Modified high molecular weight gamma-polyglutamic acid with increased dissolution rate and preparation method thereof

Abstract

The invention belongs to the field of high-molecular polymer derivation, and provides modified high-molecular-weight gamma-polyglutamic acid with an increased dissolution rate and a preparation method of the modified high-molecular-weight gamma-polyglutamic acid. According to the preparation method, the dissolution rate of gamma-PGA is improved by modifying high-molecular-weight gamma-PGA. The method comprises the following steps: respectively dissolving high molecular weight gamma-PGA, EDC, NHS and TEA in a PBS solution to obtain a solution A and a solution B; adding the solution B into the solution A, reacting at low temperature for 8-12 hours, and pre-cooling absolute ethyl alcohol at-20 DEG C to terminate the reaction; and centrifuging the reaction stop solution to obtain a precipitate, washing the precipitate for 2-3 times, re-dissolving the precipitate in deionized water, dialyzing the precipitate in a dialysis bag with the molecular weight cutoff of 100kDa, and freeze-drying to obtain the modified high-molecular-weight gamma-polyglutamic acid with the dissolution rate increased, and the dissolution rate of m-gamma-PGA is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of polymer derivatives, and in particular relates to a modified high molecular weight γ-polyglutamic acid with increased dissolution rate and its preparation method. Background Technology

[0002] γ-polyglutamic acid (γ-PGA) is a biodegradable polymer synthesized by microorganisms. γ-PGA is composed of D-glutamic acid and L-glutamic acid linked by γ-amide bonds, and its molecular weight ranges from 5 × 10⁻⁶. 4 Up to 1×10 7 Da.

[0003] γ-PGA has a wide range of applications in agriculture, industry and medicine, and is suitable for different applications depending on its molecular weight. Low molecular weight γ-PGA is suitable for drug carriers, while high molecular weight γ-PGA is suitable for thickeners, heavy metal flocculants and water-retaining agents. However, the dissolution rate of high molecular weight γ-PGA slows down with the increase of molecular weight, which limits its application as an industrial raw material.

[0004] Therefore, it is essential to provide a polymeric γ-PGA with an improved dissolution rate. Rapid dissolution can improve the production efficiency of γ-PGA in the application process, enhance the user experience, and enable γ-PGA to demonstrate great potential and value in more application areas. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a modified high molecular weight γ-polyglutamic acid with increased dissolution rate, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate, the method comprising the following steps:

[0008] 1) High molecular weight γ-polyglutamic acid was dissolved in PBS solution and stirred overnight at low temperature to obtain solution A; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC·HCl, molecular weight 155.24) was dissolved in PBS solution to obtain EDC solution; N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS, molecular weight 115.09) was added to the EDC solution, mixed evenly, and then triethanolamine (TEA) was added and stirred until completely dissolved to obtain solution B; wherein, the high molecular weight γ-polyglutamic acid is referred to as high molecular weight γ-PGA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide is referred to as EDC, N-hydroxysuccinimide is referred to as NHS, and triethanolamine is referred to as TEA;

[0009] 2) Slowly add solution B to solution A to obtain the γ-PGA derivatization reaction system;

[0010] 3) The γ-PGA derivatization reaction system was reacted at room temperature for 8-12 hours, and then the reaction was terminated with anhydrous ethanol pre-cooled at -20℃ to obtain the reaction termination solution.

[0011] 4) Centrifuge the reaction termination solution at 8000-10000 rpm for 8-10 min, wash the precipitate 2-3 times with anhydrous ethanol, and then redissolve the precipitate in deionized water to obtain the precipitate redissolved product. Dialyze the precipitate redissolved product for 48 h, and then freeze-dry it under vacuum after dialysis to obtain the modified high molecular weight γ-polyglutamic acid with increased dissolution rate, denoted as m-γ-PGA.

[0012] Furthermore, in step (1), the molecular weight range of the high molecular weight γ-PGA is 7 × 10⁻⁶. 5 -2×10 6 Da.

[0013] Furthermore, in step (1), the molar concentration of carboxyl groups in solution A is 0.30-0.43 mmol / mL.

[0014] Furthermore, in step (1), the molar concentration ratio of EDC:NHS:TEA in solution B is 1.2:0.6:2.

