Method for producing L-alanine through fermentation
By adding sodium gluconate to the fermentation medium, the difficulty of redox balance regulation and the problem of low L-alanine yield was solved, and the full anaerobic fermentation of high-yield L-alanine was achieved, which was suitable for large-scale industrial production.
Patent Information
- Application Number
- CN202510025562.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-05-06
AI Technical Summary
The methods used in the prior art for fermentation and production of L-alanine have difficulties in redox equilibrium regulation and low L-alanine yield.
By adding sodium gluconate to the fermentation medium as an auxiliary carbon source, it helps consume excessive NADH, balances the reduction force in the cell, achieves full-process anaerobic fermentation, and improves L-alanine production.
It significantly increases the production of L-alanine, simplifies production operations, reduces the risks of energy consumption and miscellaneous bacteria pollution, and is suitable for large-scale industrial fermentation production.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to a method for producing L-alanine by fermentation. Background Art
[0002] L-alanine plays an important role in the metabolism of organisms. Studies have shown that blood contains 8% of amino acids, most of which is L-alanine. As an amino acid with a special sweet taste, L-alanine is widely used in medicine, food industry, daily chemicals, etc.
[0003] At present, the main production methods of L-alanine are enzyme conversion and bio-fermentation. The starting raw material of bio-fermentation is glucose, which is a renewable resource produced from corn starch. With the depletion of chemical resources such as petroleum, bio-fermentation will become more and more competitive and strategically significant in the market.
[0004] The present application has found that the genetically engineered Escherichia coli (strain collection number: CGMCC No. 14067) used for fermentation production of L-alanine has an optimal process of fermentation production of alanine under microaerobic conditions. The dissolved oxygen value in this process is very low, and its redox balance is the key difficulty in regulation, which has a vital impact on the production metabolism and anabolism level of the bacteria; in addition, this process also has the problem of low L-alanine production. Summary of the invention
[0005] In order to solve one of the above technical problems existing in the prior art, the present invention provides a method for producing L-alanine by fermentation, which can significantly increase the yield of L-alanine.
[0006] The technical solution of the present invention is as follows:
[0007] A method for producing L-alanine by fermentation comprises inoculating Escherichia coli CGMCC No.14067 seed liquid into a fermentation medium containing glucose and gluconate for fermentation to obtain L-alanine.
[0008] Since alanine fermentation is mainly anaerobic fermentation type, although the production bacteria Escherichia coli used is a facultative anaerobic bacteria, that is, it can grow and reproduce under aerobic or anaerobic conditions, but under the condition of deep anaerobic conditions (low redox potential value), the growth and metabolic capacity of Escherichia coli are significantly weakened. Therefore, appropriately improving the redox potential level of the bacteria under anaerobic conditions plays an important role in promoting the performance of alanine fermentation.
[0009] The method of the present invention is designed based on the principle that gluconic acid has an oxidized state, and its metabolism can help consume excess NADH, generate and supplement a certain amount of NAD + , NADH / NAD +When the ratio is too high, it will inhibit the glycolysis pathway and affect the fermentation process. Therefore, when it is used as an auxiliary carbon source for glucose in a certain ratio, it can significantly reduce the intracellular NADH / NAD + ratio, thereby balancing the reducing power within the cell and realizing a new process for the full anaerobic fermentation of alanine, which can greatly simplify production operations, reduce energy consumption and reduce the risk of contamination by foreign bacteria.
[0010] In the present invention, the gluconate includes but is not limited to: one or more of sodium gluconate and calcium gluconate. In some embodiments, the gluconate includes sodium gluconate.
[0011] In some embodiments, the inoculation amount of the seed solution is 5-15% of the volume of the fermentation medium, such as 5%, 8%, 10%, 12%, 15% or any value therebetween, preferably 8-12%.
[0012] In some embodiments, the seed solution is prepared by a method comprising the following steps: taking fresh plate bacteria and inoculating them into liquid LB medium, culturing them at a temperature of 35-39°C and a rotation speed of 200-300 r / min until the OD 600nm When the value reaches 0.8-1.5, seed liquid is obtained.
