A method for extracting l-serine using chromatographic techniques

Abstract

This invention belongs to the field of microbial fermentation technology and provides a method for extracting L-serine using chromatographic technology, comprising the following steps: Step 1) fermentation broth pretreatment; Step 2) ceramic membrane filtration; Step 3) activated carbon decolorization; Step 4) chromatographic separation; Step 5) secondary decolorization; Step 6) evaporation crystallization; and Step 7) centrifugal drying. This invention does not use any chemicals in the production process, does not involve ion exchange, uses only water as the eluent, and generates no pollution, making it green and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and specifically provides a method for extracting L-serine using chromatographic technology. Background Technology

[0002] Current research on L-serine separation and extraction methods mainly includes paper chromatography, ion exchange, isoelectric potential precipitation, and membrane separation. Paper chromatography is primarily used for qualitative and quantitative analysis of simple mixtures of amino acids, but it is not effective for extracting individual components. Ion exchange separation technology offers easy adjustment of operating conditions, lower cost, and high throughput. Isoelectric potential precipitation has been less reported and is mainly used for amino acids with significantly different isoelectric points. Membrane separation is a relatively new method with advantages such as good separation efficiency and high yield. Currently, the separation and extraction of L-serine both domestically and internationally almost exclusively uses ion exchange column separation. While this method allows for large-scale production, it results in high product costs, large consumption of exchange resin, and excessive consumption of elution and regeneration acids and alkalis, causing serious environmental pollution. Summary of the Invention

[0003] The applicant's previous research, CN2024116667926, improved the fermentation yield of serine by specifically optimizing the fermentation medium and its culture conditions. Building on this foundation, further research was conducted on the isolation and purification of L-serine.

[0004] The present invention is achieved through the following technical solution.

[0005] A method for producing L-serine includes the following steps: step 1) fermentation broth pretreatment, step 2) ceramic membrane filtration, step 3) activated carbon decolorization, step 4) chromatographic separation, step 5) secondary decolorization, step 6) evaporation crystallization, and step 7) centrifugal drying.

[0006] Specifically, the method includes the following steps: Step 1) Fermentation broth pretreatment: Adjust the pH of the serine fermentation broth to 5.0-5.5 with sulfuric acid, and add polyaluminum chloride to flocculate the bacterial protein; Step 2) Ceramic membrane filtration: The pretreated serine fermentation broth from step 1) is passed through a ceramic membrane with a pore size of 50 nm to obtain ceramic membrane filtrate; Step 3) Activated carbon decolorization: Add activated carbon to the ceramic membrane filtrate obtained in step 2) for decolorization; filter to remove the activated carbon, and obtain a decolorized solution. Step 4) Chromatographic separation: The L-serine decolorized solution obtained in step 3) is introduced into the chromatographic column through a valve for adsorption. The feed amount is 20% of the resin volume, and the column flow rate is 0.4-0.6 BV / h. After the resin is saturated, it is washed with distilled water at a flow rate of 0.4-0.6 BV / h. Every 5 minutes, a sample is taken at the feed port and L-serine is detected by the ninhydrin colorimetric method. When a colorimetric reaction occurs, the L-serine aqueous solution is collected until no colorimetric reaction occurs and then the collection is stopped. Step 5) Secondary decolorization: Add activated carbon to the L-serine aqueous solution obtained in step 4) for decolorization, filter out the activated carbon, and obtain the secondary decolorized solution; Step 6) Evaporation and crystallization: The L-serine secondary decolorization solution obtained in step 5) is concentrated at 60-70℃ until the L-serine content is 400-500g / L. Then, the temperature of the concentrated solution is lowered to room temperature to precipitate L-serine crystals. Step 7) Centrifugation and drying: Centrifuge the L-serine concentrate obtained in step 6) to obtain wet L-serine, and dry it to obtain the finished L-serine product.

[0007] Preferably, the parameters of the ceramic membrane filtration are: temperature 50-55℃, working pressure 1.0-1.5 bar. Preferably, in steps 3) and 5), the decolorization temperature is 60-70℃ and the time is 50-60min.

[0008] Preferably, in step 7), the drying temperature is 70-75℃ and the time is 6 hours.

[0009] Preferably, the serine fermentation broth is obtained according to the following process: The Corynebacterium glutamicum seed culture was inoculated into a fermenter containing fermentation medium at an inoculation rate of 5-15% for fermentation culture. The fermentation culture temperature was 39℃, the tank pressure was 0.05Mpa, the stirring speed was 1000rpm, and the fermentation time was 48h.

[0010] Preferably, the fermentation medium consists of: 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 vitamin H, and 3 mg / L tetracycline.

[0011] Preferably, the fermentation medium consists of: 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 vitamin H, and 3 mg / L tetracycline.

[0012] Preferably, the accession number of the *Corynebacterium glutamicum* is CGMCC No. 18932.

