A method for producing l-citrulline by fermentation

Abstract

The application provides a method for fermentative production of L-citrulline, and belongs to the technical field of fermentation. In the fermentative production of L-citrulline, an arginine solution is used as a pH regulator, the pH value is adjusted by adding the arginine solution instead of ammonia water, and a glucose solution is added for fermentation culture. The method synchronously carries out fermentation and conversion, can omit ammonia water and sulfuric acid, maximally utilizes ammonium ions, shortens a production cycle, significantly reduces production cost, and protects the environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation, and particularly relates to a method for producing L-citrulline through fermentation. BACKGROUND

[0002] As a functional amino acid, L-citrulline has the functions of inhibiting oxidation and arteriosclerosis, scavenging free radicals, dilating blood vessels, enhancing male sexual function, improving pancreatic islet metabolism, regulating cholesterol and blood pressure, etc., and is often added to functional beverages and foods as a nutritional supplement.

[0003] In modern food industry, L-citrulline is generally produced by fermentation method. In this method, Escherichia coli producing arginine desiminase is used as the fermentation strain, glucose is used as the carbon source, and nitrogen sources such as yeast extract powder, peptone, ammonium chloride and inorganic salts such as magnesium sulfate and potassium dihydrogen phosphate are supplemented. Ammonia water is used to control the pH value, the residual sugar level is controlled during the fermentation process, and oxygen consumption fermentation is carried out. The cell secretes arginine desiminase, which catalytically converts arginine into L-citrulline in one step. The patent with publication number CN116004740A discloses a method for producing L-citrulline, and the arginine-citrulline conversion rate can reach 100%. However, the fermentation process needs to rely on ammonia water or liquid ammonia to maintain the pH value at 7.0, and the feeding medium containing yeast powder and peptone needs to be added to supplement the nitrogen source. After 50-55h of fermentation, 10-12h of enzyme conversion is still needed. The ammonium released during the conversion process is wasted, and the addition of nitrogen source increases the production cost. For example, the patent CN118931987A discloses a method for producing L-citrulline from L-arginine fermentation waste liquid. The conversion rate after fermentation and conversion is more than 98%, but the alkaline substance still needs to be added to control the stable pH value of fermentation. The enzyme solution obtained by fermentation needs to be converted for 12-16h, the production efficiency is low, and the ammonium ion released during the conversion process is wasted. It can be seen that the previous fermentation method for producing L-citrulline generally has the problems of reagent waste, low production efficiency and ammonium ion waste. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a method for producing L-citrulline through fermentation. By adding L-arginine solution instead of ammonia water to adjust the pH value, the consumption of subsequent acid reagents is omitted. The ammonium ion released when L-arginine is converted into L-citrulline by arginine desiminase is used as the inorganic nitrogen source for fermentation, which realizes the recycling of waste and shortens the fermentation period to improve the production efficiency.

[0005] The present application provides a method for producing L-citrulline through fermentation, which comprises the following steps:

[0006] inoculating a fermentation bacterium that produces arginine deiminase into a fermentation medium for fermentation and culturing;

[0007] During the fermentation, arginine solution and glucose solution were added simultaneously until the fermentation broth OD 600nm No more increase;

[0008] The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%;

[0009] The amount of the arginine solution added is such that the pH value of the fermentation system is kept neutral.

[0010] Preferably, the fermentation medium comprises the following components: yeast extract 4-6 g / L, peptone 10-12 g / L, potassium dihydrogen phosphate 6-8 g / L, disodium hydrogen phosphate dodecahydrate 18-20 g / L, ammonium sulfate 4-6 g / L, glucose 18-20 g / L, kanamycin 30-40 mg / L and IPTG 0.04-0.05 g / L.

[0011] Preferably, the flow acceleration of the glucose solution is such that the glucose concentration in the fermentation system is maintained within the range of 1-6 g / L.

[0012] Preferably, the fermentation temperature is 36-37°C; the pH value of the fermentation system is 6.9-7.1;

[0013] The fermentation pressure is 0.10-0.12 MPa; the ventilation ratio is 0.5-0.6 vvm; the rotation speed is 300-400 r / min; and the fermentation cycle is 28-32 h.

[0014] Preferably, the OD of the fermentation system 600nm When it reaches 20, add IPTG;

[0015] The fermentation temperature was lowered to 29-30° C. before IPTG addition.

