Method for preparing low-molecular-weight gamma-polyglutamic acid independent of molecular weight and concentration of substrate
Microwave-assisted hydrolysis of γ-polyglutamic acid in a controlled reactor environment addresses the inconsistency of microbial degradation, enabling consistent production of low molecular weight γ-polyglutamic acid with controlled molecular weights and distributions.
Patent Information
- Application Number
- CN202411489072.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2024-12-31
AI Technical Summary
Existing methods for preparing low molecular weight γ-polyglutamic acid are inconsistent due to variations in microbial activity, leading to unpredictable molecular weights and concentrations, complicating the degradation process.
A method involving microwave-assisted hydrolysis of γ-polyglutamic acid in a polytetrafluoroethylene-lined reactor at controlled temperatures and times, independent of substrate molecular weight and concentration, to achieve consistent low molecular weight products.
The method produces low molecular weight γ-polyglutamic acid with predictable molecular weights and distributions, simplifying operations, reducing costs, and expanding production capabilities.
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Figure CN119219917A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of high molecular weight polymer degradation, and in particular relates to a method for preparing low molecular weight gamma-polyglutamic acid which is independent of substrate molecular weight and concentration. Background Art
[0002] γ-Polyglutamic acid is a biodegradable polypeptide polymer produced by microorganisms. It has excellent water solubility, biodegradability, excellent adsorption and good biocompatibility. It can be used as antifreeze, thickener, moisturizer, nutritional additive, bio-glue, drug carrier, bio-fertilizer, bio-flocculant, etc. in food, cosmetics, medicine, agriculture, wastewater treatment and other industries. In the field of medicine, low molecular weight γ-polyglutamic acid is needed as a suitable drug carrier to achieve a suitable drug loading size to enter the target cells. In the field of cosmetics, low molecular weight γ-polyglutamic acid is needed as a moisturizer to prevent high molecular weight γ-polyglutamic acid from absorbing moisture and swelling, blocking the skin sweat glands and pores, causing local redness, swelling, pustules and other skin discomfort.
[0003] The most effective method to obtain low molecular weight γ-polyglutamic acid is to degrade high molecular weight γ-polyglutamic acid produced by microbial fermentation. However, since the conditions such as microbial activity cannot be guaranteed to be completely consistent for each batch, the high molecular weight γ-polyglutamic acid produced by microbial fermentation has different product molecular weights, which brings operational inconvenience to the subsequent degradation of low molecular weight γ-polyglutamic acid. The degradation of γ-polyglutamic acid reported in previous literature and patents, such as the use of heat, acid, and ultrasonic hydrolysis processes, did not involve the development of a method for preparing low molecular weight γ-polyglutamic acid that is independent of the substrate molecular weight and concentration. Summary of the invention
[0004] In view of the above prior art, the object of the present invention is to provide a method for preparing low molecular weight γ-polyglutamic acid which is simple to operate and can be independent of the molecular weight and concentration of the substrate. The method comprises the following steps:
[0005] a. Pour a 1-10 g / L γ-polyglutamic acid (hydrogen form) aqueous solution into a polytetrafluoroethylene-lined reactor, place the reactor into a multi-purpose microwave chemical synthesizer, heat to 100-140°C at a rate of 10°C / min, and react for 5-30 minutes. After the instrument cools down, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0006] Preferably, the weight average molecular weight M of the γ-polyglutamic acid is w 95.2~436.3KDa, number average molecular weight M n The molecular weight distribution coefficient PDI is 55.1~186.1KDa and 1.73~2.34.
[0007] Preferably, the reaction temperature is 120°C.
[0008] Preferably, the reaction time is 30 min.
[0009] The second object of the present invention is to provide a low molecular weight γ-polyglutamic acid prepared according to the above method, wherein the weight average molecular weight M of the low molecular weight γ-polyglutamic acid is w The number average molecular weight is 14.3~16.4KDa. n It is 10.7~12.6KDa, and the molecular weight distribution coefficient PDI is 1.31~1.36.
[0010] The present invention has the following beneficial effects:
[0011] The method provided by the present invention can prepare low molecular weight γ-polyglutamic acid independently of the molecular weight and concentration of the substrate. As shown in Examples 1 to 8, degradation products (M 20 ) with similar molecular weight and PDI can be obtained under the same reaction temperature of 120°C and reaction time of 30 min. w 14.3~16.4KDa, M n 10.7~12.6KDa, PDI is 1.31~1.36), regardless of the molecular weight before degradation (weight average molecular weight M w 95.2~436.3KDa, number average molecular weight M n The method can reduce the molecular weight of the degradation product by 55.1-186.1KDa, the molecular weight distribution coefficient PDI is 1.73-2.34) and the concentration of γ-polyglutamic acid (1-10g / L), and is easy to operate, does not require the addition of any reagents during the degradation process, and has a short reaction time. Compared with other methods, the method can achieve the purpose of obtaining degradation products of similar molecular weight by using γ-polyglutamic acid of different molecular weights and concentrations as substrates, thereby simplifying the operation, saving costs, and expanding production. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 is the weight average molecular weight M of γ-polyglutamic acid before and after degradation in Example 1-8 w .
[0013] Figure 2 is the weight average molecular weight M of γ-polyglutamic acid before and after degradation in Example 1-8 n .
[0014] Figure 3 It is the molecular weight distribution coefficient PDI of γ-polyglutamic acid before and after degradation in Example 1-8. DETAILED DESCRIPTION
[0015] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.
