A fermentation method for producing glutamine by fermenting Corynebacterium glutamicum

Abstract

The invention discloses a fermentation method for producing glutamine by fermentation of Corynebacterium glutamicum, and belongs to the technical field of Corynebacterium glutamicum fermentation. During the fermentation process, the content of glutamic acid is monitored in real time. When the content of glutamic acid is too low, the pH of the fermented liquid is adjusted by adjusting the mode of supplementing ammonia water or glucose, so as to adjust the content of glutamic acid and improve it. After the content of glutamic acid is improved to a certain value, the pH of the fermented liquid is adjusted by adjusting the mode of supplementing ammonia water or glucose, and the conversion of glutamic acid into glutamine is promoted. While the yield of glutamine is improved, the content of glutamic acid is reduced, and the pH is adjusted by adjusting the mode of supplementing ammonia water or glucose, and the content of glutamic acid is improved, and the cycle is adjusted 2 to 4 times. The fermentation method in the present invention is simple and easy, easy to operate, and the yield of glutamine is significantly improved, the sugar acid conversion rate can exceed 40%, the addition amount of glucose and ammonia water is significantly reduced, and the production cost is reduced, which is conducive to large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of Corynebacterium glutamicum fermentation, and particularly relates to a fermentation method for producing glutamine by fermenting Corynebacterium glutamicum. Background Art

[0002] The molecular formula of glutamine is C5H 10 N2O3, glucose is converted into pyruvate through glycolysis and hexose phosphate branch, and then oxidized into acetyl coenzyme A, and then enters the tricarboxylic acid cycle to generate α-ketoglutarate. α-ketoglutarate is catalyzed by glutamate dehydrogenase and has NH4 + Under the presence of glutamate, glutamate is generated, and then glutamine is produced under the action of glutamine synthetase.

[0003] The most important reaction processes involved in the entire biosynthetic pathway of glutamine are: the synthesis of glutamate and the conversion of glutamate into glutamine by glutamine synthetase, among which the synthesis of glutamate is easier to carry out in a slightly alkaline environment, while glutamine synthetase needs to be in a slightly acidic environment to better exert its activity. In order to take these two processes into account in the existing fermentation method, the pH value of the fermentation broth is usually controlled to 6.9, but this makes it impossible for both reaction processes to be carried out well. This compromise leads to excessive consumption of glucose and ammonia water, reducing the fermentation efficiency. In addition, the synthesis of glutamate is a dynamic process. When its content reaches a certain level, due to the effect of feedback inhibition, the synthesis of glutamate will stop. In the later stage of fermentation, glutamate will no longer be synthesized in an acidic environment, resulting in the inability to significantly improve the fermentation level of glutamine. Therefore, it is necessary to improve and optimize the existing fermentation method to improve the fermentation level of glutamine and reduce the production cost. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention discloses a method for producing glutamine by fermentation of Corynebacterium glutamicum, so as to solve the technical problems in the prior art of producing glutamine on a large scale by fermentation, such as low fermentation level and large amount of glucose and ammonia water, which leads to increased production cost.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for producing glutamine by fermentation of Corynebacterium glutamicum, which comprises the following steps:

[0006] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture;

[0007] S2: The expanded cultured Corynebacterium glutamicum was inoculated into a shake flask containing a seed culture medium and cultured in the shake flask until the seed liquid OD 600 0.6~0.8;

[0008] S3: Inoculate the seed liquid into a fermentation tank filled with fermentation medium, control the DO of the fermentation system to 30%, and ferment at 31.0-32.5°C; during the fermentation process, detect the glutamic acid content once every 2 hours, and when the glutamic acid content is lower than 0.5g / L, adjust the pH of the fermentation liquid to 7-7.5; when the glutamic acid content is higher than 3g / L, adjust the pH of the fermentation liquid to 6.2-6.8; the above operation forms a cycle; the fermentation process is repeated 2-4 times.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, the Corynebacterium glutamicum is Corynebacterium glutamicum CICC20965.