[0013] In some embodiments, the liquid LB medium comprises the following components: 10-14 g / L glycerol, 2-6 g / L yeast extract, 6-10 g / L tryptone. In some embodiments, the liquid LB medium consists of the following components: 10-14 g / L glycerol, 2-6 g / L yeast extract, 6-10 g / L tryptone, and the balance water.
[0014] In some embodiments, the content of gluconate in the fermentation medium is 8-20 g / L, for example, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L or any value therebetween, preferably 10-15 g / L.
[0015] In some embodiments, the content of glucose in the fermentation medium is 130-160 g / L, for example, 130 g / L, 135 g / L, 140 g / L, 145 g / L, 150 g / L, 155 g / L, 160 g / L or any value therebetween, preferably 140-150 g / L.
[0016] In some embodiments, the fermentation medium further contains the following components: corn steep liquor powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, FeSO4·7H2O, ZnSO4, MnSO4·H2O, CoCl2·6H2O, CuCl2·2H2O and Na2MoO4.
[0017] In some embodiments, the fermentation medium includes the following components:
[0018] Glucose 130-160g / L, sodium gluconate 8-20g / L, corn steep liquor dry powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 6.0-10.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.0-3.0g / L, CoCl2·6H2O 0.5-2.0g / L, CuCl2·2H2O 0.1-1.0g / L, Na2MoO4 0.05-0.2g / L, and the solvent is water.
[0019] In some embodiments, the fermentation medium includes the following components:
[0020] Glucose 140-150g / L, sodium gluconate 10-15g / L, corn steep liquor dry powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 7.0-9.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.5-2.5g / L, CoCl2·6H2O 0.5-1.5g / L, CuCl2·2H2O 0.3-0.8g / L, Na2MoO4 0.08-0.12g / L, and the solvent is water.
[0021] In some embodiments, the trace element-containing components include: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO4 0.1g / L, and the solvent is water.
[0022] In some embodiments, the fermentation medium includes the following components:
[0023] Glucose 140-150g / L, sodium gluconate 10-15g / L, corn steep liquor dry powder 12g / L, potassium dihydrogen phosphate 10g / L, ammonium sulfate 12g / L, magnesium sulfate heptahydrate 4g / L, components containing trace elements 4mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO4 0.1g / L, and the solvent is water.
[0024] In some embodiments, the fermentation temperature is 36-39° C. In some embodiments, the fermentation pH is 6.6-6.9. In the fermentation process of the present invention, the pH is preferably adjusted by adding ammonia water, and the ammonia water concentration is preferably 18wt%-24wt%.
[0025] In some embodiments, the fermentation is anaerobic fermentation.
[0026] In some embodiments, the fermentation comprises: continuously introducing sterile air at a ventilation ratio of 0.1 to 0.3 vvm for 0 to 8 hours of fermentation, and stopping ventilation after 8 hours of fermentation to perform anaerobic fermentation.
[0027] In some embodiments, the fermentation is terminated when the glucose concentration in the fermentation broth is below 0.2 wt %.
[0028] In some embodiments, the method comprises the steps of:
[0029] The seed liquid of Escherichia coli CGMCC No.14067 is inoculated into the fermentation medium at an inoculation rate of 8-12%, and anaerobic fermentation is performed. During the fermentation process, the fermentation temperature is controlled at 36-39° C. and the pH value is 6.6-6.9. When the glucose concentration in the fermentation liquid is below 0.2wt%, the fermentation is stopped to obtain L-alanine; wherein the fermentation medium includes the following components:
[0030] Glucose 140-150g / L, sodium gluconate 10-15g / L, corn steep liquor dry powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 7.0-9.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.5-2.5g / L, CoCl2·6H2O 0.5-1.5g / L, CuCl2·2H2O 0.3-0.8g / L, Na2MoO4 0.08-0.12g / L, and the solvent is water.
