Method for preparing alpha-pyrrolidone by fermentation method

Abstract

The invention relates to the technical field of biosynthesis, in particular to a method for preparing alpha-pyrrolidone. The method comprises the following steps: inoculating a fermentation tank with a mixed strain containing glutamate decarboxylase, and converting L-glutamic acid into gamma-aminobutyric acid through fermentation; the concentration of L-glutamic acid in the basal culture medium of the fermentation tank is 80mM, the concentration of glucose is 10-20g / L, and C / N is equal to 15-20; when the concentration of the glucose is reduced to 5g / L, feeding is started, the feeding speed of the glucose is 0.2-0.5 g / L / h, the concentration of the L-glutamic acid is kept at 40-110mM, the dissolved oxygen is kept at 30-45%, and the fermentation temperature is 37-40 DEG C; fermenting for 9-12 hours, ending the fermentation, separating thalli, and retaining supernate; the method comprises the following steps: adding a culture solution into a fermentation tank, carrying out centrifugation, carrying out sterilization treatment on a supernatant, adjusting components, continuously inoculating microorganisms with endogenous decarboxylase, carrying out fermentation, when the concentration of alpha-pyrrolidone is monitored to exceed 40g / L, dynamically exporting a fermentation broth, carrying out filtration separation through a hydrophobic microfiltration membrane, returning an retentate to the fermentation tank, supplementing the culture solution, and collecting a filtrate for separation of alpha-pyrrolidone.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and particularly relates to a method for producing α-pyrrolidone. Background Art

[0002] α-Pyrrolidone, also known as α-pyrrolidone or butyrolactam, is an important organic compound with the chemical formula C4H7NO. It has a wide range of industrial applications, especially in pharmaceuticals, pesticides, and polymer materials. Traditionally, α-pyrrolidone has mainly been prepared by chemical synthesis methods, which involve two steps: first, ammonia reacts with γ-butyrolactone to form hydroxybutyramide, and then hydroxybutyramide undergoes condensation and dehydration to form α-pyrrolidone. However, with the development of green chemistry, researchers have also been exploring more environmentally friendly production methods, such as biocatalysis or fermentation methods. The fermentation method refers to a biotechnology means that uses microorganisms or enzymes as catalysts to convert substrates into target products. For example, Chinese Patent CN103189520B relates to a method for preparing α-pyrrolidone, which includes the following steps: culturing in a medium containing L-glutamic acid or L-glutamate salt, using a microorganism with glutamate decarboxylase as a whole-cell catalyst to prepare 4-aminobutyric acid; filtering the medium to obtain the above-mentioned 4-aminobutyric acid to form a reaction composition mixed with 4-aminobutyric acid and α-pyrrolidone; performing an intramolecular cyclization reaction of 4-aminobutyric acid under a temperature condition of 118°C - 148°C or under reduced pressure at a temperature condition of 110°C - 150°C to generate α-pyrrolidone and water, and separating α-pyrrolidone. This method only uses the fermentation method to generate 4-aminobutyric acid, and the intramolecular cyclization process is still a pure chemical method, so it is not a full-process fermentation method and has the problem of high energy consumption. In addition, the substrate or product concentration during fermentation will inhibit the microbial activity and lead to premature cell senescence, and this technology does not provide a substantial solution to this problem. Summary of the Invention

[0003] (1) Technical Problems to be Solved

[0004] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides a method for preparing α-pyrrolidone by fermentation method, which generates GABA by L-glutamic acid fermentation, and then uses GABA as a substrate to generate α-pyrrolidone by fermentation method, realizing the full-process fermentation method for preparing α-pyrrolidone, improving the enzyme activity by adding auxiliary regulatory factors, increasing the reaction conversion rate, and reducing the generation of by-products.