[0011] Further, the slant culture medium includes components at the following concentrations: 4.0 g / L yeast extract, 15.0 g / L peptone, 0.5 g / L NaCl, 2.0 g / L beef extract, and 20.0 g / L agar powder.

[0012] Further, the seed culture medium includes components in the following concentrations: 20.0 g / L glucose, 5.0 g / L urea, 25.0 g / L corn steep liquor, 0.5 g / L NaCl, 0.15 g / L KH2PO4 and 2.0 g / LMgSO4·7H2O.

[0013] Further, the fermentation medium includes components in the following concentrations: 25.0 g / L glucose, 0.35 g / L cystine, 0.8 g / L ornithine, 0.08 g / L pantothenic acid, 3.5 g / L yeast extract, 1.0 g / L MgSO4·7H2O, 1.0 g / L KH2PO4, 0.001 g / L biotin, 0.0008 g / L vitamin B6, 30.0 g / L (NH4)2SO4, 5.0 g / L corn steep liquor, 0.001 g / L copper sulfate, 0.001 g / L zinc sulfate and 1.0 g / L foetida.

[0014] Furthermore, the method for detecting the glutamate content in S3 includes the following steps: aspirating 300 μL of fermentation liquid, then adding 200 μL of derivative liquid A and 200 μL of derivative liquid B, mixing and heating in a 60°C water bath for 50 min, cooling to room temperature, adding 300 μL of acetonitrile, mixing, centrifuging at 12000 rpm for 5 min, and analyzing the glutamate content in the supernatant by high performance liquid chromatography.

[0015] Furthermore, the preparation of the derivative liquid A includes the following steps: the derivative liquid A is a 2,4-dinitrofluorobenzene solution with a concentration of 10 g / L, and the solvent of the solution is anhydrous ethanol; the derivative liquid B is a 3 g / L tris(hydroxymethyl)aminomethane solution, and the solvent of the solution is a mixed solvent of water and dimethyl sulfoxide in a volume ratio of 1:4; the chromatographic column used for analysis is a Target-phenyl (II) chromatographic column, and the mobile phase A is composed of water, acetonitrile and phosphoric acid in a volume ratio of 95:5:0.01; the mobile phase B is composed of water, acetonitrile and phosphoric acid in a volume ratio of 25:75:0.01; the detection wavelength is 365 nm, the column temperature is 35°C, the flow rate is 1.2 mL / min, and the injection volume is 5 μL.

[0016] Furthermore, when the glutamate content is lower than 0.5 g / L, the pH of the fermentation liquid is adjusted by adding ammonia water and slowing down the addition of glucose; when the glutamate content is higher than 3 g / L, the pH of the fermentation liquid is adjusted by adding glucose and slowing down the addition of ammonia water.

[0017] Furthermore, when the glutamic acid content was lower than 0.5 g / L, the pH of the fermentation liquid was adjusted to 7.2; when the glutamic acid content was higher than 3 g / L, the pH of the fermentation liquid was adjusted to 6.5; the fermentation process was cycled 3 times.

[0018] The beneficial effects of the present invention are:

[0019] 1. In the fermentation method of the present invention, the content of glutamic acid in the fermentation liquid is detected every 2 hours. When the content of glutamic acid is lower than 0.5 g / L, the pH value of the fermentation liquid is 7.0-7.5 by accelerating the addition of ammonia water and slowing down the addition of glucose. This pH value is conducive to the synthesis of glutamic acid, and the content of glutamic acid is gradually increased. When the analysis shows that the content of glutamic acid is higher than 3.0 g / L, the pH value of the fermentation liquid is 6.5 by accelerating the addition of glucose and slowing down the addition of ammonia water. This pH value can promote the conversion of glutamic acid into glutamine by glutamine synthetase, and the content of glutamic acid will gradually decrease. When the analysis shows that the content of glutamic acid is lower than 0.5 g / L, the pH value of the fermentation liquid is 7.0-7.5 by accelerating the addition of ammonia water and slowing down the addition of glucose. Before the end of fermentation, it is continuously circulated 2-3 times. This fermentation method can improve the stability of the fermentation process, improve the utilization rate of glucose, and the sugar-acid conversion rate reaches more than 40%, which significantly reduces the addition amount of glucose and ammonia water, reduces the production cost, and in addition, the yield of glutamine is also significantly improved.