[0031] Compared with the prior art, the present invention has the following beneficial effects:
[0032] The method of the present invention can significantly improve the output of L-alanine by adding sodium gluconate in the fermentation medium, and the fermentation period is short, simple and effective. In addition, the method of the present invention can realize the whole anaerobic fermentation mode to obtain high-yield L-alanine. In the method of the present invention, the sodium gluconate consumption is less, the price is relatively low, and there is no obvious increase in large-scale production cost, so it is very suitable for large-scale industrial fermentation production of alanine. DETAILED DESCRIPTION
[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.
[0034] The reagents used in the following experiments of the present invention, unless otherwise specified, are all conventional commercial products or reagents prepared by conventional methods, and the methods used in the experiments, unless otherwise specified, are all conventional experimental methods. The instruments used in the experiments, unless otherwise specified, can all be obtained through commercial channels.
[0035] The E. coli genetically engineered bacteria CGMCC#No.14067 shake flask seed solution was prepared by the following method:
[0036] Take a fresh plate of bacteria and inoculate it into a sterilized liquid seed culture medium (200mL / 500mL conical flask). The seed culture medium is composed of LB culture medium (LB culture medium is composed of: 12g / L glycerol, 4g / L yeast extract powder, 8g / L trypsin and the rest is water). The culture conditions are 37°C and a shaking speed of 200r / min for 12h. The seeds are mature when the OD600nm value reaches the range of 0.8-1.5.
[0037] Example 1
[0038] Fermentation test in 50L tank with full anaerobic process and 15g / L sodium gluconate added to the fermentation medium:
[0039] A 50L fermentation tank was used to prepare a fermentation medium according to a volume of 30L of feed liquid. The medium composition was: 4.35kg (145g / L) crystalline glucose, 0.45kg (15g / L) sodium gluconate, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of components containing trace elements, and water was used as the solvent. The components containing trace elements included: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, and Na2MoO4 0.1g / L, and the solvent was water.
[0040] The culture medium was put into a 50L tank, dissolved in water and fixed to volume, and sterilized with high-temperature steam (121°C, 25min). After that, the Escherichia coli genetically engineered bacteria CGMCC#No.14067 shake bottle seed liquid was inoculated with 10% of the inoculation amount (about 3L) and accessed to the sterilized culture medium, and the initial volume after fermentation was 30L. During the fermentation process, ammonia water was used to control the pH value to 6.8±0.2, the temperature was controlled to 37±0.5°C, and anaerobic fermentation was performed without air throughout the process. The final fermentation lasted 48 hours, and the glucose concentration of the fermented liquid was detected to be less than 0.2% to meet the requirements of the tank release. At this time, the liquid chromatography detected the L-alanine content of the fermented liquid to be 140g / L, and the conversion rate was calculated to be 92%, as shown in Table 2.
[0041] Example 2
[0042] A scaled-up fermentation test in a 500L tank with full anaerobic conditions and 15g / L sodium gluconate added to the fermentation medium:
[0043] Use a 500L fermenter and prepare the fermentation medium according to the volume of 300L feed liquid. The medium composition is as follows:
[0044] 43.5kg (145g / L) crystalline glucose, 4.5kg (15g / L) sodium gluconate, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of components containing trace elements, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO4 0.1g / L, and the solvent is water.
[0045] The culture medium was put into a 50L tank, dissolved in water and fixed to volume, and sterilized with high-temperature steam (121°C, 30min). After that, the seed tank liquid of Escherichia coli genetically engineered bacteria CGMCC#No.14067 was inoculated with 10% of the inoculation amount (about 30L) and accessed to the sterilized culture medium. The initial volume after fermentation was 300L. During the fermentation process, the pH value was controlled to 6.9±0.2 with ammonia water, the temperature was controlled to 37±0.5°C, and anaerobic fermentation was performed without air throughout the process. The final fermentation lasted for 49 hours, and the glucose concentration of the fermentation liquid was detected to be less than 0.2% to meet the requirements for tank release. At this time, the L-alanine content of the fermentation liquid detected by liquid chromatography was 138g / L, and the conversion rate was calculated to be 91.4%.
