Method for producing L-arginine through escherichia coli fermentation

Abstract

The invention discloses a method for producing L-arginine through escherichia coli fermentation. The escherichia coli has the characteristics of high L-arginine yield and low heteroacid content. In the fermentation process, fed-batch sugar and sodium glutamate are adopted according to the thallus concentration OD600 and the fermentation period; the concentration of glutamic acid is controlled by feeding sodium glutamate or glutamic acid precursor sodium succinate and sodium citrate, the arginine acid production and sugar acid conversion rate is increased, the content of heteroacid is reduced, the final acid production can reach 13.9% at most, the sugar acid ratio is 57.2%, and the heteroacid content is not higher than 0.3%. The process solves the control difficulty in the industrial process of arginine, and has great commercial value.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a method for producing L-arginine by fermentation of Escherichia coli. Background Art

[0002] L-arginine is a semi-essential amino acid in the human body, which is involved in various physiological and biochemical processes such as reproductive development, immune regulation, and promoting human detoxification. Therefore, as a high-value-added amino acid, L-arginine has a wide range of applications in animal feed, health products, and pharmaceutical products. With the continuous growth of the market demand for arginine, microbial fermentation has gradually replaced protein hydrolysis to become an ideal method for large-scale industrial production of arginine. At this stage, high-yield arginine strains have become a key factor restricting the development of microbial fermentation to produce arginine.

[0003] At present, the upgrading of arginine production strains is mainly carried out through strengthening the synthetic pathway, modifying the metabolic network (modification of branch pathways, blocking the synthesis of proline, lysine, and glutamine, reducing the loss of metabolic flow; optimizing auxiliary factors, strengthening the PPP pathway) and promoting the extracellular transport of arginine. With the continuous development of synthetic biology methods, more and more metabolic engineering strategies are applied to the construction of cell factories for efficient production of arginine. However, although the rational transformation of metabolic pathways has a clear purpose and significant effect, since microorganisms are a complex system, the known metabolic network or transformation ideas cannot further achieve the purpose of rapidly increasing production. Therefore, after obtaining high-yield strains, the update of the process and the coordination with the strain become the key to technical optimization.

[0004] The following lists some patents related to arginine technology. It can be seen that the main hot spots and processes in the field of amino acid fermentation in the prior art are as follows:

[0005] A process for producing arginine by using a fed-batch culture medium fermentation, CN 112430633 B: This patent discloses a process for fermenting arginine by using a fed-batch culture medium, in which arginine is produced by feeding an aqueous ammonium sulfate solution and a glucose solution during the fermentation process;

[0006] A strain of Escherichia coli and its application in fermentation production of L-arginine, CN 116355814 A: This patent discloses an Escherichia coli, which is cultured in this way, using a 30L fermentation tank, and fermented for 48 hours to produce 136.65g / L of L-arginine with a conversion rate of 48.58%. The culture medium: contains 1g / L glucose, 1g / L yeast powder, 1g / L ammonium sulfate, 1g / L potassium dihydrogen phosphate, 0.5g / L sulfuric acid, 0.01g / L manganese sulfate, and 0.01g / L zinc sulfate.

[0007] A method for preparing gamma-aminobutyric acid by adding substrate enzyme, CN108300742A: This patent invention discloses a method for preparing gamma-aminobutyric acid by adding substrate enzyme, wherein the substrate sodium glutamate is added to a buffer solution to prepare a sodium glutamate solution, and the sodium glutamate solution is added to the bacterial slurry after filtration, and the sodium glutamate concentration in the reaction system is controlled to be 10-45g / L to carry out an enzyme synthesis reaction.

[0008] A method for producing lysine, CN104232702B: This patent invents a method for producing lysine, which includes feeding a carbon source, a nitrogen source and an inorganic salt, and controlling the concentration of a variety of key materials in the fermentation broth, and finally achieving a lysine concentration of 180 g / L and a conversion rate of 65%;

[0009] A method for regulating the fermentation process of L-arginine with high acid production, CN201510211512.7: This patent invents a method for producing arginine by Brevibacterium flavum. The temperature, stirring speed, tank pressure, pH, dissolved oxygen and other parameters in different stages of fermentation are controlled online through the FPC control system, and the final acid production is 5.8%.

