A process for producing polyglutamic acid by fermentation
By optimizing the fermentation medium and conditions, especially the addition of D-phenylalanine and ferric chloride, the problem of low fermentation of polyglutamic acid in dependent strains was solved, and a significant yield improvement was achieved, and the existing Problems of output limitations in technology.
Patent Information
- Application Number
- CN202411364970.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2044-09-29
AI Technical Summary
The prior art is difficult to effectively increase the fermentation yield of polyglutamate, especially among dependent strains, resulting in the market mainly relying on the production restrictions of L-glutamate-dependent strains.
By optimizing the composition and conditions of the fermentation medium, especially the addition of D-phenylalanine and ferrous chloride, combined with appropriate pH and temperature control, the fermentation capacity of Bacillus subtilis PGA2020-10 is enhanced and the polyglutamic acid Synthesis and secretion.
It has achieved a significant increase in the yield of polyglutamic acid fermentation. Both D-phenylalanine or ferric chloride alone have a promotion effect. The combined use of the two has a synergistic effect, significantly increasing the yield, reaching 10.3%. Increase the amount.
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Figure CN118879802B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a process for producing polyglutamic acid by fermentation. Background Art
[0002] Poly-γ-glutamic acid (γ-PGA) is a natural polyamino acid found in some specific microorganisms. It is a multifunctional peptide formed by the polymerization of glutamic acid through γ-amide bonds. It can be prepared on a large scale through microbial fermentation. The carboxyl group of γ-PGA can be combined with the hydroxyl group. It is safe and non-toxic. It has excellent water solubility, moisturizing, adsorption, sustained release, antifreeze and other biological properties. It can be used as a moisturizer, flocculant, freeze-drying protective agent, drug carrier, sustained release agent and medical dressing in many fields, generating huge social benefits and commercial application value. γ-PGA has strong water solubility, biodegradability, biocompatibility, safety, non-toxicity, edible and environmentally friendly biological characteristics. It is widely used in agriculture, environmental protection, medicine, food, industry and cosmetics. It is an ideal new biomaterial with great application prospects and economic value. In recent years, it has attracted more and more attention from domestic and foreign researchers.
[0003] The strains that produce γ-PGA are divided into two categories according to the demand for L-glutamate in biopolymer production: L-glutamate-dependent and independent strains. L-glutamate-dependent strains can biosynthesize γ-PGA at a high level by adding the precursor L-glutamate to their culture medium. Since the yield of γ-PGA by independent strains is not high, L-glutamate-dependent strains are mainly used in the current market for γ-PGA production. On the premise of obtaining high-yield strains, optimizing the fermentation medium (such as carbon source, nitrogen source, inorganic salts, etc.), optimizing the fermentation conditions (such as temperature, pH value, dissolved oxygen, etc.), and selecting the appropriate fermentation method (such as batch fermentation, continuous fermentation, batch fed fermentation, etc.) are important means to increase the yield of γ-PGA.
[0004] The Chinese patent "CN110669798A, a method for producing low-cost polyglutamic acid for agricultural use" targets a specific Bacillus subtilis CGMCC No.2108. By adding chitosan and sodium chloride to the fermentation medium, the permeability of the cell wall is changed, polyglutamic acid is secreted outside the cell, and the yield of polyglutamic acid is increased.
[0005] The Chinese patent "CN116144532A, a method for efficiently preparing γ-polyglutamic acid for agricultural use" uses ultraviolet mutagenesis technology for mutagenesis. After 24 generations of mutagenesis, the mutant strain Bacillus subtilis PGA2020-10 is obtained. The fermentation yield is increased by 16% compared with the starting strain, and glycerol can be efficiently used to ferment polyglutamic acid.
[0006] The literature "Effect of Additives on the Synthesis and Metabolism of γ-Polyglutamate in Bacillus subtilis, Bioprocess Engineering 2010" studied the metabolic network and fermentation characteristics of Bacillus subtilis HCUL-B115, and studied the synthesis and metabolism of poly-γ-glutamate fermentation by adding appropriate amounts of amino acids, organic acids and vitamins. The results showed that additives such as L-glutamic acid, L-phenylalanine, L-arginine, L-aspartic acid, L-valine, fumaric acid, oxalic acid, malonic acid, nicotinic acid and ascorbic acid significantly promoted the synthesis of poly-γ-glutamic acid by strain HCUL-B115. This strain is non-L-glutamic acid dependent and can synthesize glutamate in large quantities. By adding L-valine and L-phenylalanine, the metabolic flow is directed to the direction of pyruvate, so that more pyruvate enters the TCA cycle in the cell, increasing the accumulation of L-glutamic acid, thereby increasing the synthesis of polyglutamic acid.