[0016] Preferably, the fermentation bacteria producing arginine deiminase is Escherichia coli with a deposit number of CCTCC NO: M 2025599 ( Escherichia coli ) strain DXMC015.

[0017] Preferably, the seed liquid of the fermentation bacteria producing arginine deiminase is obtained by subjecting the fermentation bacteria producing arginine deiminase to shake flask culture, primary seed culture and secondary seed culture.

[0018] Preferably, the shake flask culture medium comprises the following components: yeast extract 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L;

[0019] The rotation speed of the shake flask culture is 180-200 r / min, the temperature of the shake flask culture is 36-37 DEG C, the pH value of the shake flask culture system is 6.9-7.1, and the cycle of the shake flask culture is 8-12 h.

[0020] Preferably, the culture medium of the primary seed culture comprises the following components: yeast extract powder 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L.

[0021] The temperature of the primary culture is 36-37 DEG C, the pressure of the primary culture is 0.08-0.10 MPa, the ventilation ratio of the primary culture is 0.5-0.6 vvm, the stirring speed of the primary culture is 300-400 r / min, and the cycle of the primary culture is 6-8 h.

[0022] Preferably, the culture medium of the secondary seed culture comprises the following components: yeast extract powder 4-6 g / L, tryptone 10-12 g / L, potassium dihydrogen phosphate 2-4 g / L, dipotassium hydrogen phosphate 11-13 g / L, magnesium sulfate heptahydrate 1-3 g / L, glucose 20-22 g / L and kanamycin 30-40 mg / L.

[0023] The temperature of the secondary seed culture is 36-37 DEG C, the pH value of the secondary seed culture system is 6.9-7.1, the pressure of the secondary seed culture is 0.08-0.10 MPa, the ventilation ratio of the secondary seed culture is 0.5-0.6 vvm, the stirring speed of the secondary seed culture is 300-400 r / min, and the cycle of the secondary seed culture is 6-8 h.

[0024] The application provides a method for producing L-citrulline by fermentation, comprising the following steps: inoculating a fermentation strain producing arginine desiminase into a fermentation medium for fermentation culture; simultaneously adding an arginine solution and a glucose solution into the fermentation liquid during the fermentation culture, and the OD 600nmNo longer increases; when the mass concentration of glucose in the fermentation system is ≤0.05%, the fermentation is terminated; the amount of the arginine solution added is such that the pH value of the fermentation system is maintained at a neutral value. The present invention aims to solve the problem that the fermentation bacteria producing arginine deiminase metabolize glucose to reduce the pH value of the fermentation system during the aerobic fermentation process, and ammonia water is needed to adjust the pH value in order to produce arginine deiminase. At the same time, arginine, as an alkaline substrate, needs to be supplemented with sulfuric acid to adjust the system to neutral or acidic in order to ensure the catalytic efficiency of arginine deiminase, and a large amount of sulfuric acid and ammonia water is wasted in the process. The present invention changes the way and timing of adding raw materials. Arginine solution is added at the beginning of fermentation culture, thereby replacing the role of ammonia water in adjusting the pH to neutrality, omitting the addition of ammonia water, and omitting the step of adding sulfuric acid to adjust the pH in the step-by-step fermentation conversion. The addition of sulfuric acid is omitted, saving the investment cost of ammonia water and sulfuric acid. At the same time, by adding arginine at the beginning of fermentation, the conversion and fermentation are carried out simultaneously, not only ensuring a 100% conversion rate of arginine, but also greatly shortening the fermentation cycle and improving production efficiency. In addition, in the fermentation method of the prior art, fermentation and conversion distribution are carried out and there is also the problem of ammonium ion removal causing waste. The technical solution of the present invention will be carried out synchronously with conversion and fermentation, and the arginine deiminase catalytic conversion L-citrulline process will remove ammonium ions as the growth of inorganic nitrogen source supply fermentation bacteria, thereby omitting the removal step of ammonium ions and making waste conversion utilization, while meeting the concept of sustainable development and green environmental protection. The results of the embodiments show that the technical solution of the present invention is used to ferment and produce L-citrulline. According to the calculation of 10,000 tons per year of an L-citrulline workshop, 3000~4000 tons of ammonia and 1500~1800 tons of sulfuric acid can be saved, reducing costs by 2.5~4.5 million yuan / year. At the same time, when the acid production level and conversion rate level are not reduced, the time required for independent conversion is saved. Relative to the prior art, each batch of production can be shortened by 4~6h. According to the calculation of 10,000 tons per year of an L-citrulline workshop, 1000~1500 tons of output can be increased, and the output value is increased by 4500~70 million yuan.