[0010] At present, domestic arginine fermentation has gradually begun to turn to genetically engineered bacteria as production strains. Most patents are mainly based on strain construction and design, with less process development. Process development patents are mainly based on conventional parameters, feed supplementation, and ammonia nitrogen addition. There is less control over intermediate products and synthetic precursors, which leads to low production of arginine engineered bacteria and poor process stability. The above shortcomings are technical problems that need to be urgently solved in this field. Summary of the invention

[0011] The present invention aims at the deficiencies in the prior art and discloses a method for producing L-arginine by fermentation of Escherichia coli. A mutant strain of Escherichia coli obtained by patenting an engineering bacterium, biomaterial and application thereof for improving arginine production (CN202311807062.9) is used as the starting bacterium for production process development, and a special arginine fermentation process is developed by controlling parameters such as dissolved oxygen and residual sugar, and further by controlling the concentration of glutamate precursor. The process has the characteristics of high arginine production, high sugar-acid conversion rate, and low impurity acid, which solves the control difficulties in the industrialization process of arginine and has great commercial value.

[0012] First, the production bacteria described in this application is a mutant strain of Escherichia coli; the preservation name is Escherichia coli IBERQ-115 (Escherichia coli) and the preservation number is CCTCC NO: M 20241820; its preservation unit is the China Center for Type Culture Collection, the address is Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University; postal code 430072; the preservation date is August 19, 2024.

[0013] Furthermore, the Escherichia coli IBERQ-115 (Escherichia coli) is constructed from Escherichia coli (MG1655) as a starting strain and is a production strain with high L-arginine production and low side acid production.

[0014] The present invention provides a bacterial agent containing the Escherichia coli IBERQ-115 (Escherichia coli), including: a bacterial flora of the Escherichia coli IBERQ-115 (Escherichia coli), and / or a culture thereof, and / or an extract thereof.

[0015] On the other hand, the present application also discloses the method for producing L-arginine by fermentation of Escherichia coli; comprising using Escherichia coli IBERQ-115 (Escherichia coli) to ferment L-arginine; more preferably, the method mainly comprises: 600 The concentration of glutamate in the fermentation broth can be controlled in stages, thereby greatly increasing the production of arginine;

[0016] For the technical scheme described above, it is further preferred that the fermentation method comprises the following steps: in the fermentation stage of L-arginine fermentation: alternately adjusting the rotation speed and ventilation to keep the dissolved oxygen at 30-40%; when the residual sugar drops to 0.05-0.15 g / L, starting to feed the sugar solution and maintaining the residual sugar concentration in the fermentation liquid at 0.05-0.15 g / L; and when the strain concentration OD 600 When the plants grow to 20-50, start adding sodium glutamate and control the glutamate level at 0.5-3.0 g / L. When the feed sugar solution is replenished and the residual sugar is exhausted, move the plants to the tank.

[0017] For the technical solution described above, it is further preferred that the glutamate precursor is sodium citrate and / or sodium succinate.

[0018] For the technical solution described above, it is further preferred that the concentration of the sodium glutamate in the feed is 8-12wt%, the sodium citrate is 8-12wt%, and the sodium succinate is 8-12wt%.

[0019] For the technical solution described above, it is further preferred that the feed sugar solution is 40-80wt% glucose solution.

[0020] For the technical solution described above, it is further preferred that when sodium glutamate is used as the feed material, the glutamate concentration in the fermentation broth is controlled at 1.0-2.0 g / L;

[0021] For the technical solution described above, it is further preferred that when sodium citrate or sodium succinate is used as the feed substance, the glutamate concentration in the fermentation broth is controlled at 0.5-1.5 g / L.

[0022] For the technical solution described above, further preferably, the fermentation method comprises the following steps: inoculating the secondary seed liquid into the fermentation tank at 5-15% (at this time, the glutamic acid concentration in the fermentation liquid is almost 0); fermenting under the conditions of 34-37°C, pH 6.6-7.0, rotation speed 150-250RPM, ventilation volume 50-150LPM, tank pressure 0.02-0.08Mpa, and the initial fermentation liquid volume is 50-70%

[0023] For the technical solution described above, it is further preferred that the fermentation stage further includes the following steps: Primary seed liquid maturity mark: OD 600 2.0-2.5; Secondary seed liquid maturity sign: OD 600 For 6-12.

[0024] For the technical solution described above, it is further preferred that the preparation of the secondary seed solution comprises the following steps: inoculating E. coli IBERQ-115 into the primary seed culture medium, 30-40°C, 100-250rpm, to obtain a shake flask seed solution, the maturity indicator of the primary seed solution is: OD 600 2.0-2.5;

[0025] The shake flask seed solution is added to the secondary seed tank culture medium at a ratio of 0.1-1.0% (V / V), and the secondary seed solution is obtained at 30-40°C, pH 6.6-7.0, and dissolved oxygen not less than 25%. The maturity indicator of the secondary seed solution is: OD 600 6-12;

[0026] For the technical solution described above, it is further preferred that the feed sugar solution is 40-80wt% glucose solution; and the feed sodium glutamate is 8-12wt%.

