CN116497075A - Method for improving fermentation conversion rate of L-proline - Google Patents
Method for improving fermentation conversion rate of L-proline Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 81
- 230000004151 fermentation Effects 0.000 title claims abstract description 81
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 title claims abstract description 34
- 229960002429 proline Drugs 0.000 title claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 29
- 229930182821 L-proline Natural products 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 13
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 36
- 229960002989 glutamic acid Drugs 0.000 claims abstract description 32
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 239000002609 medium Substances 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000011218 seed culture Methods 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 19
- 239000008103 glucose Substances 0.000 claims description 19
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 14
- 239000001301 oxygen Substances 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
- 229960002685 biotin Drugs 0.000 claims description 7
- 235000020958 biotin Nutrition 0.000 claims description 7
- 239000011616 biotin Substances 0.000 claims description 7
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 7
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 7
- 235000011164 potassium chloride Nutrition 0.000 claims description 7
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 abstract description 28
- 235000013922 glutamic acid Nutrition 0.000 abstract description 28
- 239000004220 glutamic acid Substances 0.000 abstract description 28
- 239000002253 acid Substances 0.000 abstract description 25
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 241001052560 Thallis Species 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- FBVVGPMIPAZFAW-RZVRUWJTSA-N (2S)-pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1.OC(=O)[C@@H]1CCCN1 FBVVGPMIPAZFAW-RZVRUWJTSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- SYOKIDBDQMKNDQ-XWTIBIIYSA-N vildagliptin Chemical compound C1C(O)(C2)CC(C3)CC1CC32NCC(=O)N1CCC[C@H]1C#N SYOKIDBDQMKNDQ-XWTIBIIYSA-N 0.000 description 1
- 229960001254 vildagliptin Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention discloses a method for improving the fermentation conversion rate of L-proline, which takes corynebacterium glutamicum as a production strain, firstly carries out seed culture to prepare secondary seed liquid, and then inoculates the secondary seed liquid into a fermentation culture medium according to the proportion of 10-20% for fermentation culture; the fermentation medium contains 20-30g/L glutamic acid. By adopting the method for producing the L-proline, the fermentation conversion rate is high, the quantity of the mixed acid glutamic acid is small, and the yield of the L-proline can reach more than 92.0g/L, the mixed acid glutamic acid is reduced to 2.1g/L, and the sugar acid conversion rate can reach more than 41.0 percent.
Description
Technical field:
the invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving the fermentation conversion rate of L-proline.
The background technology is as follows:
l-proline (L-proline) is a neutral amino acid and is one of the 20 amino acids constituting a protein. The L-proline has molecular weight of 115.13 and is very soluble in water, so that the L-proline is an ideal osmotic regulator and is mainly used as an important raw material for various medicines at home and abroad, such as vildagliptin.
At present, the global market demand of L-proline is only 2000 t/year, and compared with other amino acid products, the L-proline fermentation strain transformation and process development are much less researched at home and abroad. In 2018, tin-free crystal sea amino acid Co-Ltd published patent CN108841886B, glucose is fed in when dissolved oxygen in the fermentation process is raised to 25%, the dissolved oxygen in the fermentation process is controlled to be 30%, residual sugar is controlled to be low in level, fermentation is carried out for 55-65h, and acid production reaches 84.3g/L; the patent CN114107141A published by Tianjin industrial biotechnology research institute of national academy of sciences in 2022 discloses a genetically modified bacterium capable of producing L-proline at high yield, wherein the genetically modified bacterium is fermented for 49 hours in a 5L tank, the acid production rate is 142g/L, the conversion rate is 31%, the mixed acid glutamic acid is 12g/L, and the highest level is reported in China at present.
The theoretical conversion rate of L-proline is 63.9%, the conversion rate level of the prior patent technology has a large progress space, and the content of the hetero-acid glutamic acid is too high, so that huge treatment pressure is brought to the subsequent extraction and refining process, and the method is not suitable for being applied to mass production.
The invention comprises the following steps:
aiming at the problems, the invention aims to provide a method for improving the fermentation conversion rate of L-proline, which is characterized in that 10-20g/L of ammonium sulfate or ammonium chloride and 20-30g/L of glutamic acid are added into a fermentation initial culture medium, the glucose content level of 40-50g/L and the dissolved oxygen content of 5-20% are maintained in the fermentation process, and under the condition, the fermentation acid production rate and the conversion rate of L-proline are obviously improved.
The invention is implemented by the following technical scheme: a method for improving the fermentation conversion rate of L-proline, regard Corynebacterium glutamicum as the production strain, carry on the seed culture at first, prepare the second seed solution, then inoculate the second seed solution into fermentation culture medium according to the proportion of 10-20%, ferment and culture; the fermentation medium contains 20-30g/L glutamic acid.
Further, the fermentation medium also contains 10-20g/L of ammonium sulfate or ammonium chloride.
Further, the fermentation medium also contains 100 to 150g/L of glucose, 40 to 60g/L of corn steep liquor, 3 to 5g/L of potassium chloride, 1 to 3g/L of phosphoric acid, 3 to 5g/L of magnesium sulfate heptahydrate, 1 to 3mg/L of biotin and vitamin B 1 1-3 mg/L, and continuously adjusting the pH value to 7.0-7.5 by ammonia water.