[0020] 2. The present invention can optimize the synthesis conditions of glutamate and glutamine at different stages through a cyclic control pH strategy. When the glutamate content is low, the fermentation conditions are adjusted to promote the synthesis of glutamate, thereby increasing its yield. When the glutamate content is high, the conversion of glutamate to glutamine is promoted to increase the yield of glutamine. At the same time, the accumulation of glutamate is avoided to affect the synthesis of glutamate, the conversion efficiency of glutamate to glutamine and the subsequent extraction and purification of glutamine. DETAILED DESCRIPTION

[0021] Corynebacterium glutamicum ( Corynebacteriumglutamicum CICC20965) was obtained from Shanghai Institute of Industrial Microbiology.

[0022] Analysis Method of Glutamic Acid Content in Fermentation Broth

[0023] The glutamic acid in the fermentation broth was subjected to pre-column derivatization of amino acids with 2,4-dinitrofluorobenzene and then quantitatively analyzed by HPLC. The specific method is as follows:

[0024] Derivative solution A: Accurately weigh 1.0 g (accurate to 0.0001 g) of 2,4-dinitrofluorobenzene into a 100 mL volumetric flask, dilute to the mark with anhydrous ethanol, mix well to obtain derivative solution A, and store it at 4°C.

[0025] Derivative solution B: Accurately weigh 0.15 g (accurate to 0.0001 g) of tris(hydroxymethyl)aminomethane into a 10 mL volumetric flask, dissolve it with purified water and make up to the mark, shake well; use a 10 mL pipette to draw 10 mL of the solution into a 50 mL volumetric flask, make up to the mark with dimethyl sulfoxide, shake well to obtain derivative solution B, and store it at 4°C.

[0026] Take 300 μL of sample respectively, then add 200 μL of derivative solution A and 200 μL of derivative solution B, mix well and heat in a 60°C water bath for 50 min. After heating, remove from the water bath, wait for cooling to room temperature, add 300 μL of acetonitrile, mix well, centrifuge at 12000 rpm for 5 min, and take the supernatant for detection.

[0027] A Target~phenyl (II) chromatographic column was used, with mobile phase A: water: acetonitrile: phosphoric acid = 95:5:0.01 (volume ratio); mobile phase B: water: acetonitrile: phosphoric acid = 25:75:0.01 (volume ratio); the elution program was shown in Table 1, the detection wavelength was 365 nm; the column temperature was 35°C; the flow rate was 1.2 mL / min; and the injection volume was 5 μL.

[0028] Table 1 HPLC elution program

[0029]

[0030] The specific embodiments of the present invention are described in detail below in conjunction with the examples. The components of the culture medium in the examples and comparative examples are shown in Table 2.

[0031] Table 2

[0032]

[0033] The above culture media are all prepared with pure water. NaOH is used to adjust the pH of the slant culture medium and seed culture medium to 7.0 during preparation. The prepared seed culture medium is divided into 250mL shake flasks, with 30mL of liquid in each bottle to ensure that the bacteria can obtain sufficient oxygen and nutrients during the culture process, while avoiding growth inhibition caused by overcrowding.