[0007] There is relatively little research on the fermentation of polyglutamic acid, and how to increase the acid production of polyglutamic acid during fermentation is a technical issue that needs to be continuously explored. Summary of the invention
[0008] On the basis of CN116144532A, the fermentation capacity of PGA2020-10 is further studied and optimized to increase the fermentation yield of polyglutamic acid.
[0009] The present invention is achieved through the following technical solutions.
[0010] A process for producing polyglutamic acid by fermentation, comprising the following steps:
[0011] The secondary seed liquid of Bacillus subtilis is transferred into the fermentation medium at an inoculum amount of 1-5%, and the reducing sugar concentration is controlled to be not less than 0.5% by feeding glucose solution, and the pH value is adjusted to 7.0 by feeding ammonia water, and the culture time is 36 hours to obtain polyglutamic acid fermentation liquid;
[0012] The fermentation medium comprises the following components: 40 g / L glucose, 20 g / L corn steep liquor, 10 g / L yeast extract, 50 g / L sodium glutamate, 6 g / L ammonium sulfate, 5 g / L glycerol, 4 g / L D-phenylalanine, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 1 mg / L ferrous sulfate heptahydrate, and the solvent is water.
[0013] Preferably, the components of the fermentation medium are: 40 g / L glucose, 20 g / L corn steep liquor, 10 g / L yeast extract, 50 g / L sodium glutamate, 40 g / L sodium chloride, 6 g / L ammonium sulfate, 5 g / L glycerol, 4 g / L D-phenylalanine, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 1 mg / L ferrous sulfate heptahydrate, and the solvent is water.
[0014] Preferably, the culture conditions are: pH 7.0, temperature 37° C., pressure 0.05 MPa, and dissolved oxygen controlled at 10%.
[0015] Furthermore, the preparation method of the Bacillus subtilis secondary seed solution comprises:
[0016] 1) Activate Bacillus subtilis PGA2020-10 (CGMCC No. 23581), inoculate it into the slant seed culture medium, and culture it in a constant temperature box at 37°C for 48 hours;
[0017] 2) Under sterile conditions, inoculate the slant strain into the primary seed culture medium and culture at 37°C for 24 hours to obtain the primary seed solution;
[0018] 3) Inoculate the primary seed solution into the secondary seed culture medium at a 12% inoculum and culture at 37°C with shaking until the OD 600 The value is 4, and the secondary seed solution is obtained.
[0019] Specifically, the Bacillus subtilis is Bacillus subtilis PGA2020-10.
[0020] Preferably, the slant seed culture medium components are: peptone 10 g / L, yeast extract 2 g / L, glucose 5 g / L, sodium chloride 5 g / L, agar 20 g / L, potassium dihydrogen phosphate 1 g / L, and the pH is controlled at 7.0.
[0021] Preferably, the components of the primary seed culture medium and the secondary seed culture medium are: 30 g / L glucose, 10 g / L corn steep liquor, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L dipotassium hydrogen phosphate; and the pH is controlled at 7.0.
[0022] The beneficial effects achieved by the present invention mainly include the following aspects:
[0023] The synthesis and secretion of polyglutamic acid are affected by many factors such as cell permeability and efflux. The present invention studies the effects of three components, D-phenylalanine, sodium chloride and ferric chloride, on the fermentation capacity of PGA2020-10, and finds that D-phenylalanine and ferric chloride can improve the ability of PGA2020-10 to synthesize polyglutamic acid, while sodium chloride has a smaller effect. The use of D-phenylalanine in combination with sodium chloride is significantly better than using D-phenylalanine alone. The two have a certain synergistic effect. The combined use of D-phenylalanine and ferric chloride cannot achieve a mutually reinforcing technical effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 :Effect of D-phenylalanine content in culture medium on polyglutamic acid fermentation yield;
[0025] Figure 2 :Effect of sodium chloride content in culture medium on polyglutamic acid fermentation yield;
[0026] Figure 3 :Effect of ferric chloride content in culture medium on polyglutamic acid fermentation yield;
[0027] Figure 4 : The combined effects of D-phenylalanine and sodium chloride on polyglutamic acid;
[0028] Figure 5 : Effects of D-phenylalanine and ferric chloride on polyglutamic acid. DETAILED DESCRIPTION
[0029] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.