[0046] Example 3
[0047] The difference from Example 1 is that the culture medium composition is different, specifically: 4.65kg (155g / L) crystalline glucose, 0.15kg (5g / L) sodium gluconate, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of trace element-containing components, and the solvent is water; wherein the trace element-containing components include: FeSO4·7H2O8g / L, ZnSO42.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO40.1g / L, and the solvent is water.
[0048] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0049] Example 4
[0050] The difference from Example 1 is that the culture medium composition is different, specifically: 4.50 kg (150 g / L) crystalline glucose, 0.30 kg (10 g / L) sodium gluconate, corn steep liquor powder 12 g / L, potassium dihydrogen phosphate 10 g / L, ammonium sulfate 12 g / L, magnesium sulfate heptahydrate 4 g / L, trace element-containing components 2 mL / L, the solvent is water; wherein the trace element-containing components include: FeSO4 7H2O 8 g / L, ZnSO4 2.5 g / L, MnSO4 · H2O 2 g / L, CoCl2 · 6H2O 1 g / L, CuCl2 · 2H2O 0.5 g / L, Na2MoO4 0.1 g / L, the solvent is water.
[0051] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0052] Example 5
[0053] The difference from Example 1 is that the culture medium composition is different, specifically: 4.20kg (140g / L) crystalline glucose, 0.60kg (20g / L) sodium gluconate, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of trace element-containing components, and the solvent is water; wherein the trace element-containing components include: FeSO4·7H2O8g / L, ZnSO42.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO40.1g / L, and the solvent is water.
[0054] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0055] Example 6
[0056] A 50L fermentation tank was used to prepare a fermentation medium according to a volume of 30L of feed liquid. The composition of the medium was: 4.65kg (155g / L) crystalline glucose, 0.15kg (5g / L) sodium gluconate, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of components containing trace elements, and the solvent was water; wherein the components containing trace elements included: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, and Na2MoO4 0.1g / L, and the solvent was water.
[0057] The culture medium was put into a 50L tank, dissolved with water and fixed to volume, and sterilized with high-temperature steam (121°C, 25min). Then, the E. coli genetic engineering bacteria CGMCC#No.14067 shake bottle seed liquid was inoculated with 10% of the sterilized culture medium (about 3L), and the initial volume after fermentation was 30L. During the fermentation process, ammonia water was used to control the pH value to 6.8±0.2, the temperature was controlled to 37±0.5°C, and sterile air was continuously introduced at 6L / min (0.2vvm) for the first 8 hours. After 8 hours, the anaerobic fermentation was stopped until the tank was released.
[0058] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0059] Example 7
[0060] The difference from Example 6 is that the culture medium composition is different, specifically: 4.50 kg (150 g / L) crystalline glucose, 0.30 kg (10 g / L) sodium gluconate, 12 g / L corn steep liquor powder, 10 g / L potassium dihydrogen phosphate, 12 g / L ammonium sulfate, 4 g / L magnesium sulfate heptahydrate, 2 mL / L of trace element-containing components, and water as the solvent; wherein the trace element-containing components include: FeSO4·7H2O8 g / L, ZnSO42.5 g / L, MnSO4·H2O 2 g / L, CoCl2·6H2O 1 g / L, CuCl2·2H2O 0.5 g / L, Na2MoO40.1 g / L, and the solvent is water.
[0061] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0062] Example 8
[0063] The difference from Example 6 is that the culture medium composition is different, specifically: 4.20 kg (145 g / L) crystalline glucose, 0.60 kg (15 g / L) sodium gluconate, 12 g / L corn steep liquor powder, 10 g / L potassium dihydrogen phosphate, 12 g / L ammonium sulfate, 4 g / L magnesium sulfate heptahydrate, 2 mL / L of trace element-containing components, and water as the solvent; wherein the trace element-containing components include: FeSO4·7H2O8 g / L, ZnSO42.5 g / L, MnSO4·H2O 2 g / L, CoCl2·6H2O 1 g / L, CuCl2·2H2O 0.5 g / L, Na2MoO40.1 g / L, and the solvent is water.