[0005] (2) Technical Solutions

[0006] A method for preparing α-pyrrolidone by fermentation method, which includes:

[0007] S1. Inoculate a mixed strain with glutamate decarboxylase in a fermenter, and convert L-glutamic acid into γ-aminobutyric acid through fermentation; the concentration of L-glutamic acid in the basal medium of the fermenter is 80 mM, the glucose concentration is 10 - 20 g / L, and C / N = 15 - 20;

[0008] During the fermentation process, a fed-batch fermentation mode is adopted. When the glucose concentration drops to 5 g / L, feeding starts, and the glucose feeding rate is 0.2 - 0.5 g / L / h; monitor the L-glutamic acid concentration in the fermenter to make the feeding rate of L-glutamic acid reach a level that keeps the L-glutamic acid concentration in the fermenter at 40 - 110 mM; maintain the dissolved oxygen in the fermenter at 30 - 45% through dissolved oxygen feedback control, and keep the fermentation temperature at 37 - 40 °C;

[0009] S2. After 9 - 12 h of fermentation, end the fermentation, separate the cells, and retain the supernatant;

[0010] S3. Sterilize the supernatant, adjust the components to obtain the fermentation broth;

[0011] S4. Inoculate a microorganism with endogenous decarboxylase in the fermentation broth, and catalyze the production of α-pyrrolidone using γ-aminobutyric acid as a substrate; during the fermentation process, use Raman spectroscopy to monitor metabolites in real-time. When the concentration of α-pyrrolidone exceeds 40 g / L, start the export pump, dynamically export the fermentation broth and filter it through a hydrophobic microfiltration membrane for separation. Return the retentate to the fermenter and supplement the culture broth, and collect the filtrate for the separation of α-pyrrolidone.

[0012] According to a preferred embodiment of the present invention, in S1, the mixed strain is a combination of Escherichia coli (the main GABA-producing bacterium) and Lactobacillus plantarum (strong acid tolerance at pH 4.0), and the combined quantity ratio can be 1 - 5:5 - 1; Lactobacillus helps maintain a low pH environment, can balance pH fluctuations, and a stable pH is beneficial to improving enzyme activity and GABA product yield. The initial inoculation concentration of the mixed strain is 5 - 8 v / v% seed solution, and the OD600 of the seed solution is 0.3 - 0.5.

[0013] According to a preferred embodiment of the present invention, in S1, 30 - 60 μM pyridoxal phosphate is added as a coenzyme (to enhance glutamate decarboxylase activity) to the fermentation broth, and the pH of the fermentation broth is controlled at 4 - 5 using the pH-state strategy, preferably 4.5 - 5. Under this pH condition, glutamate decarboxylase has the optimal enzyme activity.

[0014] According to a preferred embodiment of the present invention, in S1, 0.3 - 0.5 M sorbitol is added to the fermenter. Sorbitol enhances glutamate decarboxylase activity and relieves high osmotic pressure.

[0015] According to a preferred embodiment of the present invention, in S2, the separation method is 8000×g for centrifugation for 15 - 20 min.

[0016] According to a preferred embodiment of the present invention, in S3, the adjusted components include supplementing carbon source and nitrogen source to the supernatant. The carbon source concentration is 10 - 15 g / L (to avoid carbon metabolism inhibition caused by excessive carbon), and the C / N ratio is 15 - 20; the preferred nitrogen source is yeast extract, which contains essential amino acids. Compared with nitrate nitrogen and ammonium nitrogen, organic nitrogen does not damage cell activity and has little effect on pH.

[0017] According to a preferred embodiment of the present invention, in S4, the microorganism with endogenous decarboxylase is from the genus Alcaligenes sp. or Pseudomonas sp., preferably Pseudomonas putida KT2440 (a commercially available strain), which has extremely high decarboxylase specific activity. The initial inoculation value is 5 - 10% v / v of the seed liquid, and the OD600 of the seed liquid is 0.3 - 0.5; 40 - 50 μM pyridoxal phosphate is also added as a coenzyme to the fermentation system (to enhance enzyme activity), and 0.2 - 0.4 M sorbitol is added to protect enzyme activity; the fermentation time is 16 - 20 h and the temperature is 30 - 32 °C; during the fermentation process, the dissolved oxygen is controlled at 10 - 20%, and the pH is 4.5 - 5.0.

