Method for improving fermentation yield and conversion rate of L-proline

Abstract

The invention belongs to the technical field of fermentation engineering, and relates to a method for improving the fermentation yield and conversion rate of L-proline. According to the method, the control process of controlling the proper residual sugar concentration in the fermentation process and combining heating fermentation and organic nitrogen source feeding is adopted, the yield of L-proline can be 150 g / L or above, the yield is 4 g / L / h or above, the conversion rate is 43% or above, the content of heteroacid glutamic acid is low and reaches the best level reported in China at present, the fermentation cost of the process is obviously reduced, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering and relates to a method for improving the fermentation yield and conversion rate of L-proline. Technical Background

[0002] L-proline is a naturally occurring non-essential amino acid for the human body and one of the 20 amino acids that make up protein. It can be used in medicine, food, agriculture, and chemical industry, especially in the pharmaceutical field.

[0003] The production methods of L-proline mainly include chemical method and fermentation method. Due to the serious pollution and high production cost of chemical method, it has been gradually replaced by fermentation method. The most commonly used strain for producing L-proline by fermentation method is Corynebacterium glutamicum, but the fermentation level reported at present is relatively low and the production cost is high. For example, patent CN108841886B uses DO-stat method to adjust the flow rate of glucose in the fermentation process, control the dissolved oxygen level at a certain value, and carry out low residual sugar fermentation. The L-proline yield reaches 84.3g / L and the yield is 2.11g / L / h; patent CN117625706A, in the fermentation process, according to the ratio of 10:1, 500g / L of glucose solution and 500g / L of ammonium citrate solution are added at the same time, and the residual sugar is controlled to be about 30g / L, and the proline yield reaches 90.3g / L, and the yield is 1.88g / L / h. The above L-proline yield and yield are relatively low, the production cost is high, and it is not conducive to industrial production. Patent CN116497075A adds 15g / L ammonium sulfate and 25g / L glutamic acid to the fermentation medium, and maintains residual sugar at 40-50g / L and dissolved oxygen at 5-20% during the fermentation process. The L-proline yield reaches 92.3g / L, the yield is 2.31g / L / h, the conversion rate is 41.0%, and the heteroacid glutamic acid is 2.1g / L. Its conversion rate is the highest level reported in China so far. However, the L-proline yield is low, and glutamic acid is added to the fermentation medium. The heteroacid glutamic acid content is high at the fermentation end point, which makes its production cost high.

[0004] Therefore, it is urgent to develop a fermentation control process with high L-proline yield and conversion rate to reduce production costs. Summary of the invention

[0005] The object of the present invention is to provide a method for improving the fermentation yield and conversion rate of L-proline. The method comprises: controlling the appropriate residual sugar concentration during the fermentation process, adopting a temperature-raising fermentation method, and simultaneously feeding a feed medium containing an organic nitrogen source, so that the fermentation yield and conversion rate of L-proline are significantly improved, the content of impure acids is significantly reduced, and the fermentation cost is reduced, which is suitable for large-scale industrial production.

[0006] The invention provides a method for improving the fermentation yield and conversion rate of L-proline. The method adopts a fermentation tank fermentation mode (activating seeds of L-proline-producing Corynebacterium glutamicum or Escherichia coli and inoculating them in a fermentation tank), culturing at 30-32°C and pH 7.0, with a rotation speed of 300-800rpm, an air volume of 0.5-1.5vvm and a tank pressure of 0.02-0.08Mpa, and controlling DO at 30-40%; and when the residual sugar concentration is reduced to 10g / L, adding 70% glucose to control the residual sugar concentration at 5-10g / L until the fermentation is finished.

[0007] In a specific example, the L-proline-producing Corynebacterium glutamicum is any one of the PRO-1 to PRO-20 strains in CN114107141B.

[0008] Further preferably, when glucose is fed, the fermentation temperature is increased to 33-37°C, such as 35°C.