[0034] Example 1

[0035] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0036] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0037] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0038] S3: Fermentation production stage (50L fermenter): fermentation volume is 27L, temperature is 31.0~32.5℃, ventilation is 1:1~2vvm, initial stirring speed is 300rpm, stirring speed during fermentation is 300~600rpm, dissolved oxygen (DO) is controlled at 30%, tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. The content of glutamate in the fermentation liquid is detected every 2h during the fermentation process. When the glutamate content is lower than 0.5g / L, the pH of the fermentation liquid is adjusted to 7.0~7.2 by slowing down the addition of glucose; when the glutamate content is higher than 3.0g / L, the pH of the fermentation liquid is adjusted to 6.4~6.6 by slowing down the addition of ammonia water; the pH is adjusted according to the glutamate content, and the pH is adjusted 3 times in a cycle. The fermentation liquid is collected after 50h of cultivation.

[0039] Example 2

[0040] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0041] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0042] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0043] S3: Fermentation production stage (50L fermenter): fermentation volume is 27L, temperature is 31.0~32.5℃, ventilation volume is 1:1~2vvm, initial stirring speed is 300rpm, stirring speed during fermentation is 300~600rpm, dissolved oxygen (DO) is controlled at 30%, tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. The content of glutamate in the fermentation liquid is detected every 2h during the fermentation process. When the glutamate content is lower than 0.5g / L, the pH of the fermentation liquid is adjusted to 7.2~7.4 by slowing down the addition of glucose; when the glutamate content is higher than 3.0g / L, the pH of the fermentation liquid is adjusted to 6.5~6.7 by slowing down the addition of ammonia water. The pH is adjusted according to the glutamate content, and the pH is adjusted twice in a cycle. The fermentation liquid is collected after 50h of cultivation.

[0044] Example 3

[0045] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0046] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0047] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0048] S3: Fermentation production stage (50L fermenter): fermentation volume is 27L, temperature is 31.0~32.5℃, ventilation volume is 1:1~2vvm, initial stirring speed is 300rpm, stirring speed during fermentation is 300~600rpm, dissolved oxygen (DO) is controlled at 30%, tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. The content of glutamate in the fermentation liquid is detected every 2h during the fermentation process. When the glutamate content is lower than 0.5g / L, the pH of the fermentation liquid is adjusted to 7.2 by accelerating the addition of ammonia water; when the glutamate content is higher than 3.0g / L, the pH of the fermentation liquid is adjusted to 6.5 by accelerating the addition of glucose. The pH is adjusted according to the glutamate content, and the pH is adjusted 4 times in a cycle. The fermentation liquid is collected after 50h of cultivation.

[0049] Example 4

[0050] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0051] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0052] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0053] S3: Fermentation production stage (50L fermenter): fermentation volume is 27L, temperature is 31.0~32.5℃, ventilation volume is 1:1~2vvm, initial stirring speed is 300rpm, stirring speed during fermentation is 300~600rpm, dissolved oxygen (DO) is controlled at 30%, tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. The content of glutamate in the fermentation liquid is detected every 2h during the fermentation process. When the glutamate content is lower than 0.5g / L, the pH of the fermentation liquid is adjusted to 7.3~7.5 by accelerating the addition of ammonia water; when the glutamate content is higher than 3.0g / L, the pH of the fermentation liquid is adjusted to 6.2~6.4 by slowing down the addition of ammonia water. The pH is adjusted according to the glutamate content, and the pH is adjusted 3 times in a cycle. The fermentation liquid is collected after 50h of cultivation.

[0054] Example 5

[0055] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0056] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0057] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0058] S3: Fermentation production stage (50L fermenter): fermentation volume is 27L, temperature is 31.0~32.5℃, ventilation is 1:1~2vvm, initial stirring speed is 300rpm, stirring speed during fermentation is 300~600rpm, dissolved oxygen (DO) is controlled at 30%, tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. The content of glutamate in the fermentation liquid is detected every 2h during the fermentation process. When the glutamate content is lower than 0.5g / L, the pH of the fermentation liquid is adjusted to 7.2 by slowing down the addition of glucose; when the glutamate content is higher than 3.0g / L, the pH of the fermentation liquid is adjusted to 6.6~6.8 by slowing down the addition of ammonia water. The pH is adjusted according to the glutamate content, and the pH is adjusted 3 times in a cycle. The fermentation liquid is collected after 50h of cultivation.