[0025] Biological deposit information

[0026] Escherichia coli ( Escherichia coli ) strain DXMC015, deposited with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 27, 2025. The deposit number is CCTCC NO: M 2025599. DETAILED DESCRIPTION

[0027] The present invention provides a method for producing L-citrulline by fermentation, comprising the following steps:

[0028] inoculating a fermentation bacterium that produces arginine deiminase into a fermentation medium for fermentation and culturing;

[0029] During the fermentation culture, the arginine solution and the glucose solution are simultaneously fed to the fermentation broth OD 600nm No longer increase;

[0030] The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%.

[0031] The feeding amount of the arginine solution is to maintain the pH value of the fermentation system to be neutral.

[0032] The present application inoculates the arginine deiminase-producing fermentation bacteria into the fermentation medium for fermentation culture.

[0033] In the present application, the type of the arginine deiminase-producing fermentation bacteria is not particularly limited, and the arginine deiminase-producing fermentation bacteria known in the art can be used, for example, the bacteria body for producing L-citrulline disclosed in the patent with publication number CN11893198A. In the embodiment of the present application, the arginine deiminase-producing fermentation bacteria is the Escherichia coli (E. coli) strain DXMC015 with the preservation number CCTCC NO: M 2025599. Escherichia coli

[0034] ​In the present application, the seed liquid of the arginine desiminase-producing fermentation bacteria is preferably obtained by shake flask culture, first-stage seed culture and second-stage seed culture of the arginine desiminase-producing fermentation bacteria. In the shake flask culture, the inoculation amount of the arginine desiminase-producing fermentation bacteria is 1-3 loops per flask, which can be 2 loops per flask. The shake flask culture medium preferably comprises the following components: yeast extract powder 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; which can be yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L and kanamycin 45 mg / L. The rotation speed of the shake flask culture is preferably 180-200 r / min, which can be 190 r / min. The temperature of the shake flask culture is preferably 36-37℃, which can be 36.5℃. The system pH value of the shake flask culture is preferably 6.9-7.1, which can be 7.0. The cycle of the shake flask culture is preferably 8-12 h, which can be 9-11 h, or 10 h. The medium of the first-stage seed culture preferably comprises the following components: yeast extract powder 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; which can be yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L and kanamycin 45 mg / L. The temperature of the first-stage seed culture is preferably 36-37℃, which can be 36.5℃. The pressure of the first-stage seed culture is preferably 0.08-0.10 MPa, which can be 0.09 MPa. The ventilation ratio of the first-stage seed culture is preferably 0.5-0.6vvm, which can be 0.55vvm. The stirring speed of the first-stage seed culture is preferably 300-400 r / min, which can be 350 r / min. The cycle of the first-stage seed culture is preferably 6-8 h, which can be 7 h. The medium of the second-stage seed culture preferably comprises the following components: yeast extract powder 4-6 g / L, peptone 10-12 g / L, potassium dihydrogen phosphate 2-4 g / L, dipotassium hydrogen phosphate 11-13 g / L, magnesium sulfate heptahydrate 1-3 g / L, glucose 20-22 g / L and kanamycin 30-40 mg / L; which can be yeast extract powder 5 g / L, peptone 11 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 2 g / L, glucose 21 g / L and kanamycin 35 mg / L. The temperature of the second-stage seed culture is preferably 36-37℃, which can be 36.5℃. The system pH value of the second-stage seed culture is preferably 6.9-7.1, which can be 7.0. The pressure of the second-stage seed culture is preferably 0.08-0.10 MPa, which can be 0.09 MPa. The ventilation ratio of the second-stage seed culture is preferably 0.5-0.6vvm, which can be 0.55vvm. The stirring speed of the second-stage seed culture is preferably 300-400 r / min, which can be 350 r / min.The period of the secondary seed culture is preferably 6-8h, and can be 7h.