[0013] The beneficial effects achieved by this invention mainly include, but are not limited to, the following: This invention adds glycine and sodium pyruvate to the fermentation medium, regulating the direction of carbon metabolic flux, thereby reducing serine metabolism and increasing serine accumulation. Using the fermentation formula and temperature control parameters of this invention, a significant increase in L-serine yield is achieved without additional equipment or manpower, making it suitable for industrial production.

[0014] This invention employs chromatographic separation technology to separate L-serine fermentation broth, yielding the L-serine product. Compared with traditional ion exchange column separation methods, the yield is improved, and the separated L-serine has higher purity. This invention's production process does not use any chemicals, does not involve ion exchange, and uses only water as the eluent, resulting in no pollution and making it environmentally friendly. Attached Figure Description

[0015] Figure 1 The effect of fermentation temperature on L-serine fermentation yield; Figure 2 The 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 Implementation

[0016] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and methods of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. To further understand this invention, the following detailed description is provided in conjunction with embodiments. Example

[0017] The applicant's previous patent technology CN112812985A screened out a strain of Corynebacterium glutamicum VJ1911-02 that produces high levels of glutamine, with accession number CGMCC No.18932; this strain can utilize methanol and uses methanol to replace part of the sugar, thus having a stronger ability to produce glutamine.

[0018] The applicant continued to study the performance of Corynebacterium glutamicum VJ1911-02 in producing serine through fermentation.

[0019] Corynebacterium glutamicum VJ1911-02 was activated and then inoculated onto LB slant medium. It was incubated in the dark at 30°C for 12 hours. The colonies were then removed and transferred to a culture flask containing primary seed medium for shake-flask seed culture. The culture was carried out at 180 r / min and 35°C for 48 hours to obtain the primary seed culture solution.

[0020] 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 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 3: sucrose 15g / L, methanol 5g / 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 4: glucose 15g / L, methanol 5g / 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.

[0021] Cultures were prepared using culture media 1-4, and the contents of glutamine and serine in the culture medium were measured. See Table 1 for details.

[0022] Table 1 Culture medium components Culture medium 1 Culture medium 2 Culture medium 3 Culture 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 highest when glucose was used as the fermentation carbon source. Therefore, glucose was selected as the fermentation carbon source in subsequent experiments. Example

[0023] Serine was prepared by fermentation of Corynebacterium glutamicum VJ1911-02.

[0024] The primary seed culture prepared in Example 1 was transferred to the secondary expansion seed culture medium at an inoculation rate of 10% and cultured until the OD600 reached about 5 to obtain the secondary seed culture. The culture temperature was 35℃ and the pH was controlled at 7.0 throughout the process. The expanded culture medium consisted of: glucose 25 g / L, corn steep liquor powder 6 g / L, yeast extract 3 g / L, ammonium sulfate 3 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, citric acid 0.6 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.1 mg / L, and manganese sulfate monohydrate 10 mg / L.

[0025] The secondary seed culture was inoculated into a fermenter containing fermentation medium at an inoculation rate of 12% for fermentation culture. The culture temperature was 35℃, the pressure was 0.05 MPa, the pH of the fermenter was maintained at 6.9 throughout the process, the stirring speed was 1000 rpm, and the fermentation time was 48 h. The residual sugar was controlled to be no less than 0.1% by adding 60% glucose, and the pH was controlled to be 6.9 by adding ammonia.

[0026] The fermentation medium consisted of: 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, and tetracycline 3 mg / L.

[0027] Under the above-mentioned L-serine fermentation medium and fermentation control conditions, the final acid production of L-serine reached 45.8 g / L, and the sugar-acid conversion rate was 16.3%. Example

[0028] 1. Effect of temperature on the fermentation of *Corynebacterium glutamicum* VJ1911-02 to prepare serine.

[0029] Different fermentation temperatures were set, and the fermentation process was the same as in Example 2. The temperatures were set at 35, 36, 37, 38, 39, and 40 degrees Celsius, for a total of six temperature gradients. Figure 1 As shown, the production of serine increases rapidly with increasing temperature. When the fermentation temperature reaches 39℃, the fermentation production of serine reaches its peak. Under this temperature condition, the activity of related enzymes such as serine hydroxymethyltransferase or phosphoserine dehydrogenase may decrease, resulting in a reduction in the consumption of serine as an intermediate metabolite, which is conducive to the accumulation of serine. However, if the temperature is further increased, the production of serine decreases. Excessively high temperatures are not conducive to the proliferation of strains and their activity is also damaged.

[0030] 2. A fermentation temperature of 39℃ was selected to verify the effect of adding regulatory factors to the fermentation medium on serine fermentation yield. Regulation by regulatory factors is a key link in microbial metabolic regulation; different types and levels of regulatory factors can alter the flow of carbon skeleton materials within the cell. Therefore, regulating carbon metabolism through regulatory factors is a very effective method to increase the yield of the target product.