Further, in the fermentation process, dissolved oxygen is controlled to be 5-20%.
Further, during the fermentation process, the glucose content is maintained at 40-50g/L, and the fermentation period is 40-44h.
The invention has the advantages that:
(1) By adopting the method for producing the L-proline, the fermentation conversion rate is high, the quantity of the mixed acid glutamic acid is small, and the yield of the L-proline can reach more than 92.0g/L, the mixed acid glutamic acid is reduced to 2.1g/L, and the sugar acid conversion rate can reach more than 41.0 percent.
(2) The invention adopts fermentation conditions of high residual sugar and low dissolved oxygen, on one hand, the invention promotes thalli to actively synthesize the permeation regulating substance, namely L-proline, and on the other hand, limits the flux of the thalli for metabolizing glutamic acid, so that the L-proline realizes a large amount of accumulation while the accumulation of the hetero-acid glutamic acid is not much, and the fermentation conversion rate is improved.
(3) According to the invention, glutamic acid is added into the fermentation medium, and the glutamic acid is a precursor of L-proline, so that the acid production and conversion rate of fermentation can be improved by directly adding the glutamic acid.
(4) The invention mainly uses corn steep liquor, ammonium sulfate or ammonium chloride and glutamic acid as nitrogen sources, the cost of the auxiliary materials is low, the investment is less, and the addition of the ammonium sulfate or ammonium chloride can create a high-ammonium environment, so that the laboratory researches find that the proline accumulation condition is better under the high-ammonium environment (the free ammonia is more than 3.0 g/L).
The specific embodiment is as follows:
the technical solutions of the present invention will be clearly and completely described below in connection with specific embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Comparative examples:
inoculating corynebacterium glutamicum into a 10L seed culture tank for culture, wherein the seed culture medium is as follows: 25g/L of sucrose, 3g/L of yeast powder, 20g/L of beet molasses, 3g/L of potassium chloride, 2g/L of phosphoric acid, 3g/L of magnesium sulfate heptahydrate, 0.5mg/L of biotin and vitamin B 1 1mg/L. The culture conditions are as follows: the temperature is 31.5 ℃, the pH value is regulated to 7.2 by ammonia water, and the dissolved oxygen is 30-60%. Stopping culturing when the OD number of the seed liquid reaches 35-40, and inoculating the seed liquid into a 60L fermentation tank according to the inoculation amount of 20% for fermentation. The fermentation medium is as follows: 120g/L glucose, 50g/L corn steep liquor, 3g/L potassium chloride, 1g/L phosphoric acid, 3g/L magnesium sulfate heptahydrate, 1mg/L biotin and vitamin B 1 1mg/L, and the pH value is continuously adjusted to 7.2 by ammonia water. The fermentation conditions are as follows: when the temperature is 32 ℃, the rotating speed is 200-700 r/min, the tank pressure is 0.06-0.08 Mpa, the air quantity is 0.5-1 v/vm, the dissolved oxygen is 20-30%, and when the glucose level in the fermentation liquid is lower than 10g/L, the glucose solution with the mass percent of 60% is fed in, the glucose level in the fermentation liquid is maintained at 10-20g/L, and the fermentation time is 40h.
The present pair of tests was repeated 3 times, and the results of the HPLC measurements of the L-proline and glutamic acid contents in the fermentation broth when the fermentation was stopped are shown in Table 1.
Example 1:
inoculating corynebacterium glutamicum into a 10L seed culture tank for culture, wherein the seed culture medium is as follows: 25g/L of sucrose, 3g/L of yeast powder, 20g/L of beet molasses, 3g/L of potassium chloride, 2g/L of phosphoric acid, 3g/L of magnesium sulfate heptahydrate, 0.5mg/L of biotin and vitamin B 1 1mg/L. The culture conditions are as follows: the temperature is 31.5 ℃, the pH value is regulated to 7.2 by ammonia water, and the dissolved oxygen is 30-60%. Stopping culturing when the OD number of the seed solution reaches 35-40, and inoculating 60L seed solution according to 20% of inoculation amountAnd fermenting in a fermentation tank. The fermentation medium is as follows: 120g/L glucose, 50g/L corn steep liquor, 25g/L glutamic acid, 3g/L potassium chloride, 1g/L phosphoric acid, 3g/L magnesium sulfate heptahydrate, 1mg/L biotin and vitamin B 1 1mg/L, and the pH value is continuously adjusted to 7.2 by ammonia water. The fermentation conditions are as follows: when the temperature is 32 ℃, the rotating speed is 200-700 r/min, the tank pressure is 0.06-0.08 Mpa, the air quantity is 0.5-1 v/vm, the dissolved oxygen is 20-30%, and when the glucose level in the fermentation liquid is lower than 10g/L, the glucose solution with the mass percent of 60% is fed in, the glucose level in the fermentation liquid is maintained at 10-20g/L, and the fermentation time is 40h.
