Fermentation production process of L-serine
By optimizing the microbial fermentation medium and conditions, especially the addition of glycine and sodium pyruvate, the carbon metabolism flow direction is adjusted, and the problem of low L-serine production is achieved, which is suitable for industrial applications.
Patent Information
- Application Number
- CN202411666792.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-01-03
AI Technical Summary
The existing industrial production methods of L-serine have problems such as environmental pollution, high production costs, limited materials and low yields, especially chemical synthesis methods and proteolysis methods, which limit their large-scale production.
By optimizing the composition and conditions of microbial fermentation medium, especially the addition of glycine and sodium pyruvate, the carbon metabolism flow direction is adjusted, the fermentation yield of L-serine is improved, and the acid production capacity of the strain is enhanced by using specific fermentation temperature and stirring parameters.
It has achieved a significant increase in L-serine production, reduced production costs, was suitable for industrial production, and reduced environmental pollution and improved the market competitiveness of the products.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial fermentation, and specifically provides a fermentation production process of L-serine. Background Art
[0002] L-serine (L-serine, L-Ser) is also known as L-2-amino-3-hydroxypropionic acid. Its crystal structure is hexagonal flakes or prisms. It is white crystals or crystalline powder at room temperature. It is a member of the acidic amino acid family. At the same time, it is also a member of the neutral aliphatic amino acids. Its chemical formula is C3H7NO3, molecular weight is 105.09, isoelectric point is 5.67, and it is easily soluble in water and almost insoluble in non-polar solvents. L-serine is a neutral amino acid with important physiological functions and is widely used in food, cosmetics, medicine and other fields.
[0003] At present, the industrial production methods of L-serine mainly include protein hydrolysis, immobilized enzyme method, chemical synthesis method and precursor fermentation method. In the production process of chemically synthesized L-serine, a large amount of wastewater containing organic reagents will be discharged, which seriously pollutes the environment. Therefore, this production technology is rarely used in amino acid production. Since the production process of L-serine by protein hydrolysis is cumbersome, a large amount of water resources and chemical reagents such as hydrochloric acid need to be consumed in the process, and the production cost is high, this production method is gradually replaced by microbial fermentation in the market. The enzyme production cost is high, and the source of materials is limited. The enzyme activity in the production process is unstable, which greatly reduces the market competitiveness of the product, thereby limiting the large-scale production of L-serine. The production of L-homoserine by microbial fermentation has the advantages of low cost, low pollution, mild conditions and easy control, and is currently the most promising production method.
[0004] The yield of serine produced by direct fermentation of sugar raw materials is generally low, mainly because L-serine is an intermediate product of amino acid metabolism and participates in the synthesis of biological substances such as glycine, cysteine, and nucleic acid bases. Although about 15% of the carbon flow substances in the glycolysis pathway enter the serine synthesis pathway, it is difficult to accumulate directly in the body. Serine is converted into glycine by serine hydroxymethyltransferase and pyruvate by phosphoserine dehydrogenase to enter the TCA cycle.
[0005] There are relatively few studies on the fermentation of serine. How to increase the acid production of serine by fermentation is a technical problem that needs to be explored continuously. Patented technology CN1966704A separates and enriches L-serine from soil, and finally screens out Corynebacterium glutamicum, with the deposit number CGMCC NO.1843. After optimizing the culture medium and culture conditions, the acid production level of this strain can reach 6g / L. Summary of the invention
[0006] Changes in microbial culture medium components and culture conditions will cause changes in microbial anabolism, thereby directly or indirectly regulating the anabolism of intracellular L-Ser. The present invention specifically optimizes the fermentation medium and its culture conditions, and can improve the fermentation yield of serine.
[0007] The present invention is achieved through the following technical solutions.
[0008] A fermentation production process for L-serine comprises the following steps: The inoculum of Corynebacterium glutamicum was inoculated into a fermentation tank containing fermentation medium at an inoculum rate of 5-15% for fermentation culture. The fermentation culture temperature was 38-39°C, the tank pressure was 0.05Mpa, the stirring speed was 1000rpm, and the fermentation time was 48h.
[0009] Preferably, the fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
[0010] Preferably, the fermentation culture temperature is 39°C.
[0011] Preferably, the preservation number of the Corynebacterium glutamicum is CGMCC No.18932.
[0012] More preferably, the fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 4 g / L glycine, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
[0013] More preferably, the fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 8 g / L sodium pyruvate, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
[0014] Most preferably, the fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 8 g / L sodium pyruvate, 4 g / L glycine, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1 g / L, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
[0015] Preferably, the method for preparing the seed solution comprises: activating Bacillus subtilis and inoculating it into a primary seed culture medium to obtain a primary seed solution; and then transferring it into a secondary seed culture medium for culturing until the OD600 reaches about 5 to obtain a Bacillus subtilis inoculum solution.
[0016] Preferably, the components of the seed culture medium are: 20 g / L glucose, 5 g / L yeast extract, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 mg / L ferrous sulfate heptahydrate, and 5 mg / L manganese sulfate monohydrate.
