the above-mentioned modified PVDF ultrafiltration membrane for separation and purification. This method utilizes the polypeptide modification on the membrane surface to achieve efficient adsorption and separation of alcohol dehydrogenase, while maintaining the high activity of the enzyme, and is applicable to the industrial-scale production of alcohol dehydrogenase. After detection, the retention rate of the ultrafiltration membrane for alcohol dehydrogenase is higher than 95%, ensuring an efficient separation effect. The activity retention rate of alcohol dehydrogenase after separation by the ultrafiltration membrane is greater than 90%, indicating that the membrane has a small impact on the enzyme activity during the separation process and can effectively maintain the biological activity of the enzyme.

[0012] In the process of separating and purifying alcohol dehydrogenase (ADH), membrane filtration technology is an efficient and widely used method. Ultrafiltration technology can effectively remove small-molecule impurities in the fermentation broth or extract, while concentrating and purifying the target enzyme, significantly improving the enzyme activity and recovery rate. The beneficial effect of the present invention is to develop a modified PVDF ultrafiltration membrane for purifying alcohol dehydrogenase. By modifying the polypeptide KGDVRDTSK on the surface of the PVDF membrane, the performance of the membrane is significantly improved. The specific features are as follows: (1) Improve the hydrophilicity of the membrane: The introduction of the polypeptide KGDVRDTSK enhances the hydrophilicity of the PVDF ultrafiltration membrane, reduces the adsorption of proteins on the membrane surface, thereby reducing membrane fouling and extending the service life of the membrane.

[0013] (2) Improve the retention rate of alcohol dehydrogenase: The modified PVDF ultrafiltration membrane of the present invention significantly improves the retention rate of the enzyme through the specific interaction between the polypeptide and alcohol dehydrogenase, while maintaining the activity of the enzyme. This technology not only improves the purification efficiency but also reduces the cost, and is suitable for large-scale industrial applications.

[0014] In summary, through the modified PVDF ultrafiltration membrane, the present invention achieves the efficient purification of alcohol dehydrogenase, while maintaining the enzyme activity and the long-term stability of the membrane, providing an efficient and economical solution for the biopharmaceutical and enzyme preparation industries. Detailed implementation mode

[0015] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0016] The polypeptide KGDVRDTSK in the embodiments of the present invention was purchased from Zhong Peptide Biotechnology Co., Ltd. Without special instructions, other reagents can be purchased commercially.

[0017] The obtained ultrafiltration membrane was measured for the rejection rate of alcohol dehydrogenase under the conditions of a pressure of 0.1 MPa and a temperature of 25 °C. The concentration changes of the original aqueous solution of alcohol dehydrogenase at 200 ppm and the protein in the permeate were measured, and calculated through the following formula: R = (Cp - Cf) / Cp × 100%, where R is the rejection rate of alcohol dehydrogenase, Cp is the concentration of alcohol dehydrogenase in the original solution, and Cf is the concentration of alcohol dehydrogenase in the permeate.

[0018] Test method for polypeptide content: The test of polypeptide content can adopt the quantitative nuclear magnetic resonance (qNMR) method. The specific steps are as follows: Cut the modified membrane into small pieces, and ultrasonically treat with an appropriate amount of deionized water to dissolve the polypeptide on the membrane surface. Take an appropriate amount of the dissolved solution and measure the polypeptide content by the qNMR method. Calculate the polypeptide content (mg / cm²) according to the area of the membrane.

[0019] Test method for contact angle: The contact angle is measured using a standard contact angle measuring instrument.

[0020] The activity of alcohol dehydrogenase (ADH) can be measured by ultraviolet spectrophotometry. The specific steps are as follows: Measure the change in absorbance of ADH at 340 nm. Calculate the change rate of absorbance (△A), and calculate the activity of ADH according to the formula: ADH activity = 1608 × △A / c (nmol / min / mg prot) Where c is the concentration of protein in the sample.

[0021] The activity retention rate can be calculated by comparing the enzyme activities before and after separation. Example 1

[0022] A preparation method of a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase, comprising the following steps: (1) Immerse the PVDF ultrafiltration membrane in 1 mol / L NaOH solution and treat it at 60 °C for 2 h.

[0023] (2) Immerse the activated membrane in a polypeptide KGDVRDTSK solution with a concentration of 0.15 mg / mL, under the condition of a pH value of 7.2, a reaction temperature of 30 °C, and a reaction time of 5 h.

[0024] (3) Immerse the membrane after the reaction in step (2) in an ethanol solution of imidazole-2-carbaldehyde with a concentration of 3 mg / g and react it at 60 °C for 7 h.

[0025] (4) Rinse the membrane surface with deionized water and obtain the modified PVDF ultrafiltration membrane after drying. Example 2

[0026] A preparation method of a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase, comprising the following steps: (1) Immerse the PVDF ultrafiltration membrane in 0.8 mol / L KOH solution and treat it at 55 °C for 2.5 h.

[0027] (2) Immerse the activated membrane in a polypeptide KGDVRDTSK solution with a concentration of 0.2 mg / mL, under the condition of a pH value of 7.4, a reaction temperature of 37 °C, and a reaction time of 6 h.

[0028] (3) Immerse the membrane after the reaction in step (2) in an ethanol solution of imidazole-2-carbaldehyde with a concentration of 5 mg / g and react it at 50 °C for 8 h.

