Method for improving fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan

Abstract

The invention provides a method for improving fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, alpha-ketoglutaric acid ferrous salt, alpha-ketoglutaric acid zinc salt, alpha-ketoglutaric acid cobalt salt and alpha-ketoglutaric acid manganese salt are added into a fermentation culture medium according to a specific proportion, alpha-ketoglutaric acid directly participates in TCA circulation, circulation operation can be accelerated, generation of NADH and FADH2 is increased, the yield of 5-hydroxytryptophan is increased, and the yield of 5-hydroxytryptophan is increased. The ATP synthesis efficiency is improved, and more energy is provided for cell growth; the alpha-ketoglutaric acid can balance amino acids required in the growth process of escherichia coli, promote the generation of proteins and nucleic acids and accelerate the growth of thalli; the alpha-ketoglutaric acid metal salt can improve the utilization efficiency of microelements by thalli; ferrous ions are cofactors of 5-hydroxytryptophan hydroxylase and can accelerate the hydroxylation process of tryptophan. Through the synergistic effect of the added components, the yield of the 5-hydroxytryptophan and the sugar-acid conversion rate are improved.

Description

Technical Field

[0001] The invention relates to the technical field of 5-hydroxytryptophan fermentation production, in particular to a method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan. Background Art

[0002] 5-Hydroxytryptophan (5-HTP) is a precursor for the synthesis of serotonin and melatonin in mammals. It plays a key role in regulating important physiological processes such as mood and behavior control, pain and appetite perception, and sleep, and has a high medical and health value. Currently, the main methods for industrial production of 5-HTP are extraction, enzymatic catalysis, and fermentation. The extraction method primarily extracts 5-HTP from natural products such as Griffonia suffruticosa seeds. However, due to the limited availability of raw materials due to regional and seasonal constraints, this method has limited production capacity and is unable to meet the growing market demand. The enzymatic method uses tryptophan as a raw material, catalyzed by hydroxylase and tetrahydropterin coenzyme, but the production cost is relatively high. The fermentation method, which uses metabolically engineered Escherichia coli and glucose as a raw material, offers advantages such as low cost, short production cycle, and relatively low environmental pollution, making it one of the most promising methods for large-scale industrial production of 5-HTP. However, this method still faces challenges such as low yield and low sugar-to-acid conversion rate, which need to be addressed. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for improving the fermentation yield of 5-hydroxytryptophan and the sugar-acid conversion rate.

[0004] In order to solve the above technical problems, the technical solution of the present invention is:

[0005] A method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan comprises producing 5-hydroxytryptophan by a microbial fermentation method, adding α-ketoglutarate ferrous salt, α-ketoglutarate zinc salt, α-ketoglutarate cobalt salt, and α-ketoglutarate manganese salt to a fermentation medium, wherein the added amounts are 20-100 mg / L of α-ketoglutarate ferrous salt, 5-30 mg / L of α-ketoglutarate zinc salt, 5-30 mg / L of α-ketoglutarate cobalt salt, and 5-30 mg / L of α-ketoglutarate manganese salt, and simultaneously feeding 10-200 mg / L of α-ketoglutarate ferrous salt during the fermentation culture process.

[0006] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, the added amount is 30-80 mg / L of α-ketoglutarate ferrous salt, 10-20 mg / L of α-ketoglutarate zinc salt, 10-15 mg / L of α-ketoglutarate cobalt salt, and 10-20 mg / L of α-ketoglutarate manganese salt.

[0007] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, the added amount is 80 mg / L of α-ketoglutarate ferrous salt, 20 mg / L of α-ketoglutarate zinc salt, 15 mg / L of α-ketoglutarate cobalt salt, and 20 mg / L of α-ketoglutarate manganese salt.

[0008] Preferably, the above method for increasing the fermentation yield of 5-hydroxytryptophan and the sugar-acid conversion rate comprises the following specific steps:

[0009] (1) Activation of the strain: streak the 5-hydroxytryptophan-producing strain onto an activation slant, culture at 36°C for 16 h, and then transfer to an eggplant-shaped flask and continue culture for 14 h;

[0010] (2) Seed culture: The activated strain was transferred to a seed culture medium for culture, with the pH maintained at 7.0-7.2, the temperature constant at 36°C, and the dissolved oxygen controlled at 20%-60%;

[0011] (3) Fermentation culture: The amount of seed bacteria (OD 600 ) reaches 20, and the seed liquid is inoculated into the fermentation medium according to the inoculation amount of 15%-25%, wherein the fermentation medium composition is glucose 15g / L, citric acid 4.0g / L, yeast powder 6.0g / L, K2HPO44.5g / L, MgSO4·7H2O 2.0g / L, V H 1mg / L, V B1 2mg / L, V B3 The invention relates to a fermentation tank comprising the following ingredients: 1) a ferrous salt of α-ketoglutarate, 2 mg / L a-ketoglutarate ferrous salt, 20-100 mg / L a-ketoglutarate zinc salt, 5-30 mg / L a-ketoglutarate cobalt salt, and 5-30 mg / L a-ketoglutarate manganese salt; 2) during the fermentation process, the pH is stably controlled at 6.7-7.0, the temperature is maintained at 36°C, the dissolved oxygen is controlled at 20%-60%, and the glucose concentration in the tank is maintained below 0.3 g / L by feeding a 700 g / L glucose solution, and 10-200 mg / L a-ketoglutarate ferrous salt is simultaneously fed along with the sugar flow.