[0030] Example 1
[0031] A process for producing polyglutamic acid by fermentation, comprising the following steps:
[0032] Bacillus subtilis PGA2020-10 (CGMCC No. 23581) was activated, inoculated into the slant seed culture medium, and cultured in a constant temperature incubator at 37°C for 48 h;
[0033] Under sterile conditions, the slant strain was inoculated into the primary seed culture medium and cultured at 37°C for 24 h to obtain the primary seed solution;
[0034] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 12%, and cultured at 37°C with shaking until the OD 600 The value is 4, and the secondary seed solution is obtained;
[0035] The components of the slant seed culture medium are: peptone 10g / L, yeast extract 2g / L, glucose 5g / L, sodium chloride 5g / L, agar 20g / L, potassium dihydrogen phosphate 1g / L, pH controlled at 7.0
[0036] The components of the primary seed culture medium and the secondary seed culture medium are: 30 g / L glucose, 10 g / L corn steep liquor, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L dipotassium hydrogen phosphate; the pH is controlled at 7.0.
[0037] The secondary seed liquid of Bacillus subtilis was transferred into the fermentation medium at an inoculum amount of 3%, and the pH value was 7.0, the temperature was 37°C, the pressure was 0.05MPa, the dissolved oxygen was controlled at 10%, the reducing sugar concentration was controlled to be not less than 0.5% by feeding 60% glucose solution, the pH value was adjusted to 7.0 by feeding ammonia water, and the culture time was 36h to obtain the γ-polyglutamic acid fermentation liquid;
[0038] The components of the fermentation medium are: 40 g / L glucose, 20 g / L corn steep liquor, 10 g / L yeast extract, 50 g / L sodium glutamate, 6 g / L ammonium sulfate, 5 g / L glycerol, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 1 mg / L ferrous sulfate heptahydrate.
[0039] Example 2
[0040] 1. Effect of D-phenylalanine on the production of polyglutamic acid by Bacillus subtilis PGA2020-10.
[0041] A certain concentration of D-phenylalanine was added to the fermentation medium, and the concentration gradient was set to 0, 0.5, 1, 2, 4, 8, 16, and the unit was g / L. The fermentation process was the same as in Example 1. Figure 1 As shown in the results, the addition concentration of D-phenylalanine was positively correlated with the yield of polyglutamic acid fermentation. When the addition concentration of D-phenylalanine reached 4g / L, the increase of polyglutamic acid slowed down. Considering the relationship between polyglutamic acid yield and D-phenylalanine cost, the addition amount of D-phenylalanine was selected as 4g / L. Under this concentration condition, the amount of polyglutamic acid produced by Bacillus subtilis PGA2020-10 fermentation increased by 10.3% compared with the group without addition. The synthesis and secretion of polyglutamic acid are affected by many factors such as cell permeability and efflux. D-phenylalanine may reshape the cell wall of Bacillus subtilis PGA2020-10 and inhibit the synthesis of cell membrane to a certain extent, which affects the efflux mechanism of cell membrane and promotes the efflux of cells, thereby increasing the fermentation amount of polyglutamic acid.
[0042] The applicant also studied the replacement of D-phenylalanine with L-phenylalanine in the culture medium and found that L-phenylalanine did not change the fermentation amount of polyglutamic acid, and there was no obvious change in the group with or without L-phenylalanine. The possible reason is that the metabolic flux of Bacillus subtilis PGA2020-10 for synthesizing polyglutamic acid has reached an optimal state, and regulating polyglutamic acid synthesis by adjusting the metabolic flux has no obvious effect. It may also be that Bacillus subtilis PGA2020-10 is a glutamate-dependent fermentation strain, which mainly relies on exogenous glutamate and synthesizes relatively little endogenous glutamate. It can be seen that the regulatory mechanisms of D-phenylalanine and L-phenylalanine on the synthesis of polyglutamic acid by Bacillus subtilis PGA2020-10 are not the same.
[0043] 2. The effect of high salt conditions on the fermentation production of polyglutamic acid by Bacillus subtilis PGA2020-10.