[0018] According to a preferred embodiment of the present invention, in S1 and S4, the microorganisms are pretreated with hydrophobic organic solvents, and the hydrophobic organic solvents are at least one of toluene and cyclohexane. Toluene pretreatment is preferably used. Toluene treatment can improve membrane permeability (GABA uptake rate +40%) and the survival rate > 85%, enabling intracellular metabolites to enter the fermentation broth more smoothly, reducing the inhibition of cell activity by metabolite accumulation, and this treatment method does not affect cell viability.

[0019] According to a preferred embodiment of the present invention, in S4, 0.1 mM cysteine or glutathione is added to the fermentation system to inhibit the oxidative damage of oxygen free radicals to decarboxylase.

[0020] According to a preferred embodiment of the present invention, in S4, the fermentation time is 16 - 20 h (the half-life of Pseudomonas decarboxylase activity is about 18 h), and the temperature is 30 - 32 °C (to balance enzyme activity and cell growth, and enzyme denaturation is likely to occur above 35 °C); during the fermentation process, the dissolved oxygen is controlled at 10 - 20%, and the pH is maintained at 4.5 - 5.0.

[0021] According to a preferred embodiment of the present invention, in S4, the hydrophobic microfiltration membrane is a pollution-resistant PTFE microfiltration membrane (pore size 0.1 μm), and the transmembrane pressure is 0.1-0.3 MPa; XAD-16N resin (using a pore size of 10 nm) is added to the filtrate to make the α-pyrrolidone recovery rate in the filtrate greater than 95%; XAD-16N resin can recover the product by thermal desorption. Microfiltration membranes (0.1 μm) are usually used to intercept bacteria (1-5 μm) or cell debris (>200 nm), while small molecule metabolites (such as α-pyrrolidone) can diffuse freely. Backwash once every 4-8 hours to prevent clogging of the membrane pores (lipids or proteins in the fermentation broth may be adsorbed on the membrane surface), and the backwashing liquid can be used to prepare supplementary culture medium.

[0022] In the present application, unless otherwise specified, the carbon source refers to glucose.

[0023] (III) Beneficial effects

[0024] The present invention generates GABA by fermenting L-glutamic acid, and then generates α-pyrrolidone by fermentation using GABA as a substrate, thereby realizing the preparation of α-pyrrolidone by a full-process fermentation method, and improving enzyme activity, reaction conversion rate and reducing by-product generation by adding auxiliary regulatory factors.

[0025] In the process of fermentation catalysis of L-glutamic acid fermentation to produce GABA, the concentration of L-glutamic acid substrate is controlled by flow addition to maintain at an appropriate level to avoid excessive L-glutamic acid concentration inhibiting fermentation bacteria; in the stage of synthesizing α-pyrrolidone with GABA catalysis, through real-time metabolite monitoring of Raman spectroscopy, when the concentration of α-pyrrolidone is monitored to exceed 40g / L, the export pump is started to dynamically export the fermentation liquid and filter and separate it through a hydrophobic microfiltration membrane, and the intercepted material is returned to the fermentation tank and supplemented with culture medium to prevent the product (α-pyrrolidone) from being too high in concentration. The inhibition of fermentation bacteria and premature aging of microbial cells are avoided. By controlling dissolved oxygen and fermentation temperature to ensure enzyme activity, improve substrate conversion rate, and promote metabolites to enter the fermentation liquid, the inhibition problem caused by the accumulation of metabolites in cells is reduced. The present invention uses the above technical means to prepare α-pyrrolidone by fermentation to achieve full process connection and improve conversion rate, providing technical support for further industrial fermentation preparation of α-pyrrolidone. DETAILED DESCRIPTION

[0026] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods.

