CN117004662A - Large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid - Google Patents
Large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid Download PDFInfo
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- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 title claims abstract description 41
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 title claims abstract description 41
- 229960005261 aspartic acid Drugs 0.000 title claims abstract description 41
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 title claims abstract description 37
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 title claims abstract description 29
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 title claims abstract description 27
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 title claims abstract description 27
- 239000011976 maleic acid Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000011031 large-scale manufacturing process Methods 0.000 title claims abstract description 18
- 230000003197 catalytic effect Effects 0.000 title claims abstract description 17
- 230000008878 coupling Effects 0.000 title claims abstract description 11
- 238000010168 coupling process Methods 0.000 title claims abstract description 11
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 241000588724 Escherichia coli Species 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 102000003813 Cis-trans-isomerases Human genes 0.000 claims abstract description 5
- 108090000175 Cis-trans-isomerases Proteins 0.000 claims abstract description 5
- NLVWBYNKMPGKRG-ODZAUARKSA-N azane;(z)-but-2-enedioic acid Chemical compound N.OC(=O)\C=C/C(O)=O NLVWBYNKMPGKRG-ODZAUARKSA-N 0.000 claims abstract description 5
- 108090000856 Lyases Proteins 0.000 claims abstract description 4
- 102000004317 Lyases Human genes 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 30
- 230000004151 fermentation Effects 0.000 claims description 30
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000012526 feed medium Substances 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 7
- 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 6
- 229960000723 ampicillin Drugs 0.000 claims description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229930027917 kanamycin Natural products 0.000 claims description 6
- 229960000318 kanamycin Drugs 0.000 claims description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 6
- 229930182823 kanamycin A Natural products 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 229960002303 citric acid monohydrate Drugs 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 12
- 239000006227 byproduct Substances 0.000 abstract description 7
- 229910021529 ammonia Inorganic materials 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 6
- 238000000746 purification Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 abstract description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 abstract description 3
- 239000001630 malic acid Substances 0.000 abstract description 3
- 235000011090 malic acid Nutrition 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 230000003834 intracellular effect Effects 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- 230000035764 nutrition Effects 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 6
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000001530 fumaric acid Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 108010011485 Aspartame Proteins 0.000 description 1
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- PMLJIHNCYNOQEQ-REOHCLBHSA-N L-aspartic 1-amide Chemical compound NC(=O)[C@@H](N)CC(O)=O PMLJIHNCYNOQEQ-REOHCLBHSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229920000805 Polyaspartic acid Polymers 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000020939 nutritional additive Nutrition 0.000 description 1
- 108010064470 polyaspartate Proteins 0.000 description 1
- 229940068988 potassium aspartate Drugs 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 235000021092 sugar substitutes Nutrition 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 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
- C12P13/20—Aspartic acid; Asparagine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12Y—ENZYMES
- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
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- C12Y502/00—Cis-trans-isomerases (5.2)
- C12Y502/01—Cis-trans-Isomerases (5.2.1)
- C12Y502/01001—Maleate isomerase (5.2.1.1)
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- 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
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses a large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid, belonging to the technical field of industrial microorganisms. The method comprises the steps of firstly constructing recombinant escherichia coli simultaneously having a maleic acid cis-trans isomerase coding gene and an L-aspartic acid lyase coding gene, and further catalyzing maleic acid ammonia by using whole cells of the recombinant escherichia coli to generate L-aspartic acid. On the basis, the nutrition condition and the culture process of the recombinant escherichia coli are optimized. The invention can efficiently catalyze maleic acid to produce L-aspartic acid by ammonia, reduces the generation of by-product malic acid, is favorable for subsequent separation and purification, has higher intracellular enzyme stability, is easy to recycle and reuse, and saves cost. The method has the advantages of simple process, high yield, less byproducts, low pollution and lower equipment requirements, and has outstanding technical advantages and large-scale application prospects.
Description
Technical Field
The invention relates to the technical field of industrial microorganisms, in particular to a large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid.