[0044] A certain concentration of sodium chloride was added to the fermentation medium, and the concentration gradient was set to 0, 5, 10, 20, 30, 40, 50, in g / L. The fermentation process was referred to Example 1. Figure 2 As shown, sodium chloride has little effect on the fermentation of polyglutamic acid produced by Bacillus subtilis PGA2020-10. There are differences between the above research results and existing literature records. For example, CN102533885A records the addition of 3-5% sodium chloride during the fermentation process, which can increase the amount of polyglutamic acid produced by Bacillus natto fermentation by more than 10%, because sodium chloride, as an osmotic pressure regulator, can change the permeability of the cell membrane, thereby leading to an increase in the secretion of polyglutamic acid. The possible reason for the above differences is that the cell membrane composition of different species of cell sources and the degree of tolerance to the environment are not the same, so a substance that affects one cell may not necessarily affect another cell. Therefore, it is necessary to select suitable chemical reagents for different specific strains.
[0045] 3. Effect of trivalent iron ions on the fermentation production of polyglutamic acid by Bacillus subtilis PGA2020-10.
[0046] On the basis of Example 1, a certain concentration of ferric chloride was added to the fermentation medium, and the concentration gradient was set to 0, 10, 20, 40, 60, 80, 100, in mg / L. The fermentation process was the same as in Example 1. Figure 3 As shown in the results, the addition of ferric chloride in the range of 0-60 mg / L has a certain promoting effect on the fermentation of polyglutamic acid by Bacillus subtilis PGA2020-10. It may be that the addition of trivalent iron ions increases the permeability of cells or increases the activity of enzymes related to polyglutamic acid synthesis. Among them, when the addition amount is 40 mg / L, it is 4% higher than that of the group without addition; but when the addition amount of ferric chloride is increased to 100 mg / L, it is reduced by 1.1% compared with the group without addition.
[0047] 4. Effect of D-phenylalanine combined with sodium chloride on the fermentation production of polyglutamic acid by Bacillus subtilis PGA2020-10.
[0048] Based on the above research, it was determined that the optimal addition amount of D-phenylalanine was 4 g / L, and the fermentation process was referred to Example 1. Since the yield of polyglutamic acid fermentation had declined to a certain extent under the condition of 50 g / L sodium chloride concentration, 40 g / L concentration was selected and used in combination with 4 g / L D-phenylalanine. Figure 4 As shown in the figure, although sodium chloride has little effect on polyglutamic acid fermentation yield when used alone, when used in combination with D-phenylalanine, it increases by 3.8g / L compared with the use of D-phenylalanine alone, and increases by 8.4g / L compared with the control group (neither D-phenylalanine nor sodium chloride is added). It can be seen that the use of D-phenylalanine combined with sodium chloride is significantly better than the use of D-phenylalanine alone, and the two have a certain synergistic effect. The use of D-phenylalanine combined with sodium chloride is beneficial to the synthesis and accumulation of polyglutamic acid, and the specific mechanism still needs to be further verified in the later stage.
[0049] 5. Effect of D-phenylalanine combined with ferric chloride on the fermentation production of polyglutamic acid by Bacillus subtilis PGA2020-10.
[0050] The optimal addition amount of D-phenylalanine was selected to be 4 g / L, the concentration of ferric chloride was 40 mg / L, and the fermentation process was referred to Example 1. Figure 5 As shown, compared with the control group (without adding D-phenylalanine and ferric chloride), the D-phenylalanine group increased by 4.6g / L, the ferric chloride group increased by 1.8g / L, and the D-phenylalanine + ferric chloride group increased by 5g / L. The combined use of D-phenylalanine and ferric chloride was only slightly better than the D-phenylalanine group alone, and no mutually reinforcing effect was achieved.
[0051] Example 3
[0052] A process for producing polyglutamic acid by fermentation, comprising the following steps:
[0053] Bacillus subtilis PGA2020-10 (CGMCC No. 23581) was activated, inoculated into the slant seed culture medium, and cultured in a constant temperature incubator at 37°C for 48 h;
[0054] Under sterile conditions, the slant strain was inoculated into the primary seed culture medium and cultured at 37°C for 24 h to obtain the primary seed solution;
[0055] The first-level seed solution was inoculated into the second-level seed culture medium at a rate of 12%, and cultured at 37°C with shaking until the OD 600 The value is 4, and the secondary seed solution is obtained;
[0056] The components of the slant seed culture medium are: peptone 10g / L, yeast extract 2g / L, glucose 5g / L, sodium chloride 5g / L, agar 20g / L, potassium dihydrogen phosphate 1g / L, and the pH is controlled at 7.0;
[0057] The components of the primary seed culture medium and the secondary seed culture medium are: 30 g / L glucose, 10 g / L corn steep liquor, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L dipotassium hydrogen phosphate; the pH is controlled at 7.0.