[0027] For the technical solution described above, further preferably, during the fermentation process, the strain concentration OD 600 When the growth reaches 30-40, start to add sodium glutamate and control the glutamate at 1-2g / L.

[0028] For the technical solution described above, it is further preferred that the fermentation condition strain concentration OD 600 When the cells grow to 35-40, start adding sodium glutamate and control the glutamate at 1-1.5g / L.

[0029] For the technical solution described above, further, in the step (1), the culture medium in the process of gradually expanding the inoculation volume of the L-arginine producing bacteria IBERQ-115 comprises:

[0030] Primary seed culture medium: glycerol 0.5-1.5wt%, peptone 0.5-1.5wt%, yeast powder 0.3-0.7wt%, KH2PO4 0.3-0.5wt%, diammonium hydrogen phosphate 0.1-0.3 / 4wt%, magnesium sulfate 0.1-0.3wt%, pH 6.5-7.0;

[0031] Secondary seed culture medium: glucose 1.5-2.5wt%, molasses 0.8-1.2wt%, yeast powder 0.05-0.15wt%, ammonium sulfate 0.2-0.6wt%, KH2PO4 0.3-0.5wt%, MgSO4·7H2O 0.03-0.1wt%, pH 6.5-7.0;

[0032] Initial fermentation medium: glucose 0.8-1.2wt%, dipotassium hydrogen phosphate 0.4-0.6wt%, potassium dihydrogen phosphate 0.5-0.7wt%, magnesium sulfate 0.03-0.07wt%, beet molasses 0.3-0.7wt%, yeast powder 0.05-0.15wt%.

[0033] For the technical solution described above, further, the culture medium in step (1) comprises:

[0034] Primary seed culture medium: glycerol 1wt%, peptone 1wt%, yeast powder 0.5wt%, KH2PO4 0.4wt%, diammonium hydrogen phosphate 0.2wt%, magnesium sulfate 0.2wt%, pH 6.6-6.8;

[0035] Secondary seed culture medium: glucose 2wt%, molasses 1.0wt%, yeast powder 0.1wt%, ammonium sulfate 0.3-0.5wt%, KH2PO4 0.4wt%, MgSO4·7H2O 0.05-0.07wt%, pH 6.6-6.8;

[0036] Initial fermentation medium: glucose 1wt%, dipotassium hydrogen phosphate 0.52wt%, potassium dihydrogen phosphate 0.6wt%, magnesium sulfate 0.05wt%, beet molasses 0.5wt%, yeast powder 0.1wt%.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention obtains a high-yield L-arginine strain through gene construction, which has high acid production and low impurity acid production. The detection results in Example 1 reached an arginine yield of 13.9%, a sugar-acid conversion rate of 57.2%, and a total impurity acid content of 0.3%; this shows that the efficiency of the strain in arginine production is much higher than that of the strains in the prior art.

[0039] 2. In the process of arginine biosynthesis, glutamate is one of the important precursors of the urea cycle, and its supply is crucial to the synthesis of arginine. In addition, the concentration of glutamate also affects the output of miscellaneous acids such as ornithine and citrulline. The present invention reduces the inhibitory effect of premature addition of glutamate on strain growth by adding glutamate precursors at critical time points of strain growth and controlling its concentration in stages, and provides sufficient fermentation precursors during critical periods, thereby greatly increasing the yield of arginine and reducing the concentration of miscellaneous acids. The data in Examples 2-4 show that at OD 600 The value is in the range of 30-45. By controlling the glutamic acid concentration at 1.0-2.0 g / L, the arginine production can be stabilized at 130-135 g / L, the sugar-acid conversion rate is between 53-56%, and the total amount of miscellaneous acids is less than 0.4%.

[0040] 3. The present invention also controls the concentration of glutamate by adding sodium succinate and sodium citrate, mainly because sodium succinate and sodium citrate are also important precursors for glutamate synthesis, and the synthesis amount of glutamate is increased to control its concentration in the fermentation liquid. The experimental results in Examples 5 and 6 show that by adding sodium succinate and sodium citrate, the arginine production reaches 125-130g / L and 128-131g / L respectively, the sugar-acid conversion rate is between 52-55%, and the total amount of assorted acids is less than 0.4%. This shows that the use of glutamate precursors can also effectively increase the production of arginine and reduce the generation of assorted acids. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with embodiments, but it should be understood that the protection scope of the present invention is not limited by the embodiments.

[0042] In the present invention, unless otherwise specified, percentages and percentage contents are all by mass. Unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from commercial sources.