Background
L-aspartic acid has wide application in the fields of medicine, food, chemical industry and the like. In the field of medicine, the amino acid preparation is a main component of an amino acid preparation and is a synthetic raw material of various medicines of L-potassium aspartate, magnesium, calcium and aspartyl ammonia; in the food industry, L-aspartic acid is a good nutritional supplement, added to various cool beverages, and is a main production raw material of sugar substitute aspartame; in the chemical industry field, the polyaspartic acid can be used as a raw material for manufacturing synthetic resin and is used for synthesizing a large amount of environment-friendly materials; can also be used as nutritional additive for cosmetics.
The L-aspartic acid can be prepared by synthesis and fermentation. The synthesis method mainly uses maleic acid or fumaric acid or their esters as raw materials, and uses ammonia treatment under pressure, then hydrolyzes. The synthesis of fumaric acid is carried out under the conditions of low pH, inorganic catalyst and the like, and after separation and purification, L-aspartic acid is synthesized under the conditions of excessive ammonia and aspartase, so that the pollution is high, the equipment requirement is high, and meanwhile, the separation and purification have loss and cost. Moreover, as step synthesis, complicated process and increased risk can be caused; in addition, the existing maleic acid cis-trans isomerase lacks stability, so that the synthesis efficiency is low, and meanwhile, the yield of byproducts is affected.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention provides a large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid, which aims to solve the technical problems of complex process, lower yield, more byproducts, high pollution, high equipment requirement and the like of the conventional synthesis method.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the large-scale production method for preparing L-aspartic acid by double enzyme coupling catalytic maleic acid is characterized by comprising the following steps: constructing recombinant escherichia coli simultaneously having a maleic acid cis-trans isomerase coding gene and an L-aspartic acid lyase coding gene, and catalyzing maleic acid ammonia by using whole cells of the recombinant escherichia coli to generate L-aspartic acid.
Preferably, the recombinant escherichia coli whole cell for catalyzing the ammonia addition of maleic acid to generate L-aspartic acid is obtained by primary seed culture, secondary seed culture and fermentation culture of the recombinant escherichia coli strain.
Preferably, the primary seed culture uses LB medium.
Preferably, each L of the LB medium contains: 10.0g of peptone, 5.0g of yeast powder and 10.0g of NaCl.
Preferably, the culture conditions for the primary seed culture include: 50. Mu.g/ml kanamycin and 50. Mu.g/ml ampicillin were added at 37℃and 200rpm, and the culture was continued for 7.5 hours with an inoculum size of 6.5%.
Preferably, the secondary seed culture uses a 2YT medium.
Preferably, each L of the 2YT medium contains: 16.0g of peptone, 10.0g of yeast powder and 5.0g of NaCl.
Preferably, the culture conditions for the secondary seed culture include: 50. Mu.g/ml kanamycin and 50. Mu.g/ml ampicillin were added at 37℃and 200rpm, and the culture was continued for 7.5 hours with an inoculum size of 6.5%.
Preferably, the fermentation medium used in the fermentation culture contains per L: glucose 8.0g, peptone 2.0g, yeast powder 2.0g, citric acid monohydrate 1.7g, mgSO 4 ·7H 2 O 1.7g，(NH 4 ) 2 HPO 4 4.0g，KH 2 PO 4 13.5g, pH is adjusted to 7.0g by ammonia water; at the saidA fermenter feed medium used in fermentation culture, comprising per L: glucose 500g, peptone 4.0g, yeast powder 4.0g, mgSO 4 ·7H 2 O 7.4g。
Preferably, the fermentation culture conditions include: adding 50 mug/mL of kanazurin at 37 ℃, inoculating 6.5% and ventilation of 2vvm, setting the dissolved oxygen in the fermentation tank to be not lower than 30% in the whole fermentation process, and after the carbon source in the initial culture medium is consumed and the dissolved oxygen rebound phenomenon occurs, starting to add a feeding culture medium into the fermentation tank in an exponential feeding manner, wherein the final concentration of the kanazurin in the feeding culture medium is also 50 mug/mL; when the cell density OD 600 When 80 is reached, the temperature is reduced to 30 ℃, and simultaneously IPTG with the final concentration of 0.5mmol/L is added for induction expression; the pH is regulated by adding 25% ammonia water in the whole fermentation process, so that the pH is maintained at 6.7-7.3; the fermentation period is 44-52 h.
