CN114875090A - Method for producing lysine and application - Google Patents
Method for producing lysine and application Download PDFInfo
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- CN114875090A CN114875090A CN202210616257.4A CN202210616257A CN114875090A CN 114875090 A CN114875090 A CN 114875090A CN 202210616257 A CN202210616257 A CN 202210616257A CN 114875090 A CN114875090 A CN 114875090A
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- fermentation
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- lysine
- glucose
- threonine
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- 239000004472 Lysine Substances 0.000 title claims abstract description 64
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- 238000000855 fermentation Methods 0.000 claims abstract description 163
- 230000004151 fermentation Effects 0.000 claims abstract description 158
- 239000004473 Threonine Substances 0.000 claims abstract description 59
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 22
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 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 37
- 239000008103 glucose Substances 0.000 claims description 37
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 24
- 230000001276 controlling effect Effects 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 18
- 240000008042 Zea mays Species 0.000 claims description 17
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 17
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 17
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 17
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 17
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 17
- 235000005822 corn Nutrition 0.000 claims description 17
- 238000011218 seed culture Methods 0.000 claims description 15
- 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
- 235000019764 Soybean Meal Nutrition 0.000 claims description 13
- 239000004455 soybean meal Substances 0.000 claims description 13
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 12
- 229910021529 ammonia Inorganic materials 0.000 claims description 12
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 11
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 229960002685 biotin Drugs 0.000 claims description 7
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- 239000011616 biotin Substances 0.000 claims description 7
- 239000000413 hydrolysate Substances 0.000 claims description 7
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- 238000005273 aeration Methods 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 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 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 238000007599 discharging Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims 2
- 239000000758 substrate Substances 0.000 abstract description 7
- 238000003912 environmental pollution Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 64
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 54
- 229960002898 threonine Drugs 0.000 description 54
- 235000018977 lysine Nutrition 0.000 description 52
- 239000002253 acid Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 230000037361 pathway Effects 0.000 description 4
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
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- 238000011160 research Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
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- 241000588724 Escherichia coli Species 0.000 description 2
- 108010064711 Homoserine dehydrogenase Proteins 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 238000009423 ventilation Methods 0.000 description 2
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- 108010055400 Aspartate kinase Proteins 0.000 description 1
- 108020004652 Aspartate-Semialdehyde Dehydrogenase Proteins 0.000 description 1
- 102100021758 E3 ubiquitin-protein transferase MAEA Human genes 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- HOSWPDPVFBCLSY-VKHMYHEASA-N L-aspartic 4-semialdehyde Chemical compound [O-]C(=O)[C@@H]([NH3+])CC=O HOSWPDPVFBCLSY-VKHMYHEASA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 108010043075 L-threonine 3-dehydrogenase Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102100026808 Mitochondrial import inner membrane translocase subunit Tim8 A Human genes 0.000 description 1
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- 230000004913 activation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 108010071598 homoserine kinase Proteins 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- -1 lysine Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000029219 regulation of pH Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000013589 supplement 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention relates to the technical field of biological fermentation, and particularly discloses a method for producing lysine and application thereof. The method for producing lysine of the invention carries out co-fermentation culture on the fermentation bacteria for producing lysine and the fermentation bacteria for producing threonine in the same fermentation medium. The method can improve the utilization rate of the substrate, save labor and energy, simplify process equipment, reduce environmental pollution and provide a new method for producing lysine.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for producing lysine and application thereof.
Background
Lysine is a basic amino acid, formula C 6 H 14 N 2 O 2 Molecular weight 146.19, which contains two amino groups in the molecule. Lysine has wide application as food enhancer and medicine, and can be used as liver cell regenerating agent for improving liver function and treating liver cirrhosisHas obvious curative effect of promoting appetite and improving nutrition status. As a feed additive, a small amount of lysine is added into the feed of livestock and poultry, and the feed additive has particularly obvious effects on the daily gain, feed conversion ratio, egg laying amount and the like of the livestock and the poultry.
The metabolic pathways for producing lysine from glucose comprise an EMP pathway, an HMP pathway, a TCA pathway, a glyoxylate pathway, a carbon dioxide fixation reaction and an oxaloacetate amination reaction to produce aspartic acid, wherein the aspartic acid generates cyclic aspartate-beta-semialdehyde under the catalysis of aspartokinase and aspartate-beta-semialdehyde dehydrogenase, and then is divided into two paths, on one hand, lysine is generated under the catalysis of a series of enzymes such as DDP synthetase, on the other hand, homoserine is generated under the catalysis of homoserine dehydrogenase, and on the other hand, threonine is generated under the catalysis of enzymes such as homoserine kinase, and on the other hand, isoleucine is generated under the catalysis of threonine dehydrogenase.