[0015] Furthermore, in step (2), the pH of the γ-PGA derivatization reaction system is controlled to be 6.5-7.5.

[0016] Furthermore, in step (2), the molar concentration ratio of γ-PGA carboxyl group: EDC: NHS: TEA in the γ-PGA derivatization reaction system is (1.2-1.5): 1.2: 0.6: 2.

[0017] Furthermore, in step (4), the molecular weight cutoff for the dialysis treatment is 100 kDa.

[0018] Furthermore, the method also includes: preparing the m-γ-PGA into a 10 mg / mL solution, performing a full wavelength scan in the range of 200-800 nm; and recording the time for complete dissolution of the m-γ-PGA.

[0019] The present invention also provides m-γ-PGA prepared by the above-mentioned method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate.

[0020] The present invention also provides the application of the above-mentioned m-γ-PGA in materials such as thickeners, heavy metal flocculants, and water-retaining agents.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0022] This invention provides modified high molecular weight γ-polyglutamic acid with increased dissolution rate and its preparation method. This method can increase the dissolution rate of high molecular weight γ-polyglutamic acid by more than 300 times, thereby reducing energy consumption and waiting time during the dissolution process of γ-polyglutamic acid, and improving the production discontinuity and application experience of γ-polyglutamic acid caused by the long dissolution time of γ-polyglutamic acid. Attached Figure Description

[0023] Figure 1 Full-wavelength scan of the modified γ-PGA (m-γ-PGA) provided in Embodiment 1 of the present invention;

[0024] Figure 2 This is an appearance diagram of m-γ-PGA provided in Embodiment 2 of the present invention;

[0025] Figure 3 This is a diagram showing the dissolution rate of m-γ-PGA provided in Example 3 of the present invention. Detailed Implementation

[0026] The inventors discovered that factors affecting the dissolution rate of substances include internal and external factors. The internal factor affecting the dissolution rate of γ-PGA is its structure and molecular weight. The γ-PGA structure contains a large number of free carboxyl groups, which can be grafted with other hydrophilic groups to form γ-PGA derivatives. Grafting hydrophilic groups can form modified (m-γ-PGA), and m-γ-PGA is expected to effectively increase the dissolution rate of γ-PGA. The ratio of γ-PGA to other derivative substrates has a significant impact on the synthesis of γ-PGA derivatives. The repeating unit in the γ-PGA molecule is Glu (molecular weight 147.13), and each Glu repeating unit contains one free carboxyl group. During the polymerization of Glu monomers through γ-amide bonds to form γ-PGA, water is lost. Therefore, the average molecular weight of each repeating unit in the γ-PGA molecule is 129 g / mol. Thus, the carboxyl group content per gram of γ-PGA is:

[0027]

[0028] The theoretical value of carboxyl group content in γ-PGA is within its molecular weight of 5 × 10⁻⁶. 4 -2×10 6 Within the Da range, the molecular weight does not affect the decomposition rate of γ-PGA; the reaction system can be designed based on the content of the carboxyl groups in γ-PGA for γ-PGA derivation. In addition, external factors such as temperature, stirring, and pressure are also important factors affecting the dissolution rate of substances. However, higher temperatures lead to the degradation of γ-PGA and a decrease in molecular weight. Stirring can increase the dissolution rate of γ-PGA to some extent, but its effect on the dissolution rate of high molecular weight or ultra-high molecular weight γ-PGA is negligible.

[0029] Therefore, the modification of high molecular weight γ-PGA by grafting hydrophilic groups is of great significance for increasing the dissolution rate of γ-PGA and its large-scale application.

[0030] The present invention will now be described in detail with reference to specific embodiments.