[0017] Preferably, the components of the secondary culture medium are: 25 g / L glucose, 6 g / L corn steep liquor powder, 3 g / L yeast extract, 3 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.6 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.1 mg / L ferrous sulfate heptahydrate, and 10 mg / L manganese sulfate monohydrate.
[0018] The beneficial effects achieved by the present invention mainly include but are not limited to the following: The regulation of regulatory factors is a key link in the regulation of microbial metabolism. Different regulatory factors can change the size of specific enzyme activity, thereby redistributing the flow of carbon skeleton substances in the cell. Therefore, regulating carbon metabolism by regulatory factors is a very effective means of operation to increase the target product. The present invention adds glycine and sodium pyruvate to the fermentation medium, adjusts the direction of the carbon metabolism flow, reduces the metabolism of serine, and increases the accumulation of serine. The fermentation formula and temperature process control parameters of the present invention are adopted to achieve a substantial increase in the output of L-serine without adding additional equipment and manpower input, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 :The effect of fermentation temperature on L-serine fermentation yield; Figure 2 :Effect of glycine concentration in fermentation medium on L-serine fermentation yield; Figure 3 :Effect of sodium pyruvate concentration in fermentation medium on L-serine fermentation yield. DETAILED DESCRIPTION
[0020] 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. Example
[0021] The applicant's previous patent technology CN112812985A screened out a high-glutamine-producing Corynebacterium glutamicum VJ1911-02, whose preservation number is CGMCC No.18932; the strain can utilize methanol and use methanol to replace part of the sugar, and has a stronger ability to produce glutamine.
[0022] The applicant continued to study the performance of Corynebacterium glutamicum VJ1911-02 in fermentation to produce serine.
[0023] The Corynebacterium glutamicum VJ1911-02 was activated and then inoculated into LB slant medium and cultured in the dark at 30°C for 12 h. The colonies were then removed and transferred to a culture bottle containing a primary seed culture medium for shake flask seed culture. The rotation speed was controlled at 180 r / min, the temperature was 35°C, and the culture was carried out for 48 h to obtain a primary seed culture solution.
[0024] Seed culture medium 1: sucrose 20g / L, yeast extract 5g / L, ammonium sulfate 5g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 20mg / L, manganese sulfate monohydrate 5mg / L; Seed culture medium 2: glucose 20 g / L, yeast extract 5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 5 mg / L; Seed culture medium 3: sucrose 15 g / L, methanol 5 g / L, yeast extract 5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 5 mg / L; Seed culture medium 4: glucose 15 g / L, methanol 5 g / L, yeast extract 5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 5 mg / L.
[0025] Culture medium 1-4 was used for culture, and the contents of glutamine and serine in the culture medium were detected. See Table 1 for details.
[0026] Table 1 Medium components Medium 1 Medium 2 Medium 3 Medium 4 Glutamine g / L 17.2 13.9 19.8 18.5 Serine g / L 8.5 10.6 7.0 7.9 As shown in Table 1, the serine yield was the highest when glucose was used as the fermentation carbon source, so glucose was used as the fermentation carbon source in subsequent experiments. Example
[0027] Serine was produced by fermentation of Corynebacterium glutamicum VJ1911-02.
[0028] The first-level seed solution prepared in Example 1 was transferred to the second-level expanded seed culture medium at a 10% inoculation rate and cultured until the OD600 reached about 5 to obtain the second-level seed solution. The culture temperature was 35° C. and the pH was controlled at 7.0 throughout the process. The components of the expansion medium are: glucose 25g / L, corn steep liquor powder 6g / L, yeast extract 3g / L, ammonium sulfate 3g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, citric acid 0.6g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.1mg / L, and manganese sulfate monohydrate 10mg / L.
[0029] The secondary seed liquid was inoculated at 12% into a fermentation tank containing a fermentation medium for fermentation culture at a culture temperature of 35°C, a tank pressure of 0.05Mpa, a pH of the fermentation tank of 6.9 throughout the process, a stirring speed of 1000rpm, and a fermentation time of 48h. The residual sugar was controlled to be no less than 0.1% by adding 60% glucose by mass fraction, and the pH was controlled to be 6.9 by adding ammonia water.
[0030] The fermentation medium components are: glucose 30 g / L, molasses 30 g / L, corn steep liquor powder 36 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, citric acid 1.2 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.3 mg / L, manganese sulfate monohydrate 50 mg / L, VH 5 mg / L, tetracycline 3 mg / L.
[0031] Under the above L-serine fermentation medium and fermentation control conditions, the final fermentation acid production of L-serine reached 45.8 g / L, and the sugar-acid conversion rate was 16.3%. Example
[0032] 1. Effect of temperature on the fermentation of serine by Corynebacterium glutamicum VJ1911-02.
[0033] Different fermentation temperatures were set, and the fermentation process was the same as in Example 2. The temperatures were set to 35, 36, 37, 38, 39, and 40, with a total of 6 temperature gradients. Figure 1 As shown in the figure, with the increase of temperature, the production of serine increased rapidly. When the fermentation temperature reached 39°C, the fermentation production of serine reached its peak. Under this temperature condition, the activity of related enzymes such as serine hydroxymethyltransferase or phosphoserine dehydrogenase may be reduced, resulting in a decrease in the consumption of serine as an intermediate metabolite, which is beneficial to the accumulation of serine. If the temperature continues to increase, the production of serine will decrease. Too high a temperature is not conducive to the proliferation of the strain and its vitality is also damaged.