[0043] The L-arginine producing strain IBERQ-115 (Escherichia coli) was deposited in the China Center for Type Culture Collection on August 19, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20241820.

[0044] Example 1

[0045] 1. The composition of the seed culture medium is as follows:

[0046] Primary seed culture medium:

[0047] Glycerol 1wt%, peptone 1wt%, yeast powder 0.5wt%, KH2PO4 0.4wt%, diammonium phosphate 0.2wt%, magnesium sulfate 0.2wt%, pH 7.0-7.2;; Packing 500ml / 3000ml; Sterilization strip 121℃ / 20min;

[0048] Secondary seed tank culture medium:

[0049] Glucose 2wt%, molasses 1.0wt%, yeast powder 0.1wt%, ammonium sulfate 0.3-0.5wt%, KH2PO4 0.4wt%, MgSO4·7H2O 0.05-0.07wt%, pH 6.8-7.2, sterilization condition 121℃ / 20min.

[0050] 2. The fermentation medium is as follows:

[0051] The initial culture medium of the standard fermenter was as follows: glucose 1wt%, dipotassium hydrogen phosphate 0.52wt%, potassium dihydrogen phosphate 0.6wt%, magnesium sulfate 0.05wt%, beet molasses 0.5wt%, yeast powder 0.1wt%, natural pH, 121°C*20min.

[0052] Feed sugar solution: 50wt% glucose + 8wt% ammonium sulfate;

[0053] Feed sodium glutamate solution: 10wt% sodium glutamate solution;

[0054] 3. Fermentation tank process (500L)

[0055] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0056] The seed solution in the shake flask was inoculated into the medium of the secondary seed tank of 50L at a ratio of 0.3% (V / V), 35℃, pH 6.8, dissolved oxygen not less than 30%, and cultured for 16 hours. The maturity indicator of the secondary seed solution was OD 6008-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 500L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 100LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the temperature was 37, sodium glutamate was added and controlled at 1.2 g / L; after the feed sugar solution was replenished and the residual sugar was exhausted, the tank was lowered, and the arginine yield was 13.9wt%, the sugar-acid conversion rate was 57.2%, and the miscellaneous acids (citrulline and ornithine) were 0.28%.

[0058] As shown in Example 1, the core of the present invention is to increase the yield of arginine and the sugar-acid ratio and reduce the content of mixed acids by controlling the concentration of the bacterial liquid and then taking glutamate, especially the content of mixed acids. Compared with the original process, the reduction of mixed acids is very obvious, especially ornithine is almost completely removed.

[0059] In the fermentation process, acid production refers to the actual concentration of arginine in the fermentation broth. The calculation formula for the sugar-acid conversion rate is as follows:

[0060] Sugar-acid conversion rate = (actual arginine concentration × actual fermentation volume) / (actual total glucose consumption × 100%)

[0061] Meanwhile, heteroacid refers to the sum of citrulline and ornithine concentrations in the fermentation broth. These two parameters are used together to evaluate the efficiency of the fermentation process and the product composition.

[0062] In summary, E. coli IBERQ-115 showed better performance in arginine production, sugar-acid conversion rate and reduction of miscellaneous acid production.

[0063] Embodiment 2

[0064] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0065] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 6008-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 30-40, start to add sodium glutamate and control it at 1.0-2.0 g / L; when the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0066] Table 1. Experimental results (g / L)

[0067] 2-1 2-2 2-3 2-4 2-5 2-6 <![CDATA[OD value of cell density during fed-batch addition of glutamate 600 value]]> 35 35 35 30 35 40 Control glutamate concentration g / L 1 1.5 2 1.2 1.2 1.2 Arginine yield g / L 130.3 133.4 130.1 129.9 135.8 133.2 Citrulline g / L 2.9 2.9 3 2.6 2.7 3.1 Ornithine acid g / L 0.5 0.1 0 0.1 0.1 0.2 Total miscellaneous acids g / L 3.4 3 3.1 2.9 2.8 3.2 Arginine sugar acid conversion rate % 53.6 54.9 53.5 53.5 55.9 54.8

[0068] As shown in the table, this embodiment includes 600 The results show that when the OD value of sodium glutamate is in the range of 30-40, the glutamate concentration is controlled to be consistent, and the OD value increases and the acid production shows a trend of first increasing and then decreasing during the flow addition, while the heteroacid citrulline shows a slow increase trend, and the closer to Example 1, the higher the acid production; when the OD value of sodium glutamate in the flow addition is consistent, the concentration of glutamate controlled is in the range of 1.0-2.0g / L, the glutamate concentration is increased, and the arginine acid production first increases and then decreases, and the heteroacid ornithine shows a slow decrease trend, and the lowest is 0;

[0069] This example shows that when sodium glutamate is added and the glutamic acid concentration is controlled at 1.0-2.0 g / L within the OD value range of 30-40, the acid production can be around 13%, the sugar-acid ratio is above 53%, and the miscellaneous acid is below 0.35%.