[0031] Example 1

[0032] Weigh out 7×10 50.5 g of Da's γ-PGA was added to 10 mL of PBS solution and stirred on a magnetic stirrer at 4 °C until completely dissolved to obtain a γ-PGA solution (solution A) with a carboxyl molar ratio of 0.388 mmol / L. 0.575 g of EDC and 0.175 g of NHS were added to 2.5 mL of PBS buffer and stirred until dissolved. Then, 0.745 g of TEA was added and stirred until dissolved to prepare mixed solution B. The molar ratio of the four substances was: γ-PGA carboxyl group: EDC: NHS: TEA = 1.5:1.2:0.6:2. While stirring, solution B was added dropwise to solution A over 15 minutes, with the pH maintained at 6.5. After the addition, the reaction was stirred at room temperature for 12 hours. The reaction was terminated by pre-cooling the solution with 5 mL of anhydrous ethanol at -20°C. The reaction system was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected and washed three times with ethanol. The washed precipitate was redissolved in 10 mL of deionized water, placed in a dialysis bag, and dialyzed for 48 hours. The solution in the dialysis bag was pre-frozen at -20°C for 20 minutes, then transferred to -80°C for 24 hours, and finally dried in a vacuum freeze dryer for 24 hours to obtain the γ-PGA modified product. The modified γ-PGA was then scanned across the entire wavelength range (200-800 nm). Figure 1 As shown in the figure, γ-PGA has a maximum absorption peak at 216 nm, TEA has a maximum absorption peak at 227 nm, and the modified γ-PGA has an absorption peak at 270 nm, indicating that γ-PGA has introduced new conjugated groups.

[0033] Example 2

[0034] Weigh out 1.1 × 10 60.4 g of Da's γ-PGA was added to 10 mL of PBS solution and stirred on a magnetic stirrer at 4 °C until completely dissolved to obtain a γ-PGA solution (solution A) with a carboxyl molar ratio of 0.30 mmol / L. 0.575 g of EDC and 0.175 g of NHS were added to 2.5 mL of PBS buffer and stirred until dissolved. Then, 0.745 g of TEA was added and stirred until dissolved to prepare mixed solution B. The molar ratio of the four substances was: γ-PGA carboxyl group: EDC: NHS: TEA = 1.5:1.2:0.6:2. While stirring, solution B was added dropwise to solution A over 15 minutes, with the pH maintained at 7.0. After the addition was complete, the reaction was stirred at room temperature for 12 hours. The reaction was terminated by pre-cooling the solution with 5 mL of anhydrous ethanol at -20°C. The reaction system was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected and washed three times with ethanol. The washed precipitate was redissolved in 10 mL of deionized water, placed in a dialysis bag, and dialyzed for 48 hours. The solution in the dialysis bag was pre-frozen at -20°C for 20 minutes, then transferred to -80°C for 24 hours, and finally dried in a vacuum freeze dryer for 24 hours to obtain the γ-PGA modified product, which was a white substance. Figure 2 As shown.

[0035] Example 3

[0036] Weigh out 7×10 5 0.5 g of Da's γ-PGA was added to 10 mL of PBS solution and stirred on a magnetic stirrer at 4 °C until completely dissolved to obtain a γ-PGA solution (solution A) with a carboxyl molar ratio of 0.388 mmol / L. 0.575 g of EDC and 0.175 g of NHS were added to 2.5 mL of PBS buffer and stirred until dissolved. Then, 0.745 g of TEA was added and stirred until dissolved to prepare mixed solution B. The molar ratio of the four substances was: γ-PGA carboxyl group: EDC: NHS: TEA = 1.5:1.2:0.6:2. While stirring, solution B was added dropwise to solution A over 15 minutes, with the pH maintained at 7.5. After the addition was complete, the mixture was stirred at room temperature for 12 hours. The reaction was terminated by pre-cooling the solution with 5 mL of anhydrous ethanol at -20°C. The reaction mixture was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected and washed three times with ethanol. The washed precipitate was redissolved in 10 mL of deionized water, placed in a dialysis bag, and dialyzed for 48 hours. The solution in the dialysis bag was then pre-frozen at -20°C for 20 minutes, transferred to -80°C for 24 hours, and then dried in a vacuum freeze dryer for 24 hours to obtain the γ-PGA modified product. 5 Da's γ-PGA and modified γ-PGA were each prepared into 5 mg / mL aqueous solutions. After 30 seconds, the modified γ-PGA completely dissolved. 7 × 105 The γ-PGA in Da was not completely dissolved, and the dissolution state of γ-PGA was recorded at 5 min, 30 min, 60 min, and 180 min. γ-PGA completely dissolved at 180 min. The dissolution rate of γ-PGA was significantly increased after modification. Figure 3 As shown.