Lysine-producing microorganisms can increase lysine production by auxotrophic mutants, such as threonine-deficient strains. Threonine can be added into the formula in the lysine fermentation process to meet the growth requirement of lysine-producing bacteria. Threonine is also an amino acid produced by microbial fermentation. The production of threonine or lysine by fermentation requires separate fermentation equipment, raw and auxiliary materials and energy, which is not favorable for saving cost and avoiding environmental pollution, so that further research on the production and fermentation of lysine is necessary.
Disclosure of Invention
The invention aims to provide a lysine production method which has the advantages of high substrate utilization rate, labor and energy saving, simplified process equipment and small environmental pollution.
The technical scheme of the invention is as follows:
a method for producing lysine comprises co-fermenting and culturing lysine-producing fermentation bacteria and threonine-producing fermentation bacteria in the same fermentation medium.
In the method, the zymocyte for producing lysine is MHZ-0914, and the preservation number of the MHZ-0914 is CGMCC No. 22648;
the fermentation bacteria for producing threonine is MHZ-0216-5.
The invention provides a novel method for producing a basic amino acid, namely lysine, which comprises the steps of inoculating a strain for producing the lysine into a culture medium for fermentation culture, and simultaneously inoculating a strain for producing threonine, and performing mixed culture with lysine bacteria. Threonine produced by the threonine production strain is used as a nitrogen source for lysine production by the lysine production strain, the lysine production strain grows and metabolizes to complete fermentation and produce lysine, so that the production of the lysine nitrogen source and the production of the lysine are carried out simultaneously in the same substrate, the threonine does not need to be produced before the lysine is produced, the fermentation process and the fermentation substrate are integrally saved, the substrate utilization rate is improved, and the industrial production cost is greatly saved.
In the method of the invention, the ratio of the inoculation amount of the lysine-producing fermentation tubes to the inoculation amount of the threonine-producing fermentation tubes is 1: (0.04-0.16), preferably 1: 0.08.
In the method, when the co-fermentation culture is carried out, the initial fermentation glucose concentration is 19-21g/L, the fermentation temperature is 36-38 ℃, the fermentation pressure is controlled to be 0.07-0.09MPa, and the ventilation volume is 0.9-1.1 vvm; continuously feeding glucose and ammonium sulfate during fermentation, controlling residual sugar concentration at 0-1g/L, and fermenting ammonia Nitrogen (NH) 4 + ) The concentration is 0.8-1.2g/L, ammonia water is used for regulating and controlling the pH value, the fermentation pH value is 6.7-7.1, and the fermentation time is 35-37 h; when the volume of the culture solution in the fermentation tank is 68-72% of the total volume of the fermentation tank, discharging the culture solution, wherein the discharge volume is 4.8-5.2% of the volume of the culture medium.
The invention takes lysine producing bacteria and threonine producing bacteria as research objects, considers the amount of threonine required to be produced during mixed culture from the amount of threonine required to be added in the single lysine fermentation process, and controls the specific inoculation proportion and the ammonium ion concentration in the fermentation process to ensure that the growth and metabolism of the lysine producing bacteria are not influenced (the mixed fermentation reaches the fermentation index level of pure fermentation lysine), and specifically controls the growth of the threonine producing bacteria, thereby realizing the control of the yield of the threonine, ensuring that the production effect of the lysine during the mixed fermentation is good, and the utilization rate of the substrate is high.
In the method, the carbon source of the fermentation medium comprises glucose and sucrose, and the nitrogen source comprises ammonium sulfate and ammonia water.
In the method of the present invention, the fermentation medium comprises:
glucose 19-21g/L, H 3 PO 4 1.2-1.7g/L，KCl 0.4-0.6g/L，MgSO 4 0.6-0.8g/L of molasses, 9-11g/L of corn steep liquor, 58-62g/L of soybean meal hydrolysate, 29-31g/L of MnSO 4 0.0018-0.0022g/L，FeSO 4 0.0018-0.0022g/L，VB 1 95-105 μ g/L, biotin 190-210 μ g/L; glucose and ammonium sulfate were fed.