[0035] In the present application, the inoculation amount of the arginine deiminase-producing fermentation bacteria in the fermentation medium is 1%-3%, and can be 2%. The fermentation medium preferably comprises the following components in the following amounts: yeast extract powder 4-6g / L, peptone 10-12g / L, potassium dihydrogen phosphate 6-8g / L, disodium phosphate dodecahydrate 18-20g / L, ammonium sulfate 4-6g / L, glucose 18-20g / L, kanamycin 30-40mg / L, and IPTG 0.04-0.05g / L; and can be yeast extract powder 5g / L, peptone 11g / L, potassium dihydrogen phosphate 7g / L, disodium phosphate dodecahydrate 19g / L, ammonium sulfate 5g / L, glucose 19g / L, kanamycin 35mg / L, and IPTG 0.045g / L. The temperature of the fermentation culture is preferably 36-37℃, and can be 36.5℃. The pH value of the fermentation system is preferably 6.9-7.1, and can be 7.0. The pressure of the fermentation culture is 0.10-0.12MPa, and can be 0.11MPa. The ventilation ratio of the fermentation culture is 0.5-0.6vvm, and can be 0.55vvm. The rotation speed of the fermentation culture is 300-400r / min, and can be 350r / min. The period of the fermentation culture is 28-32h, and can be 29-31h, or 30h.

[0036] In the present application, the feeding rate of the glucose solution is preferably such that the glucose concentration in the fermentation system is maintained in the range of 1-6g / L, and can be 2-5g / L, or 3-4g / L. The concentration of the glucose solution is preferably 60%-70%, and can be 65%. The glucose solution provides carbon source for the growth of the arginine deiminase-producing fermentation bacteria.

[0037] In the present application, the concentration of the arginine solution is preferably 35%-40%, and can be 38%. The feeding rate of the arginine solution is not specifically limited, so as to maintain the pH value of the fermentation system at neutral, for example, in the range of 6.9-7.1.

[0038] In the present application, the OD 600nm IPTG is added when the OD reaches 20. The fermentation temperature is reduced to 29-30℃ before the addition of IPTG. The purpose of adding IPTG is to induce the expression of arginine deiminase and promote secretion.

[0039] In the present application, the method for fermentatively producing L-citrulline, by using arginine solution as a pH regulator, by adding arginine solution to replace ammonia water to adjust pH, while arginine deiminase converts arginine into L-citrulline and releases ammonium ions, the ammonium ions which should be treated as waste in the subsequent extraction process can be used as inorganic nitrogen source for fermentation, thereby achieving the saving of ammonia water and effective utilization of waste ammonium ions, while the fermentation conversion is realized synchronously, which can shorten the production cycle and significantly reduce the production cost, and protect the environment.

[0040] The method for fermentatively producing L-citrulline provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0041] Example 1

[0042] The method for fermentatively producing L-citrulline comprises the following specific steps:

[0043] Step 1: culture of E. coli seed

[0044] In a sterile environment, a single colony of E. coli strain with preservation number CCTCC NO: M 2025599 is picked from a plate or slant and transferred to a shake flask containing seed culture medium for shake culture at pH 7, 37℃, 200r / min for 8h to obtain a shake flask seed; the formula of the seed culture medium is: yeast extract powder 5g / L, tryptone 10g / L, sodium chloride 9g / L, kanamycin 50mg / L.

[0045] The shake flask seed is inoculated into a primary seed tank containing culture medium for culture to obtain a primary culture seed. The formula of the primary seed tank culture medium is: yeast extract powder 5g / L, tryptone 10g / L, sodium chloride 9g / L, kanamycin 50mg / L; the culture conditions of the primary seed tank are: temperature 37℃, tank pressure 0.08MPa, ventilation ratio 0.5vvm, stirring 400r / min, culture period 6h; the end condition of the culture in the primary seed tank is: the absorbance is measured at a wavelength of 600nm using a spectrophotometer, and the seed liquid OD value reaches 3 when the culture is accepted;

[0046] The primary culture seed is inoculated into a secondary seed tank through an inoculation pipeline according to a 3% inoculation amount of the volume of the secondary seed tank culture medium for culture to obtain an E. coli seed liquid. The formula of the secondary seed tank culture medium is: yeast extract powder 5g / L, tryptone 10g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 12g / L, magnesium sulfate heptahydrate 2g / L, glucose 20g / L, and kanamycin 40mg / L; the culture conditions of the secondary seed tank are: temperature 37℃, pH value 7.1, tank pressure 0.1MPa, ventilation ratio 0.5vvm, stirring 400r / min, culture period 8h.