[0009] Specifically, the fermentation tank culture medium is: 50 g / L glucose, 3 g / L corn steep liquor powder, 3 g / L soy peptone, 3 g / L yeast powder, 15 g / L ammonium sulfate, 6 g / L KH2PO4, 0.5 g / L MgSO4, 0.05 g / L FeSO4, 0.03 g / L MnSO4, 45 μg / L biotin, and 45 μg / L vitamin B2.

[0010] Preferably, when glucose is fed, a feed medium containing an organic nitrogen source is also fed simultaneously.

[0011] More preferably, when glucose is fed, a feed medium containing an organic nitrogen source is simultaneously fed at 1 ml / h.

[0012] Specifically, the feed medium is: MgSO4 2g / L, FeSO4 0.5g / L, corn steep liquor powder 15g / L, soy peptone 15g / L, yeast powder 15g / L.

[0013] In a specific embodiment, the inoculation of the fermenter prior to fermentation is provided by culturing in a seed tank;

[0014] Preferably, the seed tank culture is carried out under the conditions of 30-32° C., pH 7.00, and DO 30-40% for 12 hours and then inoculated into a fermentation tank.

[0015] Specifically, the culture medium used in the seed tank culture is: 60g / L glucose, 15g / L yeast extract, 3g / L corn syrup powder, 5g / L ammonium sulfate, 3g / L K2HPO4, 1g / L MgSO4, 10ug / L biotin, 1mg / L vitamin B1, and 0.1mg / L vitamin B2.

[0016] In a specific embodiment, the seed tank is provided by shake flask culture before culture;

[0017] Preferably, the shake flask is cultured at 30-32°C and 200-220 rpm for 12 h and then inoculated into a seed tank;

[0018] More specifically, the culture medium used for the plate culture is: glucose 5g / L, yeast powder 5g / L, soy peptone 9g / L, urea 3g / L, succinic acid 0.5g / L, K2HPO4 1g / L, MgSO4 0.1g / L, biotin 10ug / L, vitamin B1 100ug / L, MOPS 20g / L.

[0019] Advantages of the present invention:

[0020] (1) Controlling the appropriate residual sugar concentration during the fermentation process can prevent the glucose concentration from being too low (0-0.1 g / L and below) and affecting the normal growth and metabolism of the bacteria, and can also prevent the glucose concentration from being too high (40-50 g / L and above) and causing an increase in metabolic overflow by-products, which is beneficial to increasing the L-proline yield or conversion rate.

[0021] (2) Properly increasing the temperature during the fermentation process can effectively control the growth of the strain, allowing more glucose to be used for product synthesis, which is beneficial to increasing the yield and conversion rate of L-proline.

[0022] (3) During the fermentation process, the feed medium containing organic nitrogen source is added to avoid the excessive organic nitrogen source in the initial fermentation medium, which causes the bacteria to grow vigorously in the early stage and then decrease in vitality in the later stage. It can maintain normal growth and metabolism of the bacteria throughout the whole process, which is beneficial to improve the L-proline production and conversion rate.

[0023] (4) By adopting a control process combining appropriate residual sugar concentration control during the fermentation process, temperature increase fermentation and flow addition of an organic nitrogen source, an L-proline yield of more than 150 g / L, a yield of more than 4 g / L / h, a conversion rate of more than 43%, and a low content of heteroacid glutamic acid, which is the best level reported so far. The fermentation cost of this process is significantly reduced, making it suitable for industrial production. DETAILED DESCRIPTION

[0024] The technical content of the present invention will be clearly described below in conjunction with specific embodiments. The described embodiments are only some representative embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0025] Definition of terms

[0026] The residual sugar referred to in this article refers to the concentration of glucose in the fermentation broth, in g / L.

[0027] The L-proline yield referred to herein refers to the concentration of L-proline in the fermentation broth, in g / L.

[0028] The L-proline yield referred to herein, i.e., production intensity, refers to the content of L-proline produced per unit volume of fermentation liquid per unit time, and the unit is g / L / h.