[0034] 2. Select the fermentation temperature as 39°C to verify the effect of adding regulatory factors to the fermentation medium on the serine fermentation yield. The regulation of regulatory factors is a key link in the regulation of microbial metabolism. Different types and levels of regulatory factors can change the flow of carbon skeleton substances in cells. Therefore, regulating carbon metabolism through regulatory factors is a very effective means of increasing the target product.
[0035] On the basis of the fermentation medium of Example 2, a certain concentration of glycine or sodium pyruvate was added to study the regulatory effect on serine synthesis. The fermentation temperature was selected to be 39° C., and the rest of the fermentation process was the same as in Example 2.
[0036] 1) Based on the fermentation medium of Example 2, the amount of glycine added was 0, 1, 2, 4, 8, 16, in g / L, with a total of six concentration gradients, such as Figure 2 As shown, the yield of serine is directly proportional to the concentration of glycine. As the concentration of glycine increases, the increase in serine content slows down. Considering the cost and output comprehensively, the addition amount of 4g / L glycine is the most appropriate.
[0037] 2) Based on the fermentation medium of Example 2, the addition amount of sodium pyruvate was also set to 0, 1, 2, 4, 8, 16, in g / L, with a total of six concentration gradients, such as Figure 3 As shown in the figure, the addition of low concentration of sodium pyruvate has little effect on the fermentation yield of serine. When the addition amount of sodium pyruvate reaches 8g / L, the yield of serine increases slightly, about 3% higher than that without addition. When the addition amount of sodium pyruvate continues to increase to 16g / L, the yield of serine does not change significantly.
[0038] 3) Effect of selecting 4g / L glycine + 8g / L sodium pyruvate on the serine fermentation yield. The control group was a fermentation medium without glycine and sodium pyruvate (the same fermentation medium as in Example 2). See Table 2 below for details.
[0039] Table 2 Medium components Serine yield g / L Compared with the control group, the increase in g / L Control group 50.4 —— 4g / L Glycine 58.9 8.5 8g / L Sodium pyruvate 52.1 1.7 4g / L glycine + 8g / L sodium pyruvate 65.7 15.3 As shown in Table 2, 4 g / L glycine can increase the yield of serine, sodium pyruvate has little effect on the yield of serine, and the combined use of 4 g / L glycine + 8 g / L sodium pyruvate can greatly increase the fermentation yield of serine.
[0040] Although the above-mentioned embodiments are combined with the examples to introduce the specific implementation of the present invention, they are not intended to limit the protection scope of the present invention. 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 are within the protection scope of the present invention.
Claims
1. A fermentation production process for L-serine, comprising the following steps: The inoculum of Corynebacterium glutamicum was inoculated into a fermentation tank containing fermentation medium at an inoculum rate of 5-15% for fermentation culture. The fermentation culture temperature was 38-39°C, the tank pressure was 0.05Mpa, the stirring speed was 1000rpm, and the fermentation time was 48h.
2. The process according to claim 1, characterized in that The fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
3. The process according to claim 1, characterized in that The temperature of the fermentation culture was 39°C.
4. The process according to claim 1, characterized in that The preservation number of the Corynebacterium glutamicum is CGMCC No.18932.
5. The process according to claim 1, characterized in that The fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 4 g / L glycine, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
6. The process according to claim 1, characterized in that The fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 8 g / L sodium pyruvate, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
7. The process according to claim 1, characterized in that The fermentation medium components are: 30 g / L glucose, 30 g / L molasses, 36 g / L corn steep liquor powder, 8 g / L sodium pyruvate, 4 g / L glycine, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 1.2 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.3 mg / L ferrous sulfate heptahydrate, 50 mg / L manganese sulfate monohydrate, 5 mg / L VH, and 3 mg / L tetracycline.
8. The process according to claim 1, characterized in that The preparation method of the Corynebacterium glutamicum inoculation liquid comprises: activating Bacillus subtilis, inoculating into a primary seed culture medium to obtain a primary seed liquid; and then transferring into a secondary seed culture medium for culturing until the OD600 reaches about 5 to obtain the Bacillus subtilis inoculation liquid.
9. The process according to claim 8, characterized in that The components of the primary seed culture medium are: 20 g / L glucose, 5 g / L yeast extract, 5 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 mg / L ferrous sulfate heptahydrate, and 5 mg / L manganese sulfate monohydrate.
10. The process according to claim 8, characterized in that The components of the secondary culture medium are: 25 g / L glucose, 6 g / L corn steep liquor powder, 3 g / L yeast extract, 3 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.6 g / L citric acid, 0.5 g / L magnesium sulfate heptahydrate, 0.1 mg / L ferrous sulfate heptahydrate, and 10 mg / L manganese sulfate monohydrate.