[0070] Embodiment 3

[0071] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0072] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 6008-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 25-45, start to add sodium glutamate and control it at 0.5-2.5g / L; when the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0073] Table 2. Experimental results (g / L)

[0074] 3-1 3-2 3-3 3-4 3-5 3-6 <![CDATA[OD of cell concentration when feeding glutamate 600 value]]> 25 28 42 45 35 37 Control glutamate concentration g / L 0.54 1.5 0.5 2.5 0.5 2.5 Arginine yield g / L 125.6 122.2 125.3 122.5 124.3 123.1 Citrulline g / L 2.9 2.9 3.5 3.3 3 3 Ornithine acid g / L 0.3 0.1 0.8 0.1 0.3 0 Total miscellaneous acids g / L 3.2 3 4.3 3.4 3.3 3 Arginine sugar acid conversion rate % 52.7 51.2 52.5 51.4 52.1 51.6

[0075] As shown in the table, this experiment further expanded the range of the corresponding OD value when sodium glutamate was added to 25-45, and further expanded the control range of sodium glutamate to 0.5-2.5g / L, the arginine acid production was above 12%, the sugar-acid ratio was above 51%, and the miscellaneous acid was below 0.45%;

[0076] Embodiment 4

[0077] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of the shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0078] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 600 8-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the OD value is 25-40, start to add sodium glutamate and control it at 0.5-2.5g / L, and control the glutamate concentration in stages according to the OD value; when the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0079] Table 3. Experimental results (g / L)

[0080] 4-1 4-2 4-3 4-4 4-5 4-6 OD value when sodium glutamate was added 25-35 25-35 30-40 30-40 40-50 40-50 Control glutamate concentration g / L 0.5 2 1.2 1.2 2 1 OD value ≥35 ≥35 ≥40 ≥40 ≥50 ≥50 Glutamate concentration g / L 1.5 1 0.5 2 0.5 2 Arginine yield g / L 135.2 125.5 128.6 129.6 129.5 133.2 Citrulline g / L 3.2 3.1 3.5 3.3 3 3 Ornithine acid g / L 0.1 0.2 0.5 0 0.3 0 Total miscellaneous acids g / L 3.3 3.3 4 3.3 3.3 3 Arginine sugar acid conversion rate % 56.7 52.6 53.9 54.3 54.3 55.8

[0081] As shown in the table, this experiment further enlarged the sodium glutamate flow addition and control process range, and performed segmented control as shown in the table, for example, 4-1, that is, sodium glutamate was started to be added at an OD value of 25, and the glutamate concentration was controlled at 0.5 g / L in the OD value range of 25-35. When the OD value was greater than 35, the glutamate concentration was controlled at 1.5 g / L. Other experimental groups were no longer explained in detail. This batch experiment showed that after continuing to control glutamate in segments, similar experimental results were basically achieved as in Examples 1 and 2, indicating that the glutamate control concentration basically reached a good range of 1.0-2.0 g / L. The strain itself can synthesize a part of glutamate in the early stage of fermentation. Although the addition of sodium glutamate can be utilized by the strain in part, it is not much. The rate of arginine synthesis by the strain itself in the late stage of the strain is close to the maximum. At this time, the additional increase in glutamate concentration cannot be fully utilized. In summary, the rate of arginine synthesis by the strain is limited by factors such as metabolic enzyme activity, and the excess glutamate cannot be utilized.

[0082] Embodiment 5

[0083] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0084] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 600 8-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 30-40, sodium succinate (10wt%) is added, and glutamic acid is controlled at 0.5-1.5g / L. When the feed sugar solution is replenished and the residual sugar is exhausted, the tank is lowered.

[0085] Table 4. Experimental results (g / L)

[0086] 5-1 5-2 5-3 5-4 OD value when sodium succinate was added 30 30 40 40 Control glutamate concentration g / L 0.6 1 1 1.4 Arginine yield g / L 125.5 128.3 127.1 125.2 Citrulline g / L 3.4 2.9 3.1 2.9 Ornithine acid g / L 0.3 0.1 0.3 0.1 Total miscellaneous acids g / L 3.7 3 3.4 3 Arginine sugar acid conversion rate % 52.6 53.8 53.3 52.5

[0087] As shown in the table, this batch experiment proves that the purpose of controlling glutamate concentration can also be achieved by feeding sodium succinate, a precursor of glutamate synthesis. Since there is a certain lag between the feeding of sodium succinate and glutamate synthesis, the glutamate concentration can be appropriately lowered within the range of 0.5-1.5 g / L. This batch experiment proves that similar effects can be achieved by adding glutamate precursors.