[0027] Example 1

[0028] This embodiment provides a method for preparing α-pyrrolidone by fermentation, the steps are as follows:

[0029] (1) Prepare the fermentation medium: glucose concentration is 15 g / L, the nitrogen source is yeast extract, C / N = 20, inorganic salts: 0.2 g / L of MgSO4·7H2O, 0.04 g / L of MnSO4·H2O, 0.02 g / L of FeSO4·7H2O, 2.0 g / L of K2HPO4; L-glutamic acid, 80 mM, pH = 4.5. Then prepare the glucose feeding solution respectively. The glucose feeding solution is diluted 5 times in the absence of L-glutamic acid in the aforementioned medium. The concentration of L-glutamic acid in the L-glutamic acid feeding solution is 40 mM.

[0030] (2) Prepare a 5 L fermenter, fill it with 2 L of the fermentation medium prepared in step (1), and inoculate it with a mixed strain of Escherichia coli and Lactobacillus plantarum. Before inoculation, prepare Escherichia coli (E. coli BL21(DE3)) and Lactobacillus plantarum (L. plantarum ATCC 14917) in shake flasks respectively. Seed solutions with OD600 of 0.3 and 0.4 are obtained in the shake flasks. A small amount of toluene is added dropwise to the seed solutions and shaken for pretreatment, and then inoculated into the fermenter. The total inoculation volume ratio is 6 v / v%, and the ratio of the number of Escherichia coli to Lactobacillus plantarum is 2:1. Set the fermentation temperature at 37 - 38 °C, control the dissolved oxygen at 30 - 35% for fermentation, and use the pH-state strategy to control the pH of the fermentation broth to maintain at 4.5 - 5. When the glucose concentration in the fermenter is detected to drop to 5 g / L, start to feed the glucose feeding solution at a flow rate of 0.4 g / L / h. During the fermentation process, monitor the L-glutamic acid concentration in the fermenter, and by adjusting the flow rate of the L-glutamic acid feeding solution, keep the L-glutamic acid concentration in the fermenter at 50 - 80 mM.

[0031] (3) After 12 h of fermentation, end the fermentation, centrifuge at 8000×g for 15 min, rinse the cells with sterile water, and then rinse with toluene and recycle for use; adjust the components of the supernatant obtained by centrifugation, concentrate to increase the concentration of γ-aminobutyric acid, and add glucose and yeast extract to make the glucose concentration 12 g / L and the C / N ratio 15; perform sterilization treatment to obtain the fermentation broth, and add 50 μM of pyridoxal phosphate and 0.2 M of sorbitol.

[0032] (4) Inoculate Pseudomonas putida KT2440 into 1 / 2 volume of the fermentation culture medium (the remaining fermentation culture medium is used as supplementary culture medium), with the initial inoculation value being 5% v / v of the seed solution, the OD600 of the seed solution being 0.5, and a small amount of toluene being added dropwise to the seed solution and shaken for pre-treatment before inoculating into the fermenter. Ferment at 31 - 32 °C and a dissolved oxygen of 15% for 18 h, and use the pH-state strategy to control the pH of the fermentation broth to be maintained at 4.5 - 5. During the fermentation process, Raman spectroscopy is used to monitor metabolites in real-time. When the concentration of α-pyrrolidone is monitored to exceed 40 g / L, start the export pump to dynamically export the fermentation broth and filter and separate it through a 0.1-μm hydrophobic microfiltration membrane. Return the retentate to the fermenter and supplement the culture medium, collect the filtrate, and separate and recover α-pyrrolidone.