[0064] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0065] Example 9
[0066] The difference from Example 6 is that the culture medium composition is different, specifically: 4.20 kg (140 g / L) crystalline glucose, 0.60 kg (20 g / L) sodium gluconate, corn steep liquor powder 12 g / L, potassium dihydrogen phosphate 10 g / L, ammonium sulfate 12 g / L, magnesium sulfate heptahydrate 4 g / L, trace element-containing components 2 mL / L, the solvent is water; wherein the trace element-containing components include: FeSO4 7H2O 8 g / L, ZnSO4 2.5 g / L, MnSO4 · H2O 2 g / L, CoCl2 · 6H2O 1 g / L, CuCl2 · 2H2O 0.5 g / L, Na2MoO4 0.1 g / L, the solvent is water.
[0067] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0068] Comparative Example 1
[0069] The difference from Example 6 is that sodium gluconate is not added to the culture medium, specifically:
[0070] A 50L fermentation tank was used to prepare a fermentation medium according to a volume of 30L of feed liquid. The medium composition was: 4.8kg (140g / L) of crystalline glucose, 12g / L of corn syrup dry powder, 10g / L of potassium dihydrogen phosphate, 12g / L of ammonium sulfate, 4g / L of magnesium sulfate heptahydrate, 2mL / L of components containing trace elements, and the solvent was water; wherein the components containing trace elements included: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, and Na2MoO4 0.1g / L, and the solvent was water.
[0071] The culture medium was put into a 50L tank and dissolved in water to make up the volume, and sterilized with high temperature steam (121°C, 25min). Afterwards, the Escherichia coli genetic engineering bacteria CGMCC#No.14067 shake bottle seed liquid was connected to the sterilized culture medium according to 10% inoculation amount (about 3L), and the initial volume after fermentation was 30L. The pH value was controlled to 6.8±0.2 with ammonia water during the fermentation process, the temperature was controlled to 37±0.5°C, and 6L / min (0.2vvm) of sterile air was continuously introduced for the first 8 hours, and the anaerobic fermentation was stopped after 8 hours until the tank was released. The final fermentation was 44 hours, and the glucose concentration of the fermented liquid was detected to be less than 0.1% to meet the requirements of the tank release. At this time, the liquid chromatography detected the L-alanine content of the fermented liquid to be 121g / L, and the calculated conversion rate was 84%, as shown in Table 2.
[0072] Comparative Example 2
[0073] The difference from Example 1 is that the composition of the fermentation medium is different, specifically:
[0074] 4.8kg (160g / L) crystalline glucose, 12g / L corn steep liquor powder, 10g / L potassium dihydrogen phosphate, 12g / L ammonium sulfate, 4g / L magnesium sulfate heptahydrate, 2mL / L of components containing trace elements, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 8g / L, ZnSO4 2.5g / L, MnSO4·H2O 2g / L, CoCl2·6H2O 1g / L, CuCl2·2H2O 0.5g / L, Na2MoO4 0.1g / L, and the solvent is water.
[0075] The bacterial concentration, fermentation time, L-alanine content in the fermentation liquid when the tank was released, mass conversion rate and fermentation cycle are shown in Table 2.
[0076] The culture medium types and fermentation methods of the above examples and comparative examples are listed in Table 1, and the fermentation results are listed in Table 2.
[0077] Table 1
[0078]
[0079]
[0080] Table 2
[0081]
[0082] As can be seen from the data in Table 2 above, the final fermentation conversion rate and alanine content of Example 4 and Example 1 are significantly higher than those of other embodiments, that is, 10-15g / L of sodium gluconate is added to the initial base material, and the highest fermentation index is obtained by adopting the whole process of non-ventilated anaerobic fermentation. While Comparative Example 1 is the original fermentation process, that is, the culture medium does not add sodium gluconate, and the fermentation process is ventilated in the early stage and anaerobic in the later stage. Compared with it, the new process fermentation index adopted by the present invention is significantly improved, and the alanine content is increased from 121g / L to 140g / L, and the conversion rate is increased from 84% to 92%. In addition, sodium gluconate is not added to the culture medium of Comparative Example 2, and the full anaerobic batch fermentation effect is the worst, and there is a big gap with other batches. The results of Example 3 and Example 6 show that when the addition amount of sodium gluconate is too little, the fermentation conversion rate will also be low, preferably controlled at more than 8g / L. The results of Example 5 and Example 9 show that when the addition amount of sodium gluconate reaches 20 g / L, the fermentation conversion rate is not significantly improved, so it is preferred to control the addition amount of sodium gluconate to 10-15 g / L.