[0088] Embodiment 6

[0089] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0090] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 600 8-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 35°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 30-40, sodium citrate (10 wt%) is added, and glutamic acid is controlled at 0.5-1.5 g / L; when the feed sugar solution is replenished and the residual sugar is exhausted, the tank is lowered.

[0091] Table 5. Experimental results (g / L)

[0092] 6-1 6-2 6-3 6-4 OD value when sodium citrate was added 35 35 45 45 Control glutamate concentration g / L 0.8 1.5 0.7 1 Arginine yield g / L 128.2 130.6 123.4 128.6 Citrulline g / L 3.4 2.5 2.2 3.3 Ornithine acid g / L 0.3 0.1 0.2 0.2 Total miscellaneous acids g / L 3.7 2.6 2.4 3.5 Arginine sugar acid conversion rate % 53.8 54.8 51.7 53.9

[0093] This batch of experiments was the same as Example 5, demonstrating that similar effects can be achieved by adding sodium citrate, a precursor of glutamate.

[0094] Embodiment 7

[0095] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0096] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 600 8-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 34-37°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 30-40, start to add sodium glutamate and control it at 1.0-2.0 g / L; when the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0097] Table 6. Experimental results

[0098] Serial number Dissolved oxygen% pH Residual sugar g / L Temperature ℃ Acid production g / L Sugar-acid conversion rate 7-1 30 6.6 0.05 35 129.3 54.21 7-2 30 7 0.15 35 125.6 52.66 7-3 40 6.8 0.1 34 130.9 54.88 7-4 30 6.8 0.1 37 128.8 54.00

[0099] The optimized fermentation conditions of the embodiment include temperature, pH value, dissolved oxygen level, rotation speed and ventilation volume, etc. These optimized conditions ensure that the strain grows and metabolizes in the best environment.

[0100] The batch results showed that the fermentation tank had several controlled conditions, temperature 34-37°C, dissolved oxygen 30-40%, residual sugar 0.05-

[0101] 0.15g / L, good experimental results can be achieved, the arginine yield can reach 128-131g / L, the sugar-acid conversion rate is between 52-55%, and the total amount of miscellaneous acids is less than 0.4%.

[0102] Comparative Example 1

[0103] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of the shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 It is 2.0-2.5.

[0104] The seed solution of the shake flask was added to the medium of the secondary seed tank of 5L at a ratio of 0.3% (V / V), and the temperature was 35℃, pH 6.8, and the dissolved oxygen was not less than 30%. After culturing for 16 hours, the maturity indicator of the secondary seed solution was: OD 6008-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 34-37°C, the pH was 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the pH value is 15 and 50, start to add sodium glutamate and control it at 0.5-2.0g / L; after the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0105] Table 7. Experimental results

[0106]

[0107]

[0108] As shown in the table, this batch mainly verifies the key significance of the OD value for arginine synthesis when sodium glutamate is added. When sodium glutamate is added, the OD value is low. At this time, glutamate will inhibit the growth of the strain and affect acid production. If the OD value is high when sodium glutamate is added, the glutamate added at this time cannot promote acid production.

[0109] Comparative Example 2

[0110] A loop of E. coli IBERQ-115 was inoculated into the primary seed culture medium of the shake flask. After 16 hours of rotation culture at 35°C and 180 rpm, 500 mL of shake flask seed solution was obtained. The maturity indicator of the primary seed solution was: OD 600 The seed solution in the shake flask was added to the 5L secondary seed tank culture medium at a ratio of 0.3% (V / V), 35°C, pH 6.8, dissolved oxygen not less than 30%, and after 16 hours of culture, the secondary seed solution maturity indicator: OD 600 8-10, to obtain the secondary seed solution; 10% of the secondary seed solution was inoculated into a 50L fermenter (at this time, the glutamic acid concentration in the fermentation liquid was almost 0), the initial fermentation liquid volume was 60%, the fermentation temperature was 34-37°C, pH 6.8, the speed was 200RPM, the ventilation volume was 10LPM, the tank pressure was 0.05Mpa, and the speed and ventilation were alternately adjusted to keep the dissolved oxygen at 30-40%; when the residual sugar dropped to 0.1g / L, the feed sugar solution was started to be fed, and the residual sugar concentration in the fermentation liquid was maintained at about 0.1g / L; when the strain concentration OD 600 When the temperature is 30 or 40, start to add sodium glutamate and control it at 0.1-0.4g / L or 3.0-3.5g / L. When the feed sugar solution is replenished and the residual sugar is exhausted, proceed to the tank.