[0012] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, the activated slant culture medium used in the bacterial activation comprises 1 g / L glucose, 5 g / L yeast extract powder, 10 g / L peptone, 1.2 g / L KH2PO4, 0.4 g / L MgSO4, 3.0 g / L sodium chloride, and 25 g / L agar powder, and the pH is controlled at 7.0-7.2.

[0013] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, the seed culture medium used in the seed culture comprises 35 g / L glucose, 3.0 g / L citric acid, 6 g / L yeast powder, 1 g / L MgSO4·7H2O, 2.0 g / L KH2PO4, 1.0 g / L (NH4)2SO4, V H 1mg / L, V B1 2mg / L, V B3 2mg / L.

[0014] Preferably, the method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan is as follows: the fermentation medium comprises 15 g / L glucose, 4.0 g / L citric acid, 6.0 g / L yeast extract, 4.5 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, and V H 1mg / L, V B1 2mg / L, V B3 2mg / L, α-ketoglutarate ferrous salt 80mg / L, α-ketoglutarate zinc salt 20mg / L, α-ketoglutarate cobalt salt 15mg / L, α-ketoglutarate manganese salt 20mg / L, pH 7.0-7.2.

[0015] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, 5-10 ml of fermentation liquid is sampled every 2 hours during the fermentation culture process, and the pH is maintained by adding ammonia water through a peristaltic pump.

[0016] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, 100 mg / L of ferrous α-ketoglutarate is added during the fermentation culture process.

[0017] Preferably, in the above method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, during the fermentation culture process, the addition of ferrous α-ketoglutarate is stopped 4 hours before the end of fermentation.

[0018] Beneficial effects:

[0019] The above-mentioned method for increasing the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan comprises adding α-ketoglutarate ferrous salt, α-ketoglutarate zinc salt, α-ketoglutarate cobalt salt, and α-ketoglutarate manganese salt to the fermentation medium in a specific ratio. α-ketoglutarate directly participates in the TCA cycle, can accelerate the cycle operation, increase the generation of NADH and FADH2, improve ATP synthesis efficiency, and provide more energy for cell growth; α-ketoglutarate can balance the amino acids required for the growth process of Escherichia coli, promote the production of proteins and nucleic acids, and accelerate bacterial growth; α-ketoglutarate metal salts can improve the utilization efficiency of trace elements by bacteria; ferrous ions are a cofactor of 5-hydroxytryptophan hydroxylase and can accelerate the tryptophan hydroxylation process. Through the synergistic effect of the added components, the bacterial viability and tryptophan hydroxylation rate during the fermentation process are effectively improved, thereby increasing the 5-hydroxytryptophan yield and sugar-acid conversion rate, having certain economic benefits and being suitable for industrial production. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0021] The 5-hydroxytryptophan-producing strain used in the examples is the 5-hydroxytryptophan-producing strain E. coli HTP20 in CN118979005B.

[0022] Example 1

[0023] A method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate, comprising the following steps:

[0024] (1) Activation of strains: The 5-hydroxytryptophan-producing strain was streaked onto an activated slant, cultured at 36°C for 16 h, and then transferred to an eggplant-shaped flask for further culture for 14 h. The activated slant culture medium used consisted of 1 g / L glucose, 5 g / L yeast extract powder, 10 g / L peptone, 1.2 g / L KH2PO4, 0.4 g / L MgSO4, 3.0 g / L sodium chloride, and 25 g / L agar powder, with the pH controlled at 7.0-7.2.

[0025] (2) Seed culture: The activated strain was transferred to a seed culture medium for culture, with the pH maintained at 7.0-7.2, the temperature constant at 36°C, and the dissolved oxygen controlled at 20%-60%. The seed culture medium used was composed of 35 g / L glucose, 3.0 g / L citric acid, 6 g / L yeast powder, 1 g / L MgSO4·7H2O, 2.0 g / L KH2PO4, 1.0 g / L (NH4)2SO4, and V H 1mg / L, V B1 2mg / L, V B3 2mg / L.