Preferably, the fermentation medium comprises: glucose 20g/L, H 3 PO 4 1.5g/L，KCl 0.5g/L，MgSO 4 0.7g/L of molasses, 10g/L of corn steep liquor, 60g/L of soybean meal hydrolysate, 30g/L of MnSO 4 0.002g/L，FeSO 4 0.002g/L，VB 1 100 mu g/L and biotin 200 mu g/L.
In the method of the present invention, the seed culture medium of the lysine-producing fermentation tubes comprises: 39-41g/L of glucose, KH 2 PO 4 1.4-1.6g/L，MgSO 4 1.4-1.6g/L of molasses, 11-13g/L of corn steep liquor, 24-26g/L of ammonium sulfate and MnSO 4 0.0015-0.0025g/L，FeSO 4 0.0015-0.0025g/L。
In the method of the present invention, the seed culture medium of the threonine producing zymophyte includes: 18-22g/L of glucose, 10-20g/L of corn steep liquor, 3-7g/L of soybean meal hydrolysate and MgSO 4 ·7H 2 O 0.4-0.6g/L，KH 2 PO 4 1.8-2.2g/L。
The invention also provides application of the method in reducing the cost of producing lysine.
The invention has the beneficial effects that:
the invention carries out fermentation culture on two different zymophytes under the same condition, can improve the utilization rate of the substrate, save labor and energy, simplify process equipment and reduce environmental pollution, and provides a novel method for producing lysine.
Drawings
FIG. 1 shows the results of fermentation of lysine OD and acid production in MHZ-091450L fermentor in example 1 of the present invention.
FIG. 2 shows the results of threonine fermentation OD and acid production in MHZ-0216-550L fermentation tank in example 2 of the present invention.
FIG. 3 shows the fermentation OD and acid production results of a 50L fermentor with different inoculum sizes of MHZ-0216-5 in example 3 of the present invention.
FIG. 4 shows the fermentation OD and acid production results of a 50L fermentation tank under the control of MHZ-0216-5 ammonia nitrogen concentration in different fermentation processes in example 4 of the present invention.
FIG. 5 shows the results of co-fermentation of OD and acid production in MHZ-0914 and MHZ-0216-550L fermentors in example 5 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the present invention, unless otherwise specified, the OD value is the result of the measurement at a wavelength of 600 nm.
In the embodiment of the invention, the experimental method is as follows:
(1) pure culture fermentation for producing lysine and threonine is respectively carried out;
(2) inoculating threonine bacteria into a lysine fermentation culture medium according to different inoculation amounts for pure culture fermentation;
(3) lysine bacteria and threonine bacteria are inoculated into a lysine fermentation medium according to a certain proportion for mixed fermentation.
Inoculating L-lysine and L-threonine producing strains in a bacteria-protecting tube stored at the temperature of minus 80 ℃ on a slant culture medium for activation;
secondly, inoculating the thalli on the slant culture medium into a first-class seed shake flask for culture;
inoculating the first-stage seed liquid into a 10L fermentation tank for second-stage seed culture;
and fourthly, inoculating the secondary seed liquid into a fermentation medium, and performing fermentation culture in a 50L fermentation tank.
And (3) condition control:
controlling the ammonia concentration: the ammonia concentration as a nitrogen source required for amino acid production cannot be in a low state in the medium, which would otherwise result in a decrease in the productivity of basic amino acids. While ammonia water is fed in, ammonium sulfate is fed in to keep ammonia concentration in the fermentation liquor at 0.8-1.2g/L, preferably 1 g/L.
Controlling sugar concentration and pH: the proportional relation between the two is obtained by observing the consumption conditions of the thalli in acid production, sugar and ammonia in the fermentation process. And the pH feedback signal is used as a control condition, the fermentation liquor is controlled by zero sugar, so that the pH feedback system can supplement sugar while adding ammonia into the fermentation tank, and a Kjeldahl nitrogen analyzer is used for monitoring the content of free ammonia in the fermentation process, and the record is recorded once every 6 h.
Example 1
This example uses lysine producing strains: MHZ-0914, for a separate fermentation.
Biomaterial MHZ-0914, taxonomic nomenclature: escherichia coli, deposited at CMCC on 1.6.2021 in China general microbiological culture Collection center (CMCC), was assigned: the microbial research institute of China academy of sciences, western road No. 1, 3, of Beijing, Chaoyang, and the collection number is CGMCC NO.22648, 100101.