[0059] Comparative Example 1

[0060] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0061] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0062] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0063] S3: Fermentation production stage (50L fermenter): The fermentation volume is 27L. During the fermentation process, concentrated ammonia water is used to control the pH of the fermentation liquid to 7.0, the temperature is 31.0~32.5℃, the ventilation volume is 1:1~2vvm, the initial stirring speed is 300rpm, and the stirring speed during the fermentation process is 300~600rpm. The dissolved oxygen (DO) is controlled to 30%, and the tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. When glucose is supplemented, the glucose concentration in the fermenter is controlled to be maintained at 5g / L, and the fermentation liquid is collected after 50h of cultivation.

[0064] Comparative Example 2

[0065] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0066] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0067] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9, the stirring speed was 180 rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29°C. When the seed solution OD 600 When it reaches 0.6, the seed culture is completed, and the seed liquid is transferred into a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0068] S3: Fermentation production stage (50L fermenter): The fermentation volume was 27L. Concentrated ammonia water was used to control the pH at 6.9 during the fermentation process. The temperature was 31.0℃, the ventilation volume was 1:1vvm, the initial stirring speed was 300rpm, and the stirring speed during the fermentation process was 300~600rpm. The dissolved oxygen (DO) was controlled at 30%. The tank pressure was set to 0.07MPa within 24h and adjusted to 0.09~0.1MPa after 24h. When glucose was added, the glucose concentration in the fermenter was controlled to be maintained at 5g / L. The fermentation liquid was collected after 50h of cultivation.

[0069] Comparative Example 3

[0070] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0071] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0072] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0073] S3: Fermentation production stage (50L fermenter): The fermentation volume is 27L. During the fermentation process, concentrated ammonia water is used to control the pH to 7.1, the temperature is 31.0~32.5℃, the ventilation volume is 1:1~2vvm, the initial stirring speed is 300rpm, and the stirring speed during the fermentation process is 300~600rpm. The dissolved oxygen (DO) is controlled to 30%, and the tank pressure is set to 0.07MPa within 24h, and adjusted to 0.09~0.1MPa after 24h. When glucose is supplemented, the glucose concentration in the fermenter is controlled to be maintained at 5g / L, and the fermentation liquid is collected after 50h of cultivation.

[0074] Comparative Example 4

[0075] A method for culturing Corynebacterium glutamicum to produce glutamine by fermentation, comprising the following steps:

[0076] S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture at 29°C for 24h;

[0077] S2: Seed culture stage (20L seed tank): During the culture process, the pH was controlled at 6.9-7.1, the stirring speed was 180rpm, the dissolved oxygen (DO) was controlled at 30%, and the culture temperature was 29℃. 600 When it reaches 0.6~0.8, the seed culture is completed, and the seed liquid is transferred to a 50L fermentation tank at a 5% inoculation rate for fermentation experiments.

[0078] S3: Fermentation production stage (50L fermenter): The fermentation volume was 27L. During the fermentation process, concentrated ammonia water was used to control the pH to 7.1 in the first 12 hours. After 12 hours, the pH of the fermentation liquid was controlled to 6.0 by accelerating the addition of glucose. The temperature was 31.0~32.5℃, the ventilation volume was 1:1~2vvm, the initial stirring speed was 300rpm, and the stirring speed during the fermentation process was 300~600rpm. The dissolved oxygen (DO) was controlled to 30%. The tank pressure was set to 0.07MPa within 24 hours and adjusted to 0.09~0.1MPa after 24 hours. When glucose was started to be supplemented, the glucose concentration in the fermenter was controlled to be maintained at 5g / L. The fermentation liquid was collected after 50 hours of cultivation.

[0079] Experimental example

[0080] 1. Measurement of the volume of fermented liquid in the fermentation tanks of the embodiment and the comparative example

[0081] The results are shown in Table 3.