The invention discloses a large-scale production method for preparing L-aspartic acid by double-enzyme coupling catalytic maleic acid. The method comprises the steps of firstly constructing recombinant escherichia coli simultaneously having a maleic acid cis-trans isomerase coding gene and an L-aspartic acid lyase coding gene, and further catalyzing maleic acid ammonia by using whole cells of the recombinant escherichia coli to generate L-aspartic acid. On the basis, the nutrition condition and the culture process of the recombinant escherichia coli are optimized. The invention can efficiently catalyze maleic acid to produce L-aspartic acid by ammonia, reduces the generation of by-product malic acid, is favorable for subsequent separation and purification, has higher intracellular enzyme stability, is easy to recycle and reuse, and saves cost. The method has the advantages of simple process, high yield, less byproducts, low pollution and lower equipment requirements, and has outstanding technical advantages and large-scale application prospects.
Drawings
FIG. 1 is a standard graph of L-aspartic acid concentration versus absorbance in an embodiment of the invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail. In order to avoid unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. Approximating language, as used in the following examples, may be applied to create a quantitative representation that could permissibly vary without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The large-scale production method for preparing L-aspartic acid by double enzyme coupling catalytic maleic acid comprises the following steps:
1. instrument and reagent
The reagents and apparatus used in the present invention are shown in Table 1 below.
Table 1 part of instrument and reagent list
2. Culture medium
LB medium (g.L) -1 ): peptone 10.0, yeast powder 5.0, naCl 10.0.
2YT culture medium (g.L) -1 ): peptone 16.0, yeast powder 10.0, naCl 5.0.
Fermentation medium of fermenter (g.L) -1 ): glucose 8.0, peptone 2.0, yeast powder 2.0, citric acid monohydrate 1.7, mgSO 4 ·7H 2 O 1.7，(NH 4 ) 2 HPO 4 4.0，KH 2 PO 4 13.5, pH was adjusted to 7.0 with ammonia.
Fermentation tank feed medium (g.L) -1 ): glucose 500, peptone 4.0, yeast powder 4.0, mgSO 4 ·7H 2 O7.4。
3. Culture conditions
First-stage seed: LB medium, temperature 37 ℃, rotation speed 200rpm, 50. Mu.g/ml kanamycin, 50. Mu.g/ml ampicillin, inoculum size 6.5%, and 7.5h.
Secondary seed: the culture was carried out at 37℃and 200rpm for 7.5 hours with 50. Mu.g/ml kanamycin and 50. Mu.g/ml ampicillin, respectively, and an inoculum size of 6.5%.
Fermentation culture: temperature of 37℃50. Mu.g/ml kanazurin, inoculum size 6.5%, ventilation volume 2vvm, setting dissolved oxygen in the fermentation tank not lower than 30% in the whole fermentation process, after the consumption of carbon source in the initial culture medium is completed and the rebound phenomenon of dissolved oxygen occurs, starting to feed a feed medium into the fermentation tank in an exponential feeding manner, wherein the final concentration of kanazurin in the feed medium is 50 mug/mL -1 . When the cell density OD 600 When the concentration reaches about 80 ℃, the temperature is reduced to 30 ℃, and the final concentration of 0.5 mmol.mL is added -1 Is induced to express. The pH was adjusted by feeding 25% ammonia water during the whole fermentation process to maintain it at about 7.0. The fermentation period was about 48 hours.
4. Performance testing
And measuring the content of L-aspartic acid in the product by adopting an ultraviolet spectrophotometry.
4.1 preparation of standard curve
Preparing L-aspartic acid standard solutions with different concentration gradients, wherein the concentration gradients of the L-aspartic acid standard solutions are as follows: 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL1.0mg/mL.