[0029] The conversion rate referred to in this article refers to how much L-proline the bacteria can produce using a certain amount of glucose. The calculation formula is: (L-proline production*fermentation tank volume) / total sugar consumption weight, in g / g or %.

[0030] Embodiment 1:

[0031] A glycerol tube of L-proline-producing Corynebacterium glutamicum strain (PRO-19 in L-proline-producing Corynebacterium glutamicum CN114107141B) was streaked on a plate, cultured at 30-32° C. for 24 h, and then inoculated into a shake flask; after the shake flask was cultured at 30-32° C. and 200-220 rpm for 12 h, it was inoculated into a 5 L seed tank at an inoculum rate of 1%; the seed tank was cultured at 30-32° C., pH 7.00, and DO 30-40% After culturing for 12 hours under the conditions of 10%, the inoculation volume was inoculated into a 5L fermentation tank; the fermentation tank was cultured at 30-32°C and pH 7.00, and the DO was controlled at 30-40% by adjusting the rotation speed to 300-800rpm, the air volume to 0.5-1.5vvm and the tank pressure to 0.02-0.08Mpa; when the residual sugar concentration dropped to 10g / L, 70% glucose was added to control the residual sugar concentration to 5-10g / L, and the fermentation period was 32h. The results are shown in Table 1.

[0032] Table 1 Fermentation results of Example 1

[0033] End point OD (600nm) L-Proline(g / L) Yield (g / L / h) Conversion rate (%) 136.8 119.7 3.74 35.2

[0034] As shown in Table 1, when the residual sugar concentration is controlled at 5-10 g / L during the fermentation process, the L-proline yield reaches about 120 g / L, which is higher than the levels reported in the prior art CN108841886B, CN117625706A, CN116497075A, and CN114107141A, and the conversion rate reaches about 35%. Therefore, it is shown that controlling the residual sugar concentration at 5-10 g / L during the fermentation process is beneficial to increasing the yield or conversion rate of L-proline.

[0035] Embodiment 2:

[0036] The activation, seed transfer and fermentation steps of L-proline-producing Corynebacterium glutamicum (PRO-19 in L-proline-producing Corynebacterium glutamicum CN114107141B) were the same as those in Example 1. When the residual sugar concentration of the fermentation tank was reduced to 10 g / L, 70% glucose was added to control the residual sugar concentration at 5-10 g / L, and the fermentation temperature was raised to 35°C for a fermentation period of 32 h. The results are shown in Table 2.

[0037] Table 2 Example 2 Fermentation results

[0038] End point OD (600nm) L-Proline(g / L) Yield (g / L / h) Conversion rate (%) 125.0 133.9 4.18 39.0

[0039] As shown in Table 2, when the residual sugar concentration was controlled at 5-10 g / L during the fermentation process and the fermentation temperature was increased to 35°C, the fermentation OD was significantly reduced, the L-proline yield was increased by 11.9%, the yield was increased by 11.9%, and the conversion rate was increased by 10.8% compared with Example 1. Therefore, it was shown that increasing the temperature to 35°C during the fermentation process was beneficial to increasing the L-proline yield and conversion rate.

[0040] Embodiment 3:

[0041] The activation, seed transfer and fermentation steps of L-proline-producing Corynebacterium glutamicum were the same as those in Example 1. When the residual sugar concentration in the fermentation tank was reduced to 10 g / L, 70% glucose was added to control the residual sugar concentration at 5-10 g / L, and the fermentation temperature was increased to 35°C. At the same time, a feed medium containing an organic nitrogen source was added at a rate of 1 ml / h, and the fermentation period was 36 h. The results are shown in Table 3.