[0082] Table 3

[0083]

[0084] 2. Measure the content of glutamine in the fermentation broth

[0085] The test method is consistent with the method for testing the glutamic acid content in the fermentation broth. The results are shown in Table 4.

[0086] Table 4

[0087]

[0088] Different from comparative examples 1 to 3 in which the pH of the fermentation liquid is fixedly controlled to be 6.9, 7.0 and 7.1, the embodiment of the present invention analyzes the content of glutamate in the fermentation liquid every 2 hours. When the content of glutamate is lower than 0.5 g / L, the pH of the fermentation liquid is adjusted to 7.0 to 7.5 by accelerating the addition of ammonia water and slowing down the addition of glucose, which is beneficial to the synthesis of glutamate and thus increases the content of glutamate. When the content of glutamate is higher than 3.0 g / L, the pH of the fermentation liquid is adjusted to 6.5 by accelerating the addition of glucose and slowing down the addition of ammonia water, which promotes the conversion of glutamate into glutamine by glutamine synthetase, thereby reducing the content of glutamate, avoiding excessive glutamate content that inhibits the activity of glutamine synthetase, thereby reducing the synthesis rate of glutamine. In addition, high content of glutamate also produces osmotic pressure on cells, affecting the normal physiological function of cells, thereby affecting the yield of glutamine.

[0089] Table 5

[0090]

[0091] In comparative example 4, the pH value was controlled to be 7.1 in the early stage of fermentation, and this slightly alkaline environment was conducive to the synthesis of glutamate. The pH value was controlled to be 6.0 in the late stage of fermentation, which was conducive to the conversion of glutamate into glutamine. Table 5 is a statistical table of the addition amounts of glucose and ammonia water in the embodiments and comparative examples. It can be seen from Table 5 that the fermentation method in comparative example 4 needs to consume too much glucose and ammonia water, and the sugar-acid conversion rate is also low. It can also be seen from Table 4 that the yield of glutamine in comparative example 4 is also lower than that in the embodiments. The synthesis of glutamate is a dynamic process, which is conducive to its synthesis in a slightly alkaline environment. However, when the content reaches a certain level, the synthesis stops due to feedback inhibition, which directly causes a reduction in the subsequent synthesis amount. High content of glutamate will also affect the synthesis of glutamine, causing glutamate accumulation, which is also not conducive to the subsequent extraction and purification of glutamine. In the fermentation process of the embodiment, the content of glutamate in the fermentation broth is detected every 2 hours. When the content of glutamate is lower than 0.5 g / L, the glutamine production will be reduced. Therefore, in the fermentation process, the pH of the fermentation broth is adjusted to 7.0-7.5 by accelerating the addition of ammonia water and slowing down the addition of glucose. This pH value is conducive to the synthesis of glutamate, and the content of glutamate is gradually increased. When the analysis shows that the content of glutamate is higher than 3.0 g / L, the pH of the fermentation broth is adjusted to 6.5 by accelerating the addition of glucose and slowing down the addition of ammonia water. This pH value can promote the conversion of glutamate into glutamine by glutamine synthetase. At the same time, the content of glutamate will gradually decrease. When the analysis shows that the content of glutamate is lower than 0.5 g / L, the pH of the fermentation broth is adjusted to 7.0-7.5 by accelerating the addition of ammonia water and slowing down the addition of glucose. Before the end of fermentation, the cycle is continuously repeated 2-3 times. It can be seen from Table 5 that the fermentation process in the embodiment can not only significantly reduce the amount of glucose and ammonia added, thereby reducing the production cost, but also significantly improve the glutamine fermentation level, with the sugar-acid conversion rate reaching more than 40.0%. Secondly, glutamic acid, as the main by-product in the glutamine fermentation process, is difficult to separate from glutamine during the extraction process. Therefore, controlling the content of glutamic acid in the fermentation broth can greatly reduce the difficulty of subsequent glutamine extraction and reduce costs, and has broad industrial application prospects.