This example was repeated 3 times, and the content of proline and glutamic acid in the fermentation broth when the fermentation was stopped was measured by HPLC, and the detection results are shown in Table 1.
Example 2:
this example differs from example 1 in that the fermentation medium also comprises 15g/L ammonium sulphate; the other is exactly the same as in example 1.
This example was repeated 3 times, and the content of proline and glutamic acid in the fermentation broth when the fermentation was stopped was measured by HPLC, and the detection results are shown in Table 1.
Example 3:
this example differs from example 2 in that the dissolved oxygen is 5 to 20%; the other is exactly the same as in example 2.
This example was repeated 3 times, and the content of proline and glutamic acid in the fermentation broth when the fermentation was stopped was measured by HPLC, and the detection results are shown in Table 1.
Example 4:
the difference between this example and example 3 is that when the glucose level in the fermentation broth is lower than 40g/L, a 60% glucose solution by mass is fed in, and the glucose level in the fermentation broth is maintained at 40-50 g/L; the other is exactly the same as in example 3.
This example was repeated 3 times, and the content of proline and glutamic acid in the fermentation broth when the fermentation was stopped was measured by HPLC, and the detection results are shown in Table 1.
Example 5:
this example differs from example 4 in that the fermentation medium is: 120g/L glucose, 50g/L corn steep liquor and 2 glutamic acid5g/L, 15g/L of ammonium chloride, 3g/L of potassium chloride, 1g/L of phosphoric acid, 3g/L of magnesium sulfate heptahydrate, 1mg/L of biotin and vitamin B 1 1mg/L, and continuously adjusting the pH value to 7.2 by ammonia water; the other is exactly the same as in example 4.
This example was repeated 3 times, and the content of proline and glutamic acid in the fermentation broth when the fermentation was stopped was measured by HPLC, and the detection results are shown in Table 1.
Table 1 comparative examples and summary of test results for examples 1-5
As can be seen from the results in Table 1, in example 1, after glutamic acid is added into the fermentation medium, the acid production is improved by 7.8g/L, the conversion rate is improved by 3.4 percent, and the content of the hetero-acid glutamic acid is reduced by 0.4g/L compared with the comparative example; the glutamic acid is directly added into the fermentation medium, so that the acid production and conversion rate of fermentation can be improved.
Example 2 on the basis of example 1, ammonium sulfate is further added into the fermentation medium, the acid production of example 2 is improved by 2.0g/L, the conversion rate is improved by 0.9 percent, and the content of the hetero-acid glutamic acid is reduced by 0.2g/L compared with example 1; ammonium sulfate is further added into the fermentation medium to be matched with glutamic acid, so that the acid production and conversion rate of fermentation are further improved.
Example 3 based on example 2, the dissolved oxygen is adjusted to 5-20%, compared with example 2, example 3 improves acid production by 1.9g/L, improves conversion rate by 0.9 percent, and reduces content of hetero acid glutamic acid by 0.2g/L; the dissolved oxygen is regulated to 5-20%, and the acid production and conversion rate of fermentation are further improved.
Example 4 on the basis of example 3, the glucose level in the fermentation broth is maintained at 40-50g/L, compared with example 3, example 4 has the advantages that the acid production is improved by 3.7g/L, the conversion rate is improved by 1.6 percent, and the content of the glutamic acid in the mixed acid is reduced by 0.4g/L; the invention adopts fermentation conditions of high residual sugar and low dissolved oxygen, on one hand, the invention promotes thalli to actively synthesize the permeation regulating substance, namely L-proline, and on the other hand, limits the flux of the thalli for metabolizing glutamic acid, so that the L-proline realizes a large amount of accumulation while the accumulation of the hetero-acid glutamic acid is not much, and the fermentation conversion rate is improved.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (5)
1. A method for improving the fermentation conversion rate of L-proline is characterized in that corynebacterium glutamicum is used as a production strain, seed culture is carried out firstly to prepare secondary seed liquid, and then the secondary seed liquid is inoculated into a fermentation culture medium according to the proportion of 10-20% for fermentation culture; the fermentation medium contains 20-30g/L glutamic acid.
2. The method for improving the fermentation conversion rate of L-proline according to claim 1, wherein the fermentation medium further contains 10-20g/L of ammonium sulfate or ammonium chloride.
3. The method for improving the fermentation conversion rate of L-proline according to claim 2, wherein the fermentation medium further comprises 100-150 g/L glucose, 40-60 g/L corn steep liquor, 3-5 g/L potassium chloride, 1-3 g/L phosphoric acid, 3-5 g/L magnesium sulfate heptahydrate, 1-3 mg/L biotin and vitamin B 1 1-3 mg/L, and continuously adjusting the pH value to 7.0-7.5 by ammonia water.
4. A method for increasing the fermentation conversion of L-proline according to any one of claims 1 to 3, wherein the dissolved oxygen is controlled to be 5 to 20% during the fermentation.
5. The method for improving the fermentation conversion rate of L-proline according to claim 4, wherein the glucose content is maintained at 40-50g/L and the fermentation period is 40-44h.
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