[0016] The γ-polyglutamic acid used in the following examples is γ-polyglutamic acid (hydrogen form).
[0017] The weight average molecular weight M of γ-polyglutamic acid in the following examples is w , number average molecular weight M n The molecular weight distribution index PDI was determined by gel permeation chromatography (GPC). The specific detection method is as follows: high performance liquid chromatograph: Thermo ultimate 3000; data processing software: Chromeleon7.2.10; chromatography system: Thermo U3000; chromatographic column: Waters Ultrahydrogel Linear; detector: differential refractometer RefractoMax520; mobile phase: 4.5mmol / L citric acid-5.9mmol / L sodium citrate, flow rate: 0.5mL / min, column temperature 40℃, cell temperature 40℃, injection volume 100μL. Sample treatment: The γ-polyglutamic acid sample was prepared into a 1mg / mL solution with deionized water and filtered with a 0.22μm microporous filter membrane. Molecular weight calibration: Sigma-Aldrich PEO / PEG standard (batch number BCCD474) was used for calibration.
[0018] Example 1
[0019] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0020] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0021] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 95.2KDa, 55.1KDa and 1.73 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 14.9 KDa, 11.2 KDa and 1.34 respectively.
[0022] Example 2
[0023] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0024] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0025] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 141.5 KDa, 76.0 KDa and 1.86 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 16.4KDa, 12.1KDa and 1.36 respectively.
[0026] Example 3
[0027] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0028] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0029] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 253.5 KDa, 125.6 KDa and 2.02, respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 15.5 KDa, 11.9 KDa and 1.31 respectively.
[0030] Example 4
[0031] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0032] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0033] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 273.1 KDa, 136.8 KDa and 2.00 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 16.3 KDa, 12.6 KDa and 1.29 respectively.
[0034] Example 5
[0035] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0036] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0037] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 331.7 KDa, 154.3 KDa and 2.15 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 16.0 KDa, 12.3 KDa and 1.31 respectively.
[0038] Example 6
[0039] Preparation of sample solution: 24 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 1 g / L γ-polyglutamic acid aqueous solution.
[0040] Sample hydrolysis: Pour 24 mL of 1 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene-lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0041] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 436.3 KDa, 186.1 KDa and 2.34 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 14.9 KDa, 11.1 KDa and 1.34 respectively.
[0042] Example 7
[0043] Preparation of sample solution: 120 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 5 g / L γ-polyglutamic acid aqueous solution.
[0044] Hydrolysis of the sample: Pour 24 mL of 5 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0045] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 436.3 KDa, 186.1 KDa and 2.34 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 15.5 KDa, 11.5 KDa and 1.35 respectively.
[0046] Example 8
[0047] Preparation of sample solution: 240 mg of γ-polyglutamic acid was dissolved in 24 mL of deionized water, and mixed by magnetic stirring at 350 rpm for half an hour to obtain 24 mL of 10 g / L γ-polyglutamic acid aqueous solution.
[0048] Sample hydrolysis: Pour 24 mL of 10 g / L γ-polyglutamic acid aqueous solution into a 100 mL polytetrafluoroethylene lined reactor. Place the reactor in a multi-purpose microwave chemical synthesizer, heat to 120°C at a rate of 10°C / min, and react for 30 minutes. When the instrument cools to about 60°C, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
[0049] GPC measured the M of γ-polyglutamic acid before hydrolysis w 、M n and PDI were 436.3 KDa, 186.1 KDa and 2.34 respectively. The M of low molecular weight γ-polyglutamic acid obtained after hydrolysis w 、M n and PDI are 14.3 KDa, 10.7 KDa and 1.34 respectively.
[0050] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing low molecular weight γ-polyglutamic acid independent of substrate molecular weight and concentration, characterized in that: The following steps are involved: a. preparing an aqueous solution of γ-polyglutamic acid; b. Pour the γ-polyglutamic acid aqueous solution into a polytetrafluoroethylene-lined reactor, and place the reactor into a multi-purpose microwave chemical synthesizer to react for a period of time; after the instrument cools down, turn off the instrument, take out the reactor, and the resulting product is low molecular weight γ-polyglutamic acid.
2. The method according to claim 1, characterized in that The γ-polyglutamic acid is a γ-polyglutamic acid hydrogen type.
3. The method according to claim 1, characterized in that The concentration of the γ-polyglutamic acid aqueous solution is 1-10 g / L.
4. The method according to claim 1, characterized in that: The weight average molecular weight M of the γ-polyglutamic acid w 95.2~436.3KDa, number average molecular weight M n The molecular weight distribution coefficient PDI is 55.1~186.1KDa and 1.73~2.
34.
5. The method according to claim 1, characterized in that The reaction is carried out in a microwave chemical synthesizer, with a heating rate set at 10°C / min, the temperature is raised to 100-140°C, and the reaction time is 5-30min.
6. The method according to claim 5, characterized in that The reaction was carried out in a microwave chemical synthesizer with a heating rate set at 10°C / min, the temperature was raised to 120°C, and the reaction time was 30min.
7. A low molecular weight γ-polyglutamic acid prepared by the method according to any one of claims 1 to 6.
8. The low molecular weight γ-polyglutamic acid according to claim 7, characterized in that The weight average molecular weight M of the low molecular weight γ-polyglutamic acid w The number average molecular weight is 14.3~16.4KDa. n It is 10.7~12.6KDa, and the molecular weight distribution coefficient PDI is 1.31~1.36.