[0111] Table 8. Experimental results

[0112] 2-1 2-2 2-3 2-4 OD value when glutamate was added 30 30 40 40 Control glutamate concentration g / L 0.1 3 3.0 0.3 Arginine yield g / L 114.5 100.5 105.6 100.4 Citrulline g / L 4.2 3.5 3 3 Ornithine acid g / L 0.5 0.2 0.1 0.2 Total miscellaneous acids g / L 4.7 3.7 3.1 3.2 Arginine sugar acid conversion rate % 49.9 43.8 46.0 43.7

[0113] The results of this batch show that within the OD value range of 30-40, controlling glutamate too high or too low during the flow addition of sodium glutamate will result in lower acid production than the example group. The main reason is that when glutamate is too low, it cannot promote acid production, and when glutamate is too high, it will inhibit the growth of the strain and reduce metabolic activity, thereby affecting acid production.

[0114] Comparative Example 3

[0115] Taking Example 7 as a comparative object, when the residual sugar and temperature are controlled at a relatively high or low level during the fermentation process, the fermentation results are as follows

[0116] Table 9. Experimental results

[0117] Serial number Residual sugar g / L Temperature ℃ Acid production g / L Sugar-acid conversion rate 1# 0.1 32 95.3 35.30 2# 0.1 38 70.2 26.00 3# 0.01 35 92.3 34.19 4# 0.2 35 86.9 32.19

[0118] As shown in the results of the table, when the fermentation temperature was 32°C and 38°C, the fermentation acid production decreased, proving that the strain was not suitable for growth and acid production at this temperature. In the 3-4# residual sugar control, it was proved that the acid production also decreased to varying degrees when the residual sugar was 0.01g / L and 2g / L. This comparative example proves the necessity of the residual sugar and temperature control range in Example 4.

[0119] Comparative Example 4

[0120] Taking Example 7 as a comparative object, when the dissolved oxygen and residual sugar were controlled at a relatively high or low level during the fermentation process, the fermentation results were as follows:

[0121] Table 10. Experimental results

[0122] Serial number pH Dissolved oxygen% Acid production g / L Sugar-acid conversion rate 1# 6.3 35 100.2 37.11 2# 7.1 35 95.6 35.41 3# 6.7 25 86.3 31.96 4# 6.7 50 75.5 27.96

[0123] As shown in the results of the table, when the fermentation pH is controlled at 6.3 and 7.1, the acid production begins to decrease, which indirectly reflects that the pH needs to be controlled within the suitable range of 6.4-7.0, and the 3-4# experiment shows that when the dissolved oxygen is 25% and 50%, the acid production also decreases, indicating that the dissolved oxygen should be controlled at 30-45%; this comparative example proves that the pH and dissolved oxygen in Example 3 need to be controlled within a suitable range.

[0124] Comparative Example 5

[0125] Taking Example 1 as a comparative object, during the fermentation process, only the residual sugar dissolved oxygen was controlled without glutamate control, and the fermentation result showed that the maximum acid production could reach 114 g / L, and the sugar-acid ratio was 51%;

[0126] This result indicates that controlling the glutamate concentration according to the biomass size stage of the strain can significantly increase acid production by about 20%;

[0127] In summary, through the experimental data of several comparative examples, it can be concluded that the selection of suitable bacterial species and fermentation process suitable for the strain is particularly important for arginine acid production.

[0128] Comparative experimental verification:

[0129] The effectiveness of the method of the present invention is further verified by comparing the experimental results of Examples 1 to 5.

[0130] Comparative Example 1: In an inappropriate OD 600 Glutamate was added at the OD 600 15 and 50), the arginine yield was only 109-115 g / L, the sugar-acid conversion rate was between 47-50%, and the total amount of miscellaneous acids was higher than 0.4%. This indicates that at non-optimal OD 600 Adding glutamate below the value will inhibit the growth of the strain and affect acid production.

[0131] Comparative Example 2: In OD 600 When the value is 30 and 40, sodium glutamate is added and controlled at 0.1-0.4 g / L or 3.0-3.5 g / L, the arginine production is only 100-115 g / L, the sugar acid conversion rate is between 43-50%, and the total amount of miscellaneous acids is higher than 0.4%. This shows that too high or too low glutamate concentration will lead to lower acid production than the example group.