[0033] The separation method of α-pyrrolidone in the filtrate is as follows: Adjust the pH of the filtrate to 5.5 (to improve selectivity), add XAD-16N resin particles (pore size 10 nm) to the filtrate to adsorb α-pyrrolidone in the filtrate; then place the XAD-16N resin particles in hot ethanol water at 60 °C for desorption, and the desorbed solution is concentrated and dried to obtain α-pyrrolidone, and the product yield is about 51%. The retentate separated by hydrophobic microfiltration membrane filtration is Pseudomonas putida cells, which are treated with toluene for permeability, and the toluene residue needs to be removed by nitrogen purging, and the cells can be reused several times. The hydrophobic microfiltration membrane is backwashed every 4 h, and after the backwash solution is separated and the protein is recovered, it can be used to prepare the supplementary culture medium.

[0034] Example 2

[0035] In this example, the fermentation medium formula in step (1) of Example 1 is adjusted to: glucose concentration 20 g / L, the nitrogen source is yeast extract, C / N = 20, inorganic salts: 0.3 g / L of MgSO4·7H2O, 0.03 g / L of MnSO4·H2O, 0.01 g / L of FeSO4·7H2O, 2.0 g / L of K2HPO4; L-glutamic acid, 80 mM, pH = 4.5, and 40 μM of pyridoxal phosphate is added as a coenzyme. Then, prepare the glucose feeding solution and the L-glutamic acid feeding solution respectively. The glucose feeding solution is diluted 5 times in the aforementioned medium without L-glutamic acid and pyridoxal phosphate. The concentration of L-glutamic acid in the L-glutamic acid feeding solution is 40 mM. Finally, α-pyrrolidone is recovered in this example, and the product yield is 54%.

[0036] Example 3

[0037] In this example, when preparing the fermentation medium in step (1) of Example 1, 40 μM pyridoxal phosphate was added as a coenzyme and 0.4 M sorbitol were further added on the basis of Example 1. The glucose feeding solution was diluted 5 times in the aforementioned medium in the absence of L-glutamic acid, pyridoxal phosphate and sorbitol. The concentration of L-glutamic acid in the L-glutamic acid feeding solution was 40 mM. Finally, α-pyrrolidone was recovered in this example, and the product yield was 58%.

[0038] Example 4

[0039] In this example, the operation in step (2) of Example 1 was changed to: Prepare a 5 L fermenter, load 2 L of the fermentation medium prepared in step (1), and inoculate a mixed strain of Escherichia coli and Lactobacillus plantarum. Before inoculation, Escherichia coli (E. coli BL21(DE3)) and Lactobacillus plantarum (L. plantarum CGMCC 1.2437T) were respectively prepared in shake flasks, and seed solutions with OD600 of 0.3 and 0.4 were obtained in the shake flasks and inoculated into the fermenter. The total inoculation volume ratio was 6 v / v%, and the ratio of the number of Escherichia coli to Lactobacillus plantarum was 1:3. The fermentation temperature was set at 37 - 38 °C, and the dissolved oxygen was controlled at 36 - 40% for fermentation. The pH of the fermentation broth was controlled at 4.5 - 5 using the pH-state strategy. When the glucose concentration in the fermenter decreased to 5 g / L, the glucose feeding solution was started to be fed at a flow rate of 0.5 g / L / h. During the fermentation process, the L-glutamic acid concentration in the fermenter was monitored, and by adjusting the flow rate of the L-glutamic acid feeding solution, the L-glutamic acid concentration in the fermenter was maintained at 70 - 90 mM. Finally, α-pyrrolidone was recovered in this example, and the product yield was 61%.

[0040] Example 5

[0041] In this example, the operation in step (3) of Example 1 was changed to: After fermentation for 12 h, the fermentation was ended, centrifuged at 8000 × g for 20 min, the cells were rinsed with sterile water and then with toluene and recovered for use. The supernatant obtained by centrifugation was adjusted in components and concentrated to increase the concentration of γ-aminobutyric acid (which helps to improve the separation and recovery of the target product), glucose and yeast extract were added to make the glucose concentration 12 g / L and the C / N ratio 15; sterilized to obtain the fermentation broth, and 40 μM pyridoxal phosphate was added. Finally, α-pyrrolidone was recovered in this example, and the product yield was 45%.