[0031] Based on the fermentation medium of Example 2, a certain concentration of glycine or sodium pyruvate was added to study its regulatory effect on serine synthesis. The fermentation temperature was selected as 39℃, and the rest of the fermentation process was the same as in Example 2.

[0032] 1) Based on the fermentation medium of Example 2, glycine was added at concentrations of 0, 1, 2, 4, 8, and 16 g / L, forming six concentration gradients. 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 both cost and output, the addition amount of 4 g / L glycine is the most suitable.

[0033] 2) Based on the fermentation medium of Example 2, the amount of sodium pyruvate added was also set to 0, 1, 2, 4, 8, 16 g / L, for a total of six concentration gradients, as follows: Figure 3 As shown, the addition of low concentrations of sodium pyruvate has little effect on the fermentation yield of serine. When the amount of sodium pyruvate added reaches 8 g / L, the yield of serine increases slightly, by about 3% compared to when no sodium pyruvate is added. When the amount of sodium pyruvate added is further increased to 16 g / L, the yield of serine does not change significantly.

[0034] 3) The effect of 4 g / L glycine + 8 g / L sodium pyruvate on serine fermentation yield. The control group was a fermentation medium without added glycine and sodium pyruvate (the same fermentation medium as in Example 2). See Table 2 below for details.

[0035] Table 2 Culture medium components Serine production g / L Increase in g / L compared to the control group 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, while sodium pyruvate has a relatively small effect on the yield of serine. The combined use of 4 g / L glycine and 8 g / L sodium pyruvate can significantly increase the fermentation yield of serine. Example

[0036] Based on Example 2, the optimal fermentation temperature of 39°C, optimized in Example 3, was selected, and 4 g / L glycine + 8 g / L sodium pyruvate were added to the fermentation medium. The remaining fermentation conditions were the same as in Example 2, yielding the fermentation broth. The fermentation broth was then separated and purified. The specific steps are as follows: Step 1) Fermentation broth pretreatment: Adjust the pH of the L-serine fermentation broth to 5.0 with sulfuric acid, and add 2‰ of polyaluminum chloride to flocculate the bacterial protein; Step 2) Ceramic membrane filtration: The L-serine fermentation broth pretreated in Step 1) is passed through a ceramic membrane with a pore size of 50 nm at a temperature of 50 °C and a working pressure of 1.2 bar. Step 3) Activated carbon decolorization: Add 0.9% (by mass) of activated carbon to the ceramic membrane filtrate obtained in step 2), decolorize at 65℃ for 50 min; filter out the activated carbon to obtain a decolorized solution. Step 4) Chromatographic separation: The L-serine decolorized solution obtained in step 3) is introduced into the chromatographic column through a valve for adsorption. The feed amount is 20% of the resin volume, and the column flow rate is 0.5 BV / h. After the resin is saturated, it is washed with distilled water at a flow rate of 0.5 BV / h. Every 5 minutes, samples are taken at the feed inlet and L-serine is detected by the ninhydrin colorimetric method. When a colorimetric reaction occurs, the L-serine aqueous solution is collected until no colorimetric reaction occurs and then the collection is stopped. Step 5) Secondary decolorization: Add 0.5% by weight of activated carbon to the L-serine aqueous solution obtained in step 4), decolorize at 65℃ for 50 min, filter out the activated carbon, and obtain the secondary decolorized solution; Step 6) Evaporation and crystallization: The L-serine secondary decolorization solution obtained in step 5) is concentrated at 65°C until the L-serine content is 400 g / L. Then, the temperature of the concentrated solution is lowered to room temperature to precipitate L-serine crystals. Step 7) Centrifugal drying: Centrifuge the L-serine concentrate obtained in step 6) to obtain wet L-serine, then dry it to obtain the finished L-serine product. Drying temperature: 70℃, time: 6h; Under these conditions, the content of L-serine reached 99.6%, and the extraction yield reached 76%. Example