[0042] Table 3 Example 3 Fermentation results

[0043] End point OD (600nm) L-Proline(g / L) Yield (g / L / h) Conversion rate (%) 133.0 153.0 4.25 43.5

[0044] As shown in Table 3, when the residual sugar concentration is controlled at 5-10 g / L during the fermentation process, the fermentation temperature is increased to 35°C, and the feed medium containing an organic nitrogen source is added at the same time, compared with Example 2, the fermentation OD is increased, the L-proline yield is increased by 14.3%, and the conversion rate is increased by 11.5%; compared with Example 1, the L-proline yield is increased by 27.8%, the yield is increased by 13.6%, and the conversion rate is increased by 23.6%, and the content of heteroacid glutamic acid is less than 1.5 g / L, which is significantly lower than the prior art. The results show that adding a feed medium containing an organic nitrogen source at a rate of 1 ml / h during the fermentation process is also beneficial to increasing the L-proline yield and conversion rate.

[0045] The results showed that controlling the residual sugar concentration at 5-10 g / L, raising the temperature to 35 °C and feeding a feed medium containing an organic nitrogen source during L-proline fermentation can achieve high yield, high rate and high conversion rate with low impurity acid content and low fermentation cost.

Claims

1. A method for improving the fermentation yield and conversion rate of L-proline, characterized in that: The seeds of L-proline-producing Corynebacterium glutamicum or Escherichia coli are activated and inoculated into a fermentation tank, and cultured at 30-32°C, pH 7.0, with a rotation speed of 300-800rpm, an air volume of 0.5-1.5vvm, and a tank pressure of 0.02-0.08Mpa, and the DO is controlled at 30-40%; When the residual sugar concentration drops to 10g / L, 70% glucose is added to control the residual sugar concentration at 5-10g / L until the fermentation ends.

2. The method according to claim 1, characterized in that When glucose is fed, the fermentation temperature is raised to 33-37°C, for example 35°C.

3. The method according to claim 1 or 2, characterized in that The fermentation tank culture medium is: 50 g / L glucose, 3 g / L corn steep liquor powder, 3 g / L soy peptone, 3 g / L yeast powder, 15 g / L ammonium sulfate, 6 g / L KH2PO4, 0.5 g / L MgSO4, 0.05 g / L FeSO4, 0.03 g / L MnSO4, 45 μg / L biotin, and 45 μg / L vitamin B2.

4. The method according to any one of claims 1 to 3, characterized in that When glucose was fed, a feed medium containing an organic nitrogen source was also fed simultaneously.

5. The method according to claim 4, characterized in that While glucose was being fed, a feed medium containing an organic nitrogen source was simultaneously fed at 1 ml / h.

6. The method according to claim 5, characterized in that The feed medium is: MgSO4 2g / L, FeSO4 0.5g / L, corn steep liquor powder 15g / L, soy peptone 15g / L, yeast powder 15g / L.

7. The method according to claim 1, characterized in that The inoculum before fermentation in the fermenter is provided by seed tank culture; Preferably, the seed tank culture is carried out under the conditions of 30-32° C., pH 7.00, and DO 30-40% for 12 hours and then inoculated into a fermentation tank.

8. The method according to claim 7, characterized in that The culture medium used in the seed tank culture is: 60g / L glucose, 15g / L yeast extract, 3g / L corn steep liquor powder, 5g / L ammonium sulfate, 3g / L K2HPO4, 1g / L MgSO4, 10ug / L biotin, 1mg / L vitamin B1, and 0.1mg / L vitamin B2.

9. The method according to claim 8, characterized in that Seed tank culture is provided by shake flask culture before cultivation; Preferably, the shake flask is cultured at 30-32°C and 200-220 rpm for 12 h and then inoculated into a seed tank.

10. The method according to claim 9, characterized in that The culture medium used for the plate culture is: glucose 5g / L, yeast powder 5g / L, soy peptone 9g / L, urea 3g / L, succinic acid 0.5g / L, K2HPO4 1g / L, MgSO4 0.1g / L, biotin 10ug / L, vitamin B1 100ug / L, MOPS 20g / L.