[0042] Example 6

[0043] In this example, the operation in step (4) of Example 1 was changed to: inoculate Pseudomonas putida KT2440 into 1 / 2 volume of the fermentation culture medium (the remaining fermentation culture medium was used as supplementary culture medium), with an initial inoculation value of 8% v / v of the seed liquid, OD600 of the seed liquid = 0.4. After the seed liquid was pretreated by shaking, it was then transferred to the fermenter. Fermentation was carried out at 31 - 32 °C and a dissolved oxygen of 20% for 16 h. The pH of the fermentation broth was controlled at 4.5 - 5 using the pH-state strategy. During the fermentation process, Raman spectroscopy was used to monitor metabolites in real-time. When the concentration of α-pyrrolidone was detected to exceed 40 g / L, the export pump was started to dynamically export the fermentation broth and filter and separate it through a 0.1 μm hydrophobic microfiltration membrane. The retentate was returned to the fermenter and supplemented with culture medium, the filtrate was collected, and α-pyrrolidone was separated and collected. α-Pyrrolidone was recovered by the same method as in Example 1, and the product yield was approximately 47%.

[0044] Comparative Example 1

[0045] The main difference between this comparative example and Example 1 was that: L-glutamic acid used for fermentation in step (2) was added to the fermentation medium at one time, so that the fermentation medium formula was: glucose concentration 15 g / L, nitrogen source was yeast extract, C / N = 20, inorganic salts: 0.2 g / L of MgSO4·7H2O, 0.04 g / L of MnSO4·H2O, 0.02 g / L of FeSO4·7H2O, 2.0 g / L of K2HPO4; L-glutamic acid, 300 mM, pH = 4.5. A glucose feeding solution was prepared, and the glucose feeding solution was diluted 5 times in the absence of L-glutamic acid in the aforementioned medium. During the cultivation process, the fermentation temperature was set at 37 - 38 °C, and the dissolved oxygen was controlled at 30 - 35% for fermentation. The pH of the fermentation broth was controlled at 4.5 - 5 using the pH-state strategy. When the glucose concentration in the fermenter was detected to drop to 5 g / L, the glucose feeding solution was started to be added at a feeding rate of 0.4 g / L / h. The concentration of L-glutamic acid during the fermentation process was the natural concentration. Finally, α-pyrrolidone was recovered in this example, and the product yield was 27%.

[0046] Comparative Example 2

[0047] The main difference between this comparative example and Example 1 was that: the ratio of the inoculated bacteria in step (2) was Escherichia coli BL21(DE3) and Clostridium baratii at 2:1. Seed liquids with OD600 of 0.3 and 0.4 were obtained in shake flasks respectively. A small amount of toluene was added dropwise to the seed liquids for shaking pretreatment, and then they were inoculated into the fermenter, with the total inoculation volume ratio of 6 v / v%. Finally, α-pyrrolidone was recovered in this example, and the product yield was 33%.

[0048] Comparative Example 3

[0049] The main difference between this comparative example and Example 1 is that in step (4), the concentration of α-pyrrolidone during fermentation is not monitored, and the concentration of α-pyrrolidone remains at the natural concentration all the time. Finally, α-pyrrolidone is recovered in this example, and the product yield is 29%.

[0050] From the above examples and comparative examples, it can be seen that for the process of preparing α-pyrrolidone by fermentation according to the present invention, the yield of the target product α-pyrrolidone can reach more than 45%; while the product yield in each comparative example is only about 30%, which is mainly due to the unreasonable inoculated flora in the comparative examples or the lack of targeted measures for the concentration inhibition problems of the substrate and product.

[0051] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements, or in the case where the technical features in the above examples do not conflict with each other, can be combined in the manner described in the examples, and these modifications, replacements or combinations do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the examples of the present invention.