[0026] (3) Fermentation culture: The amount of seed bacteria (OD 600 ) reaches about 20, and the seed liquid is inoculated into a fresh fermentation medium according to a 20% inoculation amount. The fermentation medium composition is glucose 15g / L, citric acid 4.0g / L, yeast powder 6.0g / L, K2HPO4 4.5g / L, MgSO4·7H2O 2.0g / L, V H 1mg / L, V B1 2mg / L, V B3 2mg / L, ferrous sulfate 40mg / L, pH 7.0-7.2. During the fermentation process, the pH was stably controlled at 6.7-7.0, the temperature was maintained at 36°C, the dissolved oxygen was controlled at 20%-60%, and the glucose concentration in the tank was maintained below 0.3g / L (0-0.3g / L) by feeding 700g / L glucose solution. 5-10ml of the fermentation broth was sampled every 2 hours for testing, and the pH was maintained by feeding ammonia water via a peristaltic pump.

[0027] After 40h of fermentation, the highest yield of 5-hydroxytryptophan and the largest bacterial volume (OD 600 ) and sugar-acid conversion rates reached 25.3 g / L, 140, and 10.1%, respectively.

[0028] This part of the examples does not use α-ketoglutaric acid trace element mixed salts, and is intended to serve as a blank control group to form a comparison with Examples 2-4.

[0029] Example 2

[0030] A method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, referring to Example 1, wherein the strain activation and seed culture are the same as in Example 1, and the fermentation culture is different from that in Example 1 in that the fermentation medium comprises 15 g / L glucose, 4.0 g / L citric acid, 6.0 g / L yeast powder, 4.5 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, and V H 1mg / L, V B1 2mg / L, V B3 2mg / L, α-ketoglutarate ferrous salt 30mg / L, α-ketoglutarate zinc salt 10mg / L, α-ketoglutarate cobalt salt 10mg / L, α-ketoglutarate manganese salt 10mg / L.

[0031] After 40h of fermentation, the highest yield of 5-hydroxytryptophan and the largest bacterial volume (OD 600 ) and sugar-acid conversion rates reached 28.6 g / L, 149, and 12.2%, respectively.

[0032] Example 3

[0033] A method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, referring to Example 1, wherein the strain activation and seed culture are the same as in Example 2, and the fermentation culture is different from that in Example 2 in that the fermentation medium comprises 15 g / L glucose, 4.0 g / L citric acid, 6.0 g / L yeast powder, 4.5 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, and V H 1mg / L, V B1 2mg / L, V B3 2mg / L, α-ketoglutarate ferrous salt 80mg / L, α-ketoglutarate zinc salt 20mg / L, α-ketoglutarate cobalt salt 15mg / L, α-ketoglutarate manganese salt 20mg / L.

[0034] After 40h of fermentation, the highest yield of 5-hydroxytryptophan and the largest bacterial volume (OD 600 ) and sugar-acid conversion rates reached 31.3 g / L, 153, and 13.1%, respectively.

[0035] Example 4

[0036] A method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, referring to Example 1, wherein the strain activation and seed culture are the same as in Example 2, and the fermentation culture differs from Example 2 in that the fermentation medium components are the same as in Example 3, and 100 mg / L of α-ketoglutarate ferrous salt is added along with the sugar during the fermentation process (a mother liquor of α-ketoglutarate ferrous salt solution with a concentration of 100 g / L is prepared with deionized water, 2 mL of the solution is taken, aliquoted, added to a 700 g / L glucose solution, and shaken before feeding). The feeding is stopped after fermentation for 36 hours.

[0037] The fermentation was completed after 40h. The highest yield of 5-hydroxytryptophan and the largest bacterial volume (OD 600 ) and sugar-acid conversion rates reached 35.2 g / L, 162, and 15.2%, respectively.

[0038] As can be seen from the results of Examples 1-4, α-ketoglutaric acid trace element mixed salt has a more obvious promoting effect on the final yield and maximum bacterial volume of 5-hydroxytryptophan. The method for improving the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan of the present invention, a specific amount of α-ketoglutaric acid trace element mixed salt is added during the fermentation process, α-ketoglutaric acid directly participates in the TCA cycle, increases the generation of NADH and FADH2, improves ATP synthesis efficiency, and provides more energy for cell growth; α-ketoglutaric acid can balance the amino acids required for the growth process of Escherichia coli, promotes the generation of proteins and nucleic acids, and accelerates bacterial growth; α-ketoglutaric acid metal salts can improve the utilization efficiency of trace elements by bacteria. Ferrous ion is a cofactor of 5-hydroxytryptophan hydroxylase and can accelerate the tryptophan hydroxylation process. Ultimately, the bacterial viability and tryptophan hydroxylation rate during the fermentation process are effectively improved. Fermentation 40h, maximum bacterial volume (OD 600 ) reached 162, the maximum 5-hydroxytryptophan production reached 35.2 g / L, and the sugar-acid conversion rate reached 15.2%, which were increased by 15.7%, 39.1% and 50.5% respectively compared with the non-optimized ones, achieving a significant increase in 5-hydroxytryptophan production and sugar-acid conversion rate, improving economic efficiency, and being suitable for industrial production.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for increasing the fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, characterized in that: 5-hydroxytryptophan is produced by a microbial fermentation method. α-ketoglutarate ferrous salt, α-ketoglutarate zinc salt, α-ketoglutarate cobalt salt and α-ketoglutarate manganese salt are added to the fermentation medium. The added amounts are 20-100 mg / L of α-ketoglutarate ferrous salt, 5-30 mg / L of α-ketoglutarate zinc salt, 5-30 mg / L of α-ketoglutarate cobalt salt and 5-30 mg / L of α-ketoglutarate manganese salt. Simultaneously, 10-200 mg / L of α-ketoglutarate ferrous salt is fed during the fermentation culture.

2. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 1, wherein: The added amounts are 30-80 mg / L of α-ketoglutarate ferrous salt, 10-20 mg / L of α-ketoglutarate zinc salt, 10-15 mg / L of α-ketoglutarate cobalt salt, and 10-20 mg / L of α-ketoglutarate manganese salt.

3. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 1 or 2, characterized in that: The added amounts are 80 mg / L of α-ketoglutarate ferrous salt, 20 mg / L of α-ketoglutarate zinc salt, 15 mg / L of α-ketoglutarate cobalt salt, and 20 mg / L of α-ketoglutarate manganese salt.

4. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 1, wherein: The specific steps are as follows: (1) Activation of strains: streak the 5-hydroxytryptophan-producing strain onto an activated slant culture plate and transfer the plate to an eggplant-shaped flask for further culture. (2) Seed culture: The activated strain was transferred to a seed culture medium for culture, with the pH maintained at 7.0-7.2, the temperature constant at 36°C, and the dissolved oxygen controlled at 20%-60%; (3) Fermentation culture: When the seed cell count reaches 20, the seed liquid is inoculated into the fermentation medium at a rate of 15%-25%. The fermentation medium comprises 15 g / L glucose, 4.0 g / L citric acid, 6.0 g / L yeast powder, 4.5 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, V H 1mg / L, V B1 2mg / L, V B3 The invention relates to a fermentation tank comprising the following ingredients: 1) a ferrous salt of α-ketoglutarate, 2 mg / L a-ketoglutarate ferrous salt, 20-100 mg / L a-ketoglutarate zinc salt, 5-30 mg / L a-ketoglutarate cobalt salt, and 5-30 mg / L a-ketoglutarate manganese salt; 2) during the fermentation process, the pH is stably controlled at 6.7-7.0, the temperature is maintained at 36°C, the dissolved oxygen is controlled at 20%-60%, and the glucose concentration in the tank is maintained below 0.3 g / L by feeding a 700 g / L glucose solution, and 10-200 mg / L a-ketoglutarate ferrous salt is simultaneously fed along with the sugar flow.

5. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 4, characterized in that: The activation slant culture medium used in the bacterial activation comprises 1 g / L glucose, 5 g / L yeast extract powder, 10 g / L peptone, 1.2 g / L KH2PO4, 0.4 g / L MgSO4, 3.0 g / L sodium chloride, and 25 g / L agar powder, and the pH is controlled at 7.0-7.

2.

6. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 4, characterized in that: The seed culture medium used in the seed culture comprises 35 g / L glucose, 3.0 g / L citric acid, 6 g / L yeast powder, 1 g / L MgSO4·7H2O, 2.0 g / L KH2PO4, 1.0 g / L (NH4)2SO4, and 1.0 g / L V H 1mg / L, V B1 2mg / L, V B3 2mg / L.

7. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 4, characterized in that: The fermentation medium comprises 15 g / L glucose, 4.0 g / L citric acid, 6.0 g / L yeast extract, 4.5 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, V H 1mg / L, V B1 2mg / L, V B3 2mg / L, α-ketoglutarate ferrous salt 80mg / L, α-ketoglutarate zinc salt 20mg / L, α-ketoglutarate cobalt salt 15mg / L, α-ketoglutarate manganese salt 20mg / L, pH 7.0-7.

2.

8. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 4, characterized in that: During the fermentation process, 5-10 ml of the fermentation liquid was sampled every 2 hours for testing, and the pH was maintained by adding ammonia water through a peristaltic pump.

9. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 1 or 4, characterized in that: During the fermentation culture, 100 mg / L of α-ketoglutarate ferrous salt was added.

10. The method for increasing 5-hydroxytryptophan fermentation yield and sugar-acid conversion rate according to claim 1 or 4, characterized in that: During the fermentation culture process, the addition of ferrous α-ketoglutarate was stopped 4 hours before the end of the fermentation.