[0132] Comparative Example 3: Under the control of high or low residual sugar (0.01-0.2 g / L) and temperature (32-38°C), the arginine yield was only 70-95 g / L, and the sugar-acid conversion rate was between 26-35%. This shows that the fermentation temperature and residual sugar control have a significant effect on the arginine yield and conversion rate.

[0133] Comparative Example 4: Under the control of high or low pH (6.3-7.1) and dissolved oxygen (25-50%), the arginine production was only 75-100 g / L, and the sugar-acid conversion rate was between 28-37%. This shows that pH and dissolved oxygen levels also have a significant effect on arginine production and conversion rate.

[0134] Comparative Example 5: Only residual sugar and dissolved oxygen were controlled, and glutamate was not controlled. The maximum arginine production was only 114 g / L, and the sugar-acid conversion rate was about 51%. This shows that controlling the glutamate concentration according to the biomass size stage of the strain can significantly increase the acid production by about 20%.

[0135] Through the above method and technical means, the present invention not only improves the yield and purity of L-arginine, but also significantly reduces the production cost and the generation of by-products, and has important industrial application value. The results of the comparative experiment further confirm the superiority of the present invention in increasing the yield of arginine and reducing the generation of heteroacids.

[0136] The above contents are further elaborations of the present invention in conjunction with the specific preferred embodiments of the present invention, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Any technician familiar with the profession, without departing from the technical scope of the present invention, makes slight changes or modifications, which will be deemed to be covered by the claims of the present invention.

Claims

1. A method for producing L-arginine by fermentation, characterized in that: L-arginine was produced by fermentation using Escherichia coli IBERQ-115 (CCTCC NO: M 20241820).

2. The method according to claim 1, characterized in that: According to the bacterial concentration OD 600 The concentration of glutamate in the fermentation broth is controlled according to the situation and stage.

3. The method according to claim 2, characterized in that: During the fermentation phase of L-arginine fermentation: alternately adjust the speed and aeration to keep the dissolved oxygen at 30-40%; When the residual sugar content drops to 0.05-0.15 g / L, start feeding the feed sugar solution and maintain the residual sugar concentration in the fermentation broth at 0.05-0.15 g / L. 600 When the growth reaches 20-50, start to feed sodium glutamate or glutamate precursor, and control the glutamate at 0.5-3.0g / L; when the feed sugar solution is replenished and the residual sugar is exhausted, put it into the tank.

4. The method according to claim 3, characterized in that: The glutamate precursor is sodium citrate and / or sodium succinate.

5. The method according to claim 4, characterized in that: The concentration of the feed sodium glutamate is 8-12wt%, the concentration of the sodium citrate is 8-12wt%, and the concentration of the sodium succinate is 8-12wt%.

6. The method according to claim 3, characterized in that: The feed sugar solution is 40-80wt% glucose solution.

7. The method according to claim 3, characterized in that: The fermentation stage also includes the following steps: inoculating 5-15% of the secondary seed liquid into the fermentation tank; fermenting at 34-37°C, pH 6.6-7.0, rotation speed 150-250RPM, ventilation volume 50-150LPM, tank pressure 0.02-0.08Mpa, and the initial fermentation liquid volume is 50-70%.

8. The method according to claim 3, characterized in that: The fermentation stage also includes the following steps: the first seed liquid maturity mark: OD 600 2.0-2.5; Secondary seed liquid maturity sign: OD 600 For 6-12.

9. The method according to claim 3, characterized in that: During the fermentation process, the strain concentration OD 600 When the growth reaches 30-40, start to add sodium glutamate and control the glutamate at 1-2g / L.

10. The method according to claim 3, characterized in that: The culture medium used in step (1) includes: primary seed culture medium: 0.5-1.5wt% glycerol, 0.5-1.5wt% peptone, 0.3-0.7wt% yeast powder, 0.3-0.5wt% KH2PO4, 0.1-0. 3 / 4wt%, magnesium sulfate 0.1-0.3wt%, pH 6.5-7.0; Secondary seed culture medium: glucose 1.5-2.5wt%, molasses 0.8-1.2wt%, yeast powder 0.05-0.15wt%, ammonium sulfate 0.2-0.6wt%, KH2PO4 0.3-0.5wt%, MgSO4·7H2O 0.03-0.1wt%, pH 6.5-7.0; Initial fermentation medium: glucose 0.8-1.2wt%, dipotassium hydrogen phosphate 0.4-0.6wt%, potassium dihydrogen phosphate 0.5-0.7wt%, magnesium sulfate 0.03-0.07wt%, beet molasses 0.3-0.7wt%, yeast powder 0.05-0.15wt%.

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