Claims

1. A method for preparing α-pyrrolidone by fermentation method, characterized in that, Including: S1. Inoculate a mixed strain with glutamate decarboxylase in a fermenter, and convert L-glutamate into γ-aminobutyric acid through fermentation. The concentration of L-glutamate in the basal medium of the fermenter is 80 mM, the glucose concentration is 10 - 20 g / L, and C / N = 15 - 20; During the fermentation process, a fed-batch fermentation mode is adopted. When the glucose concentration drops to 5 g / L, feeding starts, and the glucose feeding rate is 0.2 - 0.5 g / L / h. Monitor the L-glutamate concentration in the fermenter to keep the L-glutamate concentration in the fermenter at 40 - 110 mM. Keep the dissolved oxygen in the fermenter at 30 - 45% through dissolved oxygen feedback control, and keep the fermentation temperature at 37 - 40°C; S2. After 9 - 12 h of fermentation, end the fermentation, separate the cells, and retain the supernatant; S3. Sterilize the supernatant, adjust the components, and obtain the fermentation culture solution; S4. Inoculate a microorganism with endogenous decarboxylase in the fermentation culture solution, and catalyze the generation of α-pyrrolidone using γ-aminobutyric acid as a substrate. During the fermentation process, use Raman spectroscopy to monitor metabolites in real-time. When the concentration of α-pyrrolidone exceeds 40 g / L, start the export pump, dynamically export the fermentation broth and filter and separate it through a hydrophobic microfiltration membrane. Return the retentate to the fermenter and supplement the culture solution, and collect the filtrate for separating α-pyrrolidone.

2. The method according to claim 1, characterized in that In S1, 30 - 60 μM pyridoxal phosphate is added to the fermentation broth as a coenzyme, and the pH of the fermentation broth is controlled at 4 - 5 using the pH-state strategy.

3. The method according to claim 1 or 2, characterized in that, In S1, 0.3 - 0.5 M sorbitol is added to the fermenter.

4. The method according to claim 1, wherein In S1, the mixed strain is a combination of Escherichia coli and Lactobacillus plantarum; the initial inoculation concentration of the mixed strain is 5 - 8 v / v% of the seed liquid, and the OD600 of the seed liquid is 0.3 - 0.

5.

5. The method according to claim 1, characterized in that, In S2, the separation method is centrifugation at 8000×g for 15 - 20 min.

6. The method according to claim 1, wherein In S3, adjusting the components includes supplementing carbon source and nitrogen source to the supernatant. The carbon source concentration is 10 - 15 g / L, and the C / N ratio is 15 - 20; the nitrogen source is yeast extract.

7. The method according to claim 1, characterized in that In S4, the microorganism with endogenous decarboxylase is Pseudomonas putida KT2440, and the initial inoculation value is 5 - 10% v / v of the seed liquid, and the OD600 of the seed liquid is 0.3 - 0.5; in the fermentation system, 40 - 50 μM pyridoxal phosphate is added as a coenzyme, and 0.2 - 0.4 M sorbitol is added to protect the enzyme activity; the fermentation time is 16 - 20 h and the temperature is 30 - 32°C; during the fermentation process, control the dissolved oxygen at 10 - 20% and maintain the pH at 4.5 - 5.

0.

8. The method according to claim 1, wherein The microorganisms in S1 and S4 are microorganisms pretreated with a hydrophobic organic solvent, and the hydrophobic organic solvent is at least one of toluene and cyclohexane.

9. The method according to claim 1, characterized in that, In S4, 0.1 mM cysteine or glutathione is added to the fermentation system to inhibit the oxidative damage of oxygen free radicals to decarboxylase.

10. The method according to claim 1, wherein In S4, the hydrophobic microfiltration membrane is a fouling-resistant PTFE microfiltration membrane with a pore size of 0.1 μm and a transmembrane pressure of 0.1 - 0.3 MPa; XAD-16N resin is added to the filtrate to recover α-pyrrolidone in the filtrate.