[0029] (4) Rinse the membrane surface with deionized water and obtain the modified PVDF ultrafiltration membrane after drying. Example 3

[0030] A preparation method of a modified PVDF ultrafiltration membrane for the separation and purification of alcohol dehydrogenase, comprising the following steps: (1) Immerse the PVDF ultrafiltration membrane in 0.5 mol / L NaOH solution and treat it at 50 °C for 3 h.

[0031] (2) Immerse the activated membrane in a polypeptide KGDVRDTSK solution with a concentration of 0.1 mg / mL, under the condition of a pH value of 7.0, a reaction temperature of 25 °C, and a reaction time of 4 h.

[0032] (3) Immerse the membrane after the reaction in step (2) in an ethanol solution of imidazole-2-carbaldehyde with a concentration of 1 mg / g and react it at 70 °C for 6 h.

[0033] (4) Rinse the membrane surface with deionized water and obtain the modified PVDF ultrafiltration membrane after drying.

[0034] Comparative Example 1 A preparation method of a PVDF ultrafiltration membrane includes the following steps: (1) Immerse the unactivated PVDF ultrafiltration membrane in a polypeptide KGDVRDTSK solution with a concentration of 0.15 mg / mL, at a pH value of 7.2, a reaction temperature of 30 °C, and a reaction time of 5 h.

[0035] (2) Immerse the membrane after the above reaction in an ethanol solution of imidazole-2-carboxaldehyde with a concentration of 3 mg / g and react at 60 °C for 7 h.

[0036] (3) Rinse the membrane surface with deionized water and obtain the unactivated PVDF ultrafiltration membrane after drying.

[0037] Comparative Example 2 A preparation method of a PVDF ultrafiltration membrane includes the following steps: (1) Immerse the PVDF ultrafiltration membrane in a 1 mol / L NaOH solution and treat it at 60 °C for 2 h.

[0038] (2) Immerse the activated membrane in a polypeptide KGDVRDTSK solution with a concentration of 0.15 mg / mL, at a pH value of 7.2, a reaction temperature of 30 °C, and a reaction time of 5 h.

[0039] (3) Rinse the membrane surface with deionized water and obtain the PVDF ultrafiltration membrane without cross-linking reaction after drying.

[0040] Comparative Example 3 A preparation method of a PVDF ultrafiltration membrane includes the following steps: (1) Immerse the PVDF ultrafiltration membrane in a 1 mol / L NaOH solution and treat it at 60 °C for 2 h.

[0041] (2) Immerse the activated membrane in an ethanol solution of imidazole-2-carboxaldehyde with a concentration of 3 mg / g and react at 60 °C for 7 h.

[0042] (3) Rinse the membrane surface with deionized water and obtain the PVDF ultrafiltration membrane without polypeptide modification after drying.

[0043] The following Table 1 shows the performance test results of the PVDF ultrafiltration membranes prepared in Examples 1-3 and Comparative Examples 1-3: Table 1

[0044] As can be seen from Table 1 above, the water contact angle of the modified PVDF ultrafiltration membrane of the present invention is significantly lower than that of the unmodified membrane, and the retention rate of alcohol dehydrogenase and the enzyme activity retention rate after separation are both significantly improved; therefore, for the modified PVDF ultrafiltration membrane for separating and purifying alcohol dehydrogenase of the present invention, by modifying the polypeptide KGDVRDTSK on the surface of the PVDF membrane, the hydrophilicity of the membrane and the retention rate of alcohol dehydrogenase are significantly improved, while the activity of the enzyme is maintained.

[0045] The above specific embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modification and change made to the present invention within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

[0046] Taking the above ideal embodiments based on the present invention as inspiration, through the above invention content, relevant staff can completely make various changes and modifications without departing from the technical idea of this invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A modified PVDF ultrafiltration membrane for separation and purification of alcohol dehydrogenase, characterized in that: The polypeptide KGDVRDTSK is fixed on the surface of the ultrafiltration membrane by covalent bonds, and the polypeptide content is 0.05-0.2 mg / cm².

2. The modified PVDF ultrafiltration membrane according to claim 1, characterized in that The water contact angle of the ultrafiltration membrane is less than or equal to 32°.

3. A method for preparing a modified PVDF ultrafiltration membrane for separation and purification of alcohol dehydrogenase, characterized in that: The following steps are involved: a. Immersing the PVDF ultrafiltration membrane in an alkaline solution for surface activation; b. immersing the activated membrane in a polypeptide solution for reaction; c The film after the reaction in step b was immersed in an ethanol solution of imidazole-2-carboxaldehyde for reaction; d. Rinse the membrane surface with deionized water and obtain the modified PVDF ultrafiltration membrane after drying.

4. The preparation method according to claim 3, characterized in that: The alkaline solution in step a is an inorganic strong alkaline solution, the concentration of the alkaline solution is 0.5-1 mol / L, the treatment temperature is 50-60° C., and the treatment time is 2-3 h.

5. The preparation method according to claim 3, characterized in that: The polypeptide in step b is KGDVRDTSK.

6. The preparation method according to claim 3, characterized in that: The concentration of the polypeptide solution in step b is 0.1-0.2 mg / mL, the reaction pH is 7.0-7.4, the reaction temperature is 25-37° C., and the reaction time is 4-6 h.

7. The preparation method according to claim 3, characterized in that: In step c, the concentration of imidazole-2-carboxaldehyde is 1-5 mg / g, the reaction temperature is 50-70° C., and the reaction time is 6-8 h.

8. A modified PVDF ultrafiltration membrane prepared by the preparation method according to any one of claims 3 to 7.

9. Use of the modified PVDF ultrafiltration membrane according to claim 1, 2 or 8 in the separation and purification of alcohol dehydrogenase.

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