[0047] Step two, L-citrulline fermentation

[0048] The E. coli seed liquid was inoculated into a 7 m³ fermenter containing 3 m³ fermentation medium for cultivation. The fermentation medium formula was as follows: yeast extract powder 5 g / L, peptone 10 g / L, potassium dihydrogen phosphate 6 g / L, sodium hydrogen phosphate dodecahydrate 18 g / L, ammonium sulfate 5 g / L, glucose 20 g / L, kanamycin 30 mg / L, and IPTG 0.048 g / L. The inoculation conditions were as follows: the absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the seed liquid OD value reached 3 when inoculation was performed; the inoculation amount of the secondary seed culture was 2% of the volume of the fermentation medium in the fermenter; and the fermentation culture conditions were as follows: the initial temperature was 37 °C, the pH value was 7.0, the tank pressure was 0.1 MPa, the ventilation ratio was 0.5 vvm, and the stirring speed was 300 r / min.

[0049] During the fermentation process, a 35% arginine solution was fed to maintain the pH value to 7.0, and a 65% glucose solution was fed to control the glucose concentration to 2-5 g / L. When the OD value of the fermentation broth was 20.25 at 8 h, the temperature was lowered to 30 °C, an IPTG solution was pumped into the fermenter to a final concentration of 0.048 g / L, and the culture was continued. The culture period was 30 h, and when the OD value began to decrease, the feeding of the glucose solution and the arginine solution was stopped. The culture period was 32 h, the glucose concentration in the fermentation broth was 0.022%, and the fermentation was completed. L-citrulline was obtained. 600nm

[0050] After the fermentation was completed, the contents of the obtained L-citrulline and L-arginine were determined, and the details are described in the following prior art ([1] Li W, Tao QY, Li WL. L-glutamine and L-arginine fermentation production [M]. Beijing: Chemical Industry Press, 2021: 191-192. [2] Ma Y, Su LQ, Wu D, et al. Optimization of process conditions for preparing L-citrulline by recombinant arginine deiminase [J]. Biotechnology Bulletin, 2015, 31(8): 180-18.)

[0051] The L-citrulline concentration in the fermentation broth was 110.20 g / L, the L-arginine concentration was 0, and the L-citrulline conversion rate was 100%. No ammonia water and sulfuric acid were consumed during the fermentation process.

[0052] Example 2

[0053] A method for fermenting L-citrulline, and the specific steps are as follows:

[0054] Step one, cultivation of E. coli seed

[0055] ​In a sterile environment, a single colony of E. coli strain with preservation number CCTCC No: M 2025599 was picked from a plate or slant and transferred to a shake flask containing seed culture medium to obtain a shake flask seed; the seed culture medium formula is: yeast extract powder 4 g / L, tryptone 9 g / L, sodium chloride 9 g / L, kanamycin 45 mg / L; the shake flask culture conditions are: temperature 37°C, rotation speed 180 r / min, pH 7, culture period 10 h.

[0056] The shake flask seed was transferred to a first-stage seed tank for culture until the OD value reached 3 to obtain a first-stage culture seed. The first-stage seed tank culture medium formula is: yeast extract powder 6 g / L, tryptone 10 g / L, sodium chloride 10 g / L, kanamycin 45 mg / L; the first-stage seed tank culture conditions are: temperature 37°C, tank pressure 0.08 MPa, ventilation ratio 0.6 vvm, stirring 350 r / min, culture period 7 h. 600nm

[0057] The first-stage culture seed was transferred to a second-stage seed tank through a transfer pipeline for culture to obtain an E. coli seed liquid, wherein the transfer amount of the first-stage culture seed is 2.5% of the volume of the second-stage seed tank culture medium. The second-stage seed tank culture medium formula is: yeast extract powder 4 g / L, tryptone 12 g / L, potassium dihydrogen phosphate 2.5 g / L, dipotassium hydrogen phosphate 11 g / L, magnesium sulfate heptahydrate 2.5 g / L, glucose 22 g / L, kanamycin 30 mg / L; the second-stage seed tank culture conditions are: temperature 37°C, pH 7.0, tank pressure 0.09 MPa, ventilation ratio 0.55 vvm, stirring 350 r / min, culture period 6 h.