[0058] The secondary seed liquid of Bacillus subtilis was transferred into the fermentation medium at an inoculum amount of 3%, and the pH value was 7.0, the temperature was 37°C, the pressure was 0.05MPa, the dissolved oxygen was controlled at 10%, the reducing sugar concentration was controlled to be not less than 0.5% by feeding 60% glucose solution, the pH value was adjusted to 7.0 by feeding ammonia water, and the culture time was 36h to obtain the γ-polyglutamic acid fermentation liquid;
[0059] The components of the fermentation medium are: 40 g / L glucose, 20 g / L corn steep liquor, 10 g / L yeast extract, 50 g / L sodium glutamate, 40 g / L sodium chloride, 6 g / L ammonium sulfate, 5 g / L glycerol, 4 g / L D-phenylalanine, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 1 mg / L ferrous sulfate heptahydrate.
[0060] Example 4
[0061] In the process of preparing glutamic acid by biological fermentation, the extraction waste liquid is a high-concentration wastewater that is difficult to treat and has a high cost for wastewater treatment. It still contains a certain amount of glutamic acid, protein, sugar, ammonium sulfate and other nutrients, which can be used as an important raw material for the production of polyglutamic acid. In line with the concept of improving economic benefits and reducing sewage treatment and discharge, the use of glutamic acid extraction waste liquid to produce γ-polyglutamic acid not only reduces the production cost of γ-PGA, but also creates economic benefits and has the comprehensive benefits of utilizing glutamic acid extraction wastewater.
[0062] Taking the extracted wastewater from the glutamic acid production workshop as an example, the glutamic acid content is 3.6g / L.
[0063] During the preparation of the fermentation medium, glutamate extraction wastewater was used to replace the culture medium water, and then the glutamate content was supplemented to 50 g / L, and the other nutrient components were also supplemented as in Example 1. The fermentation process was the same as in Example 1, and the final yield of fermented polyglutamic acid was only 4.7% lower than that in Example 1. Considering the comprehensive cost, using glutamate extraction wastewater to replace the culture medium water can bring certain economic benefits.
[0064] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.
Claims
1. A process for producing polyglutamic acid by fermentation, characterized in that: The process comprises the following steps: The secondary seed liquid of Bacillus subtilis is transferred into the fermentation medium at an inoculum amount of 1-5%, and the reducing sugar concentration is controlled to be not less than 0.5% by feeding glucose solution, and the pH value is adjusted to 7.0 by feeding ammonia water, and the culture time is 36 hours to obtain polyglutamic acid fermentation liquid; The components of the fermentation medium are: 40 g / L glucose, 20 g / L corn steep liquor, 10 g / L yeast extract, 50 g / L sodium glutamate, 40 g / L sodium chloride, 6 g / L ammonium sulfate, 5 g / L glycerol, 4 g / L D-phenylalanine, 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 1 mg / L ferrous sulfate heptahydrate, and the solvent is water; The Bacillus subtilis is Bacillus subtilis PGA2020-10, and its preservation number is CGMCC No.23581.
2. The process according to claim 1, characterized in that The culture conditions are: temperature 37° C., pressure 0.05 MPa, and dissolved oxygen controlled at 10%.
3. The process according to claim 1, characterized in that The preparation method of the Bacillus subtilis secondary seed liquid comprises: 1) Activate Bacillus subtilis, inoculate it into the slant seed culture medium, and culture it in a constant temperature box at 37°C for 48 hours; 2) Under sterile conditions, inoculate the slant strain into the primary seed culture medium and culture at 37°C for 24 hours to obtain the primary seed solution; 3) Inoculate the primary seed solution into the secondary seed culture medium at a 12% inoculum and culture at 37°C with shaking until the OD 600 The value is 4, and the secondary seed solution is obtained.
4. The process according to claim 3, characterized in that The slant seed culture medium comprises: 10 g / L peptone, 2 g / L yeast extract, 5 g / L glucose, 5 g / L sodium chloride, 20 g / L agar, and 1 g / L potassium dihydrogen phosphate, and the pH is controlled at 7.
0.
5. The process according to claim 3, characterized in that The components of the primary seed culture medium and the secondary seed culture medium are: 30 g / L glucose, 10 g / L corn steep liquor, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L dipotassium hydrogen phosphate; and the pH is controlled at 7.0.