[0037] Example 4

[0038] Weigh out 2×10 6 0.55 g of Da's γ-PGA was added to 10 mL of PBS solution and stirred on a magnetic stirrer at 4 °C until completely dissolved to obtain γ-PGA solution (solution A), with a carboxyl molar ratio of 0.43 mmol / L. 0.575 g of EDC and 0.175 g of NHS were added to 2.5 mL of PBS buffer and stirred until dissolved. Then, 0.745 g of TEA was added and stirred until dissolved to prepare mixed solution B. The molar ratio of the four substances was: γ-PGA carboxyl group: EDC: NHS: TEA = 1.5:1.2:0.6:2. While stirring, mixed solution B was added dropwise to solution A over 15 minutes, with the pH controlled at 7.0. After the addition was complete, the reaction was stirred at room temperature for 12 hours. The reaction was terminated by pre-cooling the solution with 5 mL of anhydrous ethanol at -20°C. The reaction system was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected and washed three times with ethanol. The washed precipitate was redissolved in 10 mL of deionized water, placed in a dialysis bag, and dialyzed for 48 hours. The solution in the dialysis bag was pre-frozen at -20°C for 20 minutes, then transferred to -80°C for 24 hours, and finally dried in a vacuum freeze dryer for 24 hours to obtain the γ-PGA modified product.

[0039] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate, characterized in that, The method includes the following steps: 1) Dissolve high molecular weight γ-polyglutamic acid in PBS solution and stir overnight at low temperature to obtain solution A; 1-Ethyl-(3-dimethylaminopropyl)carbodiimide was dissolved in PBS solution to obtain EDC solution; N-hydroxysuccinimide was added to the EDC solution, and after mixing well, triethanolamine was added and stirred until completely dissolved to obtain solution B; Wherein, the high molecular weight γ-polyglutamic acid is referred to as high molecular weight γ-PGA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide is referred to as EDC, N-hydroxysuccinimide is referred to as NHS, and triethanolamine is referred to as TEA; 2) Slowly add solution B to solution A to obtain the γ-PGA derivatization reaction system; 3) The γ-PGA derivatization reaction system was reacted at room temperature for 8-12 hours, and then the reaction was terminated with anhydrous ethanol pre-cooled at -20℃ to obtain the reaction termination solution. 4) Centrifuge the reaction termination solution at 8000-10000 rpm for 8-10 min, wash the precipitate 2-3 times with anhydrous ethanol, and then redissolve the precipitate in deionized water to obtain the precipitate redissolved product. The precipitate was dialyzed for 48 hours, and then freeze-dried under vacuum after dialysis to obtain the modified high molecular weight γ-polyglutamic acid with increased dissolution rate, denoted as m-γ-PGA.

2. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, In step (1), the molecular weight range of the high molecular weight γ-PGA is 7 × 10⁻⁶. 5 -2×10 6 Da.

3. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 2, characterized in that, In step (1), the molar concentration of carboxyl groups in solution A is 0.30-0.43 mmol / mL.

4. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, In step (1), the molar ratio of EDC:NHS:TEA in solution B is 1.2:0.6:

2.

5. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, In step (2), the pH of the γ-PGA derivatization reaction system is controlled to be 6.5-7.

5.

6. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, In step (2), the molar ratio of γ-PGA carboxyl group: EDC: NHS: TEA in the γ-PGA derivatization reaction system is (1.2-1.5): 1.2: 0.6:

2.

7. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, In step (4), the molecular weight cutoff for the dialysis treatment is 100 kDa.

8. The method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate as described in claim 1, characterized in that, The method further includes: preparing the m-γ-PGA into a 10 mg / mL solution, performing a full wavelength scan in the range of 200-800 nm; and recording the time for complete dissolution of the m-γ-PGA.

9. m-γ-PGA prepared by a method for preparing modified high molecular weight γ-polyglutamic acid with increased dissolution rate according to any one of claims 1 to 8.

10. The application of m-γ-PGA according to claim 9 in materials that are thickeners, heavy metal flocculants, and water-retaining agents.

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