[0092] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A fermentation method for producing glutamine by fermentation of Corynebacterium glutamicum, characterized in that: The following steps are involved: S1: Inoculate Corynebacterium glutamicum into slant culture medium for expansion culture; S2: The expanded cultured Corynebacterium glutamicum was inoculated into a shake flask containing a seed culture medium and cultured in the shake flask until the seed liquid OD 600 0.6~0.8; S3: Inoculate the seed liquid into the fermentation tank filled with fermentation medium, control the DO of the fermentation system to 30%, and ferment at 31.0-32.5℃; during the fermentation process, detect the glutamic acid content once every 2 hours. When the glutamic acid content is lower than 0.5g / L, adjust the pH of the fermentation liquid to 7-7.5 by adding ammonia water and slowing down the addition of glucose; when the glutamic acid content is higher than 3g / L, adjust the pH of the fermentation liquid to 6.2-6.8 by adding glucose and slowing down the addition of ammonia water; the above operation forms a cycle; the fermentation process is repeated 2-4 times.

2. The fermentation method according to claim 1, characterized in that: The Corynebacterium glutamicum is Corynebacterium glutamicum CICC20965.

3. The fermentation method according to claim 1, characterized in that The slant culture medium includes components with the following concentrations: 4.0 g / L yeast extract, 15.0 g / L peptone, 0.5 g / L NaCl, 2.0 g / L beef extract and 20.0 g / L agar powder.

4. The fermentation method according to claim 1, characterized in that The seed culture medium includes components with the following concentrations: 20.0 g / L glucose, 5.0 g / L urea, 25.0 g / L corn steep liquor, 0.5 g / L NaCl, 0.15 g / L KH2PO4 and 2.0 g / LMgSO4·7H2O.

5. The fermentation method according to claim 1, characterized in that The fermentation medium includes the following components at the following concentrations: 25.0g / L glucose, 0.35g / L cystine, 0.8g / L ornithine, 0.08g / L pantothenic acid, 3.5g / L yeast extract, 1.0g / LMgSO4·7H2O, 1.0g / L KH2PO4, 0.001g / L biotin, 0.0008g / L vitamin B6, 30.0g / L (NH4)2SO4, 5.0g / L corn steep liquor, 0.001g / L copper sulfate, 0.001g / L zinc sulfate and 1.0g / L foetida.

6. The fermentation method according to claim 1, characterized in that: The method for detecting the glutamic acid content in S3 includes the following steps: absorbing 300 μL of fermentation liquid, then adding 200 μL of derivative liquid A and 200 μL of derivative liquid B, mixing and heating in a 60°C water bath for 50 minutes, cooling to room temperature, adding 300 μL of acetonitrile, mixing and centrifuging at 12000 rpm for 5 minutes, and analyzing the glutamic acid content in the supernatant by high performance liquid chromatography; the derivative liquid A is a 2,4-dinitrofluorobenzene solution with a concentration of 10 g / L, and the solvent of the solution is anhydrous ethanol; the derivative liquid B is a 3 g / L tris(hydroxymethyl)aminomethane solution, and the solvent of the solution is a mixed solvent of water and dimethyl sulfoxide in a volume ratio of 1:

4.

7. The fermentation method according to claim 6, characterized in that: The chromatographic column used for analysis was a Target-phenyl (II) chromatographic column. Mobile phase A was composed of water, acetonitrile and phosphoric acid in a volume ratio of 95:5:0.01; mobile phase B was composed of water, acetonitrile and phosphoric acid in a volume ratio of 25:75:0.

01. The detection wavelength was 365 nm, the column temperature was 35°C, the flow rate was 1.2 mL / min, and the injection volume was 5 μL.

8. The fermentation method according to claim 1, characterized in that: When the glutamic acid content was lower than 0.5 g / L, the pH of the fermentation liquid was adjusted to 7.2; when the glutamic acid content was higher than 3 g / L, the pH of the fermentation liquid was adjusted to 6.5; the fermentation process was cycled 3 times.

Images