[0058] Step two, L-citrulline fermentation

[0059] The E. coli seed liquid was transferred to a 7 m³ fermentation tank containing 3 m³ fermentation culture medium for culture. The fermentation culture medium formula includes: yeast extract powder 6 g / L, tryptone 10 g / L, potassium dihydrogen phosphate 7 g / L, sodium hydrogen phosphate dodecahydrate 19 g / L, ammonium sulfate 4 g / L, glucose 19 g / L, kanamycin 32 mg / L, and IPTG 0.045 g / L. The transfer conditions are: the absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the seed liquid was transferred when the OD value reached 3; the transfer amount of the second-stage seed culture is 2.5% of the volume of the fermentation tank culture medium; the fermentation culture conditions are: initial temperature 37°C, pH 6.9, tank pressure 0.12 MPa, ventilation ratio 0.55 vvm, stirring 350 r / min;

[0060] ​The fermentation process is carried out by adding 38% arginine solution to maintain pH value to 6.9, and adding 60% glucose solution to control glucose concentration to 3-5 g / L. When the OD value of the fermentation liquid is 21.1 after 8 h of fermentation, the temperature is reduced to 29℃, IPTG is pumped into the fermentation tank to a final concentration of 0.045 g / L, and the culture is continued. The culture period is 28 h, the OD value decreases, the addition of glucose solution and arginine solution is stopped, the culture period is 30 h, the glucose concentration of the fermentation liquid is 0.035%, and the fermentation is completed to obtain L-citrulline.

[0061] After the fermentation is completed, the concentration of L-citrulline in the fermentation liquid is 109.80 g / L, the concentration of L-arginine is 0, and the conversion rate of L-citrulline is 100%. The fermentation process does not involve the use of ammonia and sulfuric acid.

[0062] Example 3

[0063] A method for producing L-citrulline by fermentation, the specific steps are as follows:

[0064] Step 1: Culture of E. coli seed

[0065] In a sterile environment, a single colony of E. coli strain with preservation number CCTCC No: M 2025599 is picked from a plate or slant and transferred to a shake flask containing seed culture medium to obtain a shake flask seed. The seed culture medium formula is: yeast extract powder 6 g / L, tryptone 11 g / L, sodium chloride 11 g / L, and kanamycin 42 mg / L. The shake flask culture conditions are: temperature 36.5℃, rotation speed 190 r / min, and culture period 8 h.

[0066] The shake flask culture seed is transferred to a primary seed tank for culture to obtain a primary culture seed. The primary seed tank culture medium composition is: yeast extract powder 6 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and kanamycin 42 mg / L. The primary seed tank culture conditions are: temperature 36.5℃, tank pressure 0.09 MPa, ventilation ratio 0.45 vvm, stirring 300 r / min, and culture period 8 h. The transfer conditions of the primary seed tank are: the absorbance is measured at a wavelength of 600 nm using a spectrophotometer, and the seed liquid OD value reaches 3 when the transfer is performed.

[0067] The primary culture seed is transferred to a secondary seed tank through a transfer pipeline for culture to obtain an E. coli seed solution. The transfer amount of the primary culture seed is 2.5% of the volume of the secondary seed tank culture medium. The secondary seed tank culture medium comprises 4 g / L of yeast extract powder, 11 g / L of peptone, 2.5 g / L of potassium dihydrogen phosphate, 11 g / L of dipotassium hydrogen phosphate, 2.5 g / L of magnesium sulfate heptahydrate, 22 g / L of glucose, and 30 mg / L of kanamycin. The culture conditions of the secondary seed tank are as follows: a temperature of 37°C, a pH of 6.9, a tank pressure of 0.09 MPa, a ventilation ratio of 0.55 vvm, stirring at 350 r / min, and a culture period of 7 h.