4mL of standard solution with different concentrations are respectively filled into 10mL of test tubes, then 2mL of ninhydrin color reagent is filled into the test tubes for display reaction, the absorbance values of the standard solution with different concentrations of L-aspartic acid are measured at the wavelength of 510nm, and a standard curve is prepared according to the measurement result. The standard curve is shown in figure 1. The standard curve formula is: y=0.0678x+0.0135; r is (r) 2 ＝0.9995。
4.2 test of samples to be tested
Transferring 4mL of the liquid to be detected into a 10mL test tube by using a pipette, adding 2mL of ninhydrin color former for color development, measuring the absorbance value at 510nm, and calculating the L-aspartic acid content in the fermentation liquor according to the absorbance value of the standard curve and the sample.
The test results are shown in table 2 below:
TABLE 2 detection results of L-aspartic acid and by-product malic acid content in the product
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the scope of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The large-scale production method for preparing L-aspartic acid by double enzyme coupling catalytic maleic acid is characterized by comprising the following steps: constructing recombinant escherichia coli simultaneously having a maleic acid cis-trans isomerase coding gene and an L-aspartic acid lyase coding gene, and catalyzing maleic acid ammonia by using whole cells of the recombinant escherichia coli to generate L-aspartic acid.
2. The large-scale production method for preparing L-aspartic acid by double enzyme coupling catalytic maleic acid according to claim 1, wherein the recombinant escherichia coli whole cells for catalyzing maleic acid ammonia to produce L-aspartic acid are obtained by primary seed culture, secondary seed culture and fermentation culture of the recombinant escherichia coli strain.
3. The large-scale production method for preparing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 2, wherein the primary seed culture adopts LB culture medium.
4. The large-scale production method for producing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 3, wherein each L of the LB medium contains: 10.0g of peptone, 5.0g of yeast powder and 10.0g of NaCl.
5. The large-scale production method for preparing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 3, wherein the culture conditions of the primary seed culture include: 50. Mu.g/ml kanamycin and 50. Mu.g/ml ampicillin were added at 37℃and 200rpm, and the culture was continued for 7.5 hours with an inoculum size of 6.5%.
6. The large-scale production method for preparing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 2, wherein the secondary seed culture adopts a 2YT culture medium.
7. The large-scale production method for preparing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 6, wherein each L of the 2YT medium contains: 16.0g of peptone, 10.0g of yeast powder and 5.0g of NaCl.
8. The large-scale production method for preparing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 6, wherein the culture conditions of the secondary seed culture include: 50. Mu.g/ml kanamycin and 50. Mu.g/ml ampicillin were added at 37℃and 200rpm, and the culture was continued for 7.5 hours with an inoculum size of 6.5%.
9. The large-scale production method for producing L-aspartic acid by double enzyme-coupled catalytic maleic acid according to claim 2, wherein the fermentation medium used in the fermentation culture contains per L: glucose 8.0g, peptone 2.0g, yeast powder 2.0g, citric acid monohydrate 1.7g, mgSO 4 ·7H 2 O1.7g，(NH 4 ) 2 HPO 4 4.0g，KH 2 PO 4 13.5g, pH is adjusted to 7.0g by ammonia water; a fermenter feed medium used in the fermentation culture, comprising per L: glucose 500g, peptone 4.0g, yeast powder 4.0g, mgSO 4 ·7H 2 O 7.4g。
10. The large-scale production method for preparing L-aspartic acid by double enzyme coupling catalysis of maleic acid according to claim 2, wherein the fermentation culture conditions comprise: adding kanazurin 50 μg/ml at 37deg.C, inoculating 6.5%, ventilating 2vvm, setting dissolved oxygen in the fermentation tank at not lower than 30% in the whole fermentation process, and after the carbon in the initial culture medium is consumed,After the dissolved oxygen rebound phenomenon occurs, feeding a feed medium into the fermentation tank in an exponential feeding manner, wherein the final concentration of the kanapigenin in the feed medium is 50 mug/mL; when the cell density OD 600 When 80 is reached, the temperature is reduced to 30 ℃, and simultaneously IPTG with the final concentration of 0.5mmol/L is added for induction expression; the pH is regulated by adding 25% ammonia water in the whole fermentation process, so that the pH is maintained at 6.7-7.3; the fermentation period is 44-52 h.
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