[0037] The fermentation broth was obtained using the process described in Example 2, and then the broth was separated and purified. The specific steps are as follows: Step 1) Pretreatment of fermentation broth: Adjust the pH of the L-serine fermentation broth after fermentation to 5.2 with sulfuric acid, and add 2‰ of polyaluminum chloride to flocculate the bacterial protein; Step 2) Ceramic membrane filtration: The L-serine fermentation broth pretreated in Step 1) is passed through a ceramic membrane with a pore size of 50 nm at a temperature of 55 °C and a working pressure of 1.5 bar. Step 3) Activated carbon decolorization: Add 0.9% (by mass) of activated carbon to the ceramic membrane filtrate obtained in step 2), decolorize at 65℃ for 50 min; filter out the activated carbon to obtain a decolorized solution. Step 4) Chromatographic separation: The L-serine decolorized solution obtained in step 3) is introduced into the chromatographic column through a valve for adsorption. The feed amount is 25% of the resin volume, and the column flow rate is 0.5 BV / h. After the resin is saturated, it is washed with distilled water at a flow rate of 0.5 BV / h. Every 5 minutes, a sample is taken at the feed inlet and L-serine is detected by the ninhydrin colorimetric method. When a colorimetric reaction occurs, the L-serine aqueous solution is collected until no colorimetric reaction occurs and then the collection is stopped. Step 5) Secondary decolorization: Add 0.5% activated carbon (by mass of the aqueous solution) to the L-serine aqueous solution obtained in step 4), decolorize at 65℃ for 50 min, filter out the activated carbon, and obtain the secondary decolorized solution; Step 6) Evaporation and crystallization: The L-serine secondary decolorization solution obtained in step 5) is concentrated at 70°C until the L-serine content is 450 g / L. Then, the temperature of the concentrated solution is lowered to room temperature to precipitate L-serine crystals. Step 7) Centrifugal drying: Centrifuge the L-serine concentrate obtained in step 6) to obtain wet L-serine, and dry it to obtain the finished L-serine product; the drying temperature is 72℃ and the time is 6h.

[0038] Under these conditions, the content of L-serine reached 99.4%, and the extraction yield reached 73%.

[0039] While the specific embodiments of the present invention have been described above in conjunction with examples, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A method for producing L-serine, comprising the following steps: step 1) fermentation broth pretreatment, step 2) ceramic membrane filtration, step 3) activated carbon decolorization, step 4) chromatographic separation, step 5) secondary decolorization, step 6) evaporation crystallization, and step 7) centrifugal drying.

2. The method according to claim 1, characterized in that, The method includes the following steps: Step 1) Fermentation broth pretreatment: Adjust the pH of the serine fermentation broth to 5.0-5.5 with sulfuric acid, and add polyaluminum chloride to flocculate the bacterial protein; Step 2) Ceramic membrane filtration: The pretreated serine fermentation broth from step 1) is passed through a ceramic membrane with a pore size of 50 nm to obtain ceramic membrane filtrate; Step 3) Activated carbon decolorization: Add activated carbon to the ceramic membrane filtrate obtained in step 2) for decolorization; Filter to remove activated carbon, obtaining a primary decolorizing solution; Step 4) Chromatographic separation: The decolorized solution obtained in step 3) is introduced into the chromatographic column through a valve for adsorption. The feed amount is 20% of the resin volume, and the column flow rate is 0.4-0.6 BV / h. After the resin is saturated, it is washed with distilled water at a flow rate of 0.4-0.6 BV / h. Every 5 minutes, a sample is taken at the feed inlet and L-serine is detected by the ninhydrin colorimetric method. When a colorimetric reaction occurs, the L-serine aqueous solution is collected until no colorimetric reaction occurs and then the collection is stopped. Step 5) Secondary decolorization: Add activated carbon to the L-serine aqueous solution collected in step 4) for decolorization, filter out the activated carbon, and obtain the secondary decolorized solution; Step 6) Evaporation and crystallization: The L-serine secondary decolorization solution obtained in step 5) is concentrated at 60-70℃, and the temperature of the concentrated solution is lowered to room temperature to precipitate L-serine crystals. Step 7) Centrifugation and drying: After centrifugation, wet L-serine is obtained, and after drying, L-serine product is obtained.

3. The method according to claim 2, characterized in that, The parameters for the ceramic membrane filtration are: temperature 50-55℃, working pressure 1.0-1.5 bar.

4. The method according to claim 2, characterized in that, In steps 3) and 5), the decolorization temperature is 60-70℃ and the time is 50-60min.

5. The method according to claim 2, characterized in that, In step 7), the drying temperature is 70-75℃ and the time is 6 hours.

6. The method according to claim 2, characterized in that, The serine fermentation broth was obtained according to the following process: The Corynebacterium glutamicum seed culture was inoculated into a fermenter containing fermentation medium at an inoculation rate of 5-15% for fermentation culture. The fermentation culture temperature was 39℃, the tank pressure was 0.05Mpa, the stirring speed was 1000rpm, and the fermentation time was 48h.

7. The method according to claim 6, characterized in that, The fermentation medium consists of the following components: 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, and tetracycline 3 mg / L.

8. The method according to claim 6, characterized in that, The fermentation medium consists of the following components: glucose 30 g / L, molasses 30 g / L, corn steep liquor powder 36 g / L, sodium pyruvate 8 g / L, glycine 4 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, and tetracycline 3 mg / L.

9. The method according to claim 6, characterized in that, The accession number of the Corynebacterium glutamicum is CGMCC No. 18932.

Images