[0083] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.
Claims
1. A method for producing L-alanine by fermentation, comprising inoculating seed liquid of Escherichia coli CGMCC No.14067 into a fermentation medium containing glucose and gluconate for fermentation to obtain L-alanine.
2. The method according to claim 1, characterized in that The gluconate includes one or more of sodium gluconate and calcium gluconate, preferably sodium gluconate.
3. The method according to claim 1 or 2, characterized in that: The inoculation amount of the seed liquid is 5-15% of the volume of the fermentation medium, preferably 8-12%.
4. The method according to any one of claims 1 to 3, characterized in that: In the fermentation medium, the content of gluconate is 8-20 g / L, preferably 10-15 g / L; and / or the content of glucose is 130-160 g / L, preferably 140-150 g / L.
5. The method according to any one of claims 1 to 4, characterized in that: The fermentation medium also contains the following components: corn steep liquor dry powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, FeSO4·7H2O, ZnSO4, MnSO4·H2O, CoCl2·6H2O, CuCl2·2H2O and Na2MoO4.
6. The method according to any one of claims 1 to 5, characterized in that: The fermentation medium comprises the following components: Glucose 130-160g / L, sodium gluconate 8-20g / L, corn steep liquor powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 6.0-10.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.0-3.0g / L, CoCl2·6H2O 0.5-2.0g / L, CuCl2·2H2O 0.1-1.0g / L, Na2MoO4 0.05-0.2g / L, and the solvent is water; Preferably, the fermentation medium comprises the following components: Glucose 140-150g / L, sodium gluconate 10-15g / L, corn steep liquor dry powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 7.0-9.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.5-2.5g / L, CoCl2·6H2O 0.5-1.5g / L, CuCl2·2H2O 0.3-0.8g / L, Na2MoO4 0.08-0.12g / L, and the solvent is water.
7. The method according to any one of claims 1 to 6, characterized in that: The fermentation conditions include: a temperature of 36-39° C., and / or a pH of 6.6-6.9; Preferably, during the fermentation process, the pH is adjusted by adding aqueous ammonia, and the concentration of the aqueous ammonia is preferably 18 wt%-24 wt%.
8. The method according to any one of claims 1 to 7, characterized in that: The fermentation is anaerobic fermentation, or Sterile air was continuously introduced at a ventilation ratio of 0.1 to 0.3 vvm during fermentation for 0-8 hours. After 8 hours of fermentation, ventilation was stopped and anaerobic fermentation was carried out.
9. The method according to any one of claims 1 to 8, characterized in that: The fermentation was terminated when the glucose concentration in the fermentation broth was below 0.2 wt %.
10. The method according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: The seed liquid of Escherichia coli CGMCC No.14067 is inoculated into the fermentation medium at an inoculation rate of 8-12%, and anaerobic fermentation is performed. During the fermentation process, the fermentation temperature is controlled at 36-39° C. and the pH value is 6.6-6.
9. When the glucose concentration in the fermentation liquid is below 0.2wt%, the fermentation is stopped to obtain L-alanine; wherein the fermentation medium includes the following components: Glucose 140-150g / L, sodium gluconate 10-15g / L, corn steep liquor dry powder 10-14g / L, potassium dihydrogen phosphate 8-12g / L, ammonium sulfate 10-14g / L, magnesium sulfate heptahydrate 2-6g / L, components containing trace elements 2-6mL / L, and the solvent is water; wherein the components containing trace elements include: FeSO4·7H2O 7.0-9.0g / L, ZnSO4 2.0-3.0g / L, MnSO4·H2O 1.5-2.5g / L, CoCl2·6H2O 0.5-1.5g / L, CuCl2·2H2O 0.3-0.8g / L, Na2MoO4 0.08-0.12g / L, and the solvent is water.