[0068] Step two, L-citrulline fermentation

[0069] The E. coli seed solution is transferred to a 7 m³ fermentation tank containing 3 m³ of fermentation medium for culture. The fermentation medium formula is as follows: 6 g / L of yeast extract powder, 12 g / L of peptone, 8 g / L of potassium dihydrogen phosphate, 20 g / L of sodium hydrogen phosphate dodecahydrate, 4.5 g / L of ammonium sulfate, 20 g / L of glucose, 35 mg / L of kanamycin, and 0.04 g / L of IPTG. The transfer conditions are as follows: the absorbance is measured at a wavelength of 600 nm by using a spectrophotometer, and the seed solution is transferred when the OD value reaches 3; the transfer amount of the secondary seed culture is 1.5% of the volume of the fermentation tank culture medium; and the fermentation culture conditions are as follows: an initial temperature of 37°C, a pH of 7.0, a tank pressure of 0.11 MPa, a ventilation ratio of 0.6 vvm, and stirring at 400 r / min.

[0070] During the fermentation process, 36% arginine solution is added to maintain a pH of 7.0, and a 70% glucose solution is added to control the glucose concentration to 1-3 g / L. When the OD value of the fermentation solution is 20.6 after 9 h of fermentation, the temperature is reduced to 30°C, IPTG is pumped into the fermentation tank to a final concentration of 0.040 g / L, and the culture is continued. When the OD value starts to decrease after 31 h of culture, the addition of the glucose solution and the arginine solution is stopped. After 32 h of culture, the glucose concentration in the fermentation solution is 0.046%, and the fermentation is completed to obtain L-citrulline.

[0071] After the fermentation is completed, the L-citrulline concentration in the obtained fermentation solution is 111.20 g / L, the L-arginine concentration is 0, and the L-citrulline conversion rate is 100%. No ammonia water and sulfuric acid are consumed during the fermentation process.

[0072] Comparative Example 1

[0073] Based on Example 1, 20% ammonia water is used to maintain a fermentation pH of 7.0 during the fermentation process. After the fermentation is completed, an arginine solution with the same amount as in Example 1 is prepared, and concentrated sulfuric acid is used to adjust the pH to 7.0 and add it to the fermentation solution. The fermentation conditions remain unchanged, and the conversion is performed for 5 h. The L-arginine concentration is 0, and the conversion is stopped.

[0074] After 32h fermentation, the concentration of L-citrulline in the obtained fermentation liquor was 107.80g / L, the concentration of L-arginine was 0, and the conversion rate of L-citrulline was 100%. The volume of ammonia water consumed in the fermentation process was 148L, and the volume of sulfuric acid consumed was 55L.

[0075] Comparative Example 2

[0076] Based on Example 2, the fermentation process used 20% ammonia water to maintain the fermentation pH, and after the fermentation was completed, an arginine solution with the same amount as in Example 2 was prepared, and concentrated sulfuric acid was used to adjust the pH to 6.9 and added to the fermentation liquor. The fermentation conditions were maintained unchanged, and the conversion was carried out for 4.5h, the concentration of L-arginine was 0, and the conversion was stopped.

[0077] After 30h fermentation, the concentration of L-citrulline in the obtained fermentation liquor was 106.20g / L, the concentration of L-arginine was 0, and the conversion rate of L-citrulline was 100%. The volume of ammonia water consumed in the fermentation process was 127L, and the volume of concentrated sulfuric acid consumed was 51L.

[0078] Comparative Example 3

[0079] Based on Example 3, the fermentation process used 20% ammonia water to maintain the fermentation pH, and after the fermentation was completed, an arginine solution with the same amount as in Example 2 was prepared, and concentrated sulfuric acid was used to adjust the pH to 6.9 and added to the fermentation liquor. The fermentation conditions were maintained unchanged, and the conversion was carried out for 5.5h, the concentration of L-arginine was 0, and the conversion was stopped.

[0080] After 32h fermentation, the concentration of L-citrulline in the obtained fermentation liquor was 108.60g / L, the concentration of L-arginine was 0, and the conversion rate of L-citrulline was 100%. The volume of ammonia water consumed in the fermentation process was 135L, and the volume of concentrated sulfuric acid consumed was 57L.

[0081] The fermentation results of the three examples and Comparative Examples 1-3 can be seen as follows:

[0082] 1. The present application can save ammonia water and sulfuric acid without reducing the acid production level and conversion rate by adding an arginine solution instead of ammonia water to adjust the pH. According to an L-citrulline workshop with an annual output of 10,000 tons, 3,000-4,000 tons of ammonia water and 1,500-1,800 tons of sulfuric acid can be saved, and the cost can be reduced by 25-45 million yuan per year.

[0083] 2. The present application can save the time required for separate conversion by adding an arginine solution instead of ammonia water to adjust the pH without reducing the acid production level and conversion rate. Compared with the prior art, each batch can be shortened by 4-6h, and according to an L-citrulline workshop with an annual output of 10,000 tons, the output can be increased by 1,000-1,500 tons, and the production value can be increased by 45-70 million yuan.

[0084] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for producing L-citrulline by fermentation, characterized in that: The following steps are involved: The fermentation bacteria producing arginine deiminase is inoculated into the fermentation medium for fermentation and culture; the fermentation bacteria producing arginine deiminase is Escherichia coli with a preservation number of CCTCC NO: M 2025599 ( Escherichia coli ) strain DXMC015; During the fermentation, arginine solution and glucose solution were added simultaneously until the fermentation broth OD 600nm No longer increases; the flow acceleration of the glucose solution is such that the glucose concentration of the fermentation system is maintained within the range of 1~6g / L; The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%; The amount of the arginine solution added is such as to maintain the pH value of the fermentation system at neutral; The OD of the fermentation system 600nm When the pH reaches 20, add IPTG.

2. The method for producing L-citrulline by fermentation according to claim 1, wherein The fermentation medium comprises the following components: 4-6 g / L yeast extract, 10-12 g / L peptone, 6-8 g / L potassium dihydrogen phosphate, 18-20 g / L disodium hydrogen phosphate dodecahydrate, 4-6 g / L ammonium sulfate, 18-20 g / L glucose, 30-40 mg / L kanamycin, and 0.04-0.05 g / L IPTG.

3. The method for producing L-citrulline by fermentation according to claim 1, wherein The fermentation culture temperature is 36-37°C; the pH value of the fermentation system is 6.9-7.1; The fermentation pressure is 0.10-0.12 MPa; the ventilation ratio is 0.5-0.6 vvm; the rotation speed is 300-400 r / min; and the fermentation cycle is 28-32 h.

4. The method for producing L-citrulline by fermentation according to claim 1, wherein The fermentation temperature was lowered to 29-30° C. before IPTG addition.

5. The method for producing L-citrulline by fermentation according to claim 1, wherein The seed liquid of the fermentation bacteria producing arginine deiminase is obtained by subjecting the fermentation bacteria producing arginine deiminase to shaking flask culture, primary seed culture and secondary seed culture.

6. The method for producing L-citrulline by fermentation according to claim 5, wherein: The shake flask culture medium comprises the following components: yeast extract 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; The rotation speed of the shake flask culture is 180-200 r / min, the temperature of the shake flask culture is 36-37° C., the pH value of the shake flask culture system is 6.9-7.1, and the cycle of the shake flask culture is 8-12 hours.

7. The method for producing L-citrulline by fermentation according to claim 5, wherein: The culture medium for primary seed culture comprises the following components: 4-6 g / L yeast extract, 9-11 g / L tryptone, 9-11 g / L sodium chloride, and 40-50 mg / L kanamycin; The temperature of the primary culture is 36~37°C, the pressure of the primary culture is 0.08~0.10MPa, the ventilation ratio of the primary culture is 0.5~0.6vvm, the stirring speed of the primary culture is 300~400r / min, and the cycle of the primary culture is 6~8h.

8. The method for producing L-citrulline by fermentation according to claim 5, wherein: The medium for secondary seed culture comprises the following components: 4-6 g / L yeast extract, 10-12 g / L peptone, 2-4 g / L potassium dihydrogen phosphate, 11-13 g / L dipotassium hydrogen phosphate, 1-3 g / L magnesium sulfate heptahydrate, 20-22 g / L glucose, and 30-40 mg / L kanamycin. The temperature of the secondary seed culture is 36~37°C, the system pH value of the secondary seed culture is 6.9~7.1, the pressure of the secondary seed culture is 0.08~0.10MPa, the ventilation ratio of the secondary seed culture is 0.5~0.6vvm, the stirring speed of the secondary seed culture is 300~400r / min, and the cycle of the secondary seed culture is 6~8h.