Seed medium (g/L): glucose 40, KH 2 PO 4 1.5，MgSO 4 1.5 molasses 12, corn steep liquor 25, ammonium sulfate 12, MnSO 4 0.002，FeSO 4 0.002。
Fermentation medium: glucose 20g/L, H 3 PO 4 1.5g/L，KCl 0.5g/L，MgSO 4 0.7g/L of molasses, 10g/L of corn steep liquor, 60g/L of soybean meal hydrolysate, 30g/L of MnSO 4 0.002g/L，FeSO 4 0.002g/L，VB 1 100 mu g/L and biotin 200 mu g/L. Fedding (flow adding)Glucose 600g/L, ammonium sulfate 500g/L, total nitrogen: threonine 4 g/L. Sterilizing at 121 deg.C for 20 min.
Respectively inoculating the lysine producing strains into a seed tank, inoculating into a fermentation tank for fermentation when OD value reaches 0.8, wherein the fermentation inoculation ratio is 20%, the initial fermentation glucose concentration is 20g/L, the fermentation temperature is 36-38 ℃, the fermentation pressure is controlled at 0.08MPa, the ventilation volume is 1vvm, culturing is carried out for 36h, continuously feeding fermentable sugar into the fermentation tank, the fermentable sugar is high-concentration glucose liquid with the concentration of 600g/L, the fed ammonium sulfate concentration is 500g/L, the fed total nitrogen is threonine, the total nitrogen addition accounts for 22% of the volume of the glucose liquid addition, the residual sugar in the fermentation process is 1g/L, and fermenting ammonia nitrogen (inorganic free ammonia NH (NH)) is added 4 + ) The concentration is 0.8-1.2g/L, ammonia water is used for pH regulation, the fermentation pH value is 6.7-7.1, when the volume of the culture solution in the fermentation tank is 70% of the volume of the fermentation tank, the culture solution is discharged, the discharge volume is 5% of the volume of the culture medium, and the fermentation time is 36 h. During the fermentation process, acid and free ammonia during the fermentation process are measured.
The fermentation results are shown in FIG. 1, from which it can be seen that the fermentation time is 36h and the lysine-terminal acid content is 190 g/L. The total sugar content in fermentation is 10050g, the conversion rate is 65.2%, the theoretical acid is 6552g, the maximum OD value is 34, the threonine consumption is 14.5g, and the ammonia nitrogen in the fermentation process is controlled to be 0.8-1.2 g/L.
Example 2
This example uses threonine producing strains: MHZ-0216-5, and carrying out single fermentation.
The L-threonine-producing strain in this example is Escherichia coli MHZ-0216-5 (see Chinese patent CN113846132A for strain MHZ-0216-5).
Seed culture medium: 20g/L of glucose, 15g/L of corn steep liquor, 5g/L of soybean meal hydrolysate and MgSO 4 ·7H 2 O0.5g/L，KH 2 PO 4 2g/L。
Fermentation medium: 40g/L glucose, 7g/L corn steep liquor, 7g/L soybean meal hydrolysate and MgSO 4 ·7H 2 O0.5g/L，KH 2 PO 4 2g/L。
The sterilized L-threonine seed culture medium is added into a sterile seed tank 10L, water is added to adjust the initial volume of the seed culture medium to 6L, and 200mL of seed solution is inoculated. The culture conditions are as follows: aeration quantity is 0.8vvm, rotation speed is 300rpm, culture pH is 7.0, temperature is 37 ℃, and dissolved oxygen is 20%. And stopping culturing when the seeds grow to the OD value of 10.
Inoculating the sterilized L-threonine fermentation medium into a sterile fermentation tank 50L, adding sterile water to a constant volume of 15L; taking 3L of seed liquid in the seed tank and inoculating the seed liquid into the fermentation tank. Controlling conditions in the fermentation process: 0.5-0.8vvm, 300-700rpm, 0.07MPa of tank pressure, 7.0 of pH value, 37 ℃ of temperature, 20 percent of dissolved oxygen and 36 hours of fermentation period.
When the residual sugar content in the fermentation liquid is 0.1g/L, feeding carbon source, using ammonia water as pH regulator to control pH7.0, preparing 50% glucose solution as feeding carbon source, using 25% ammonia water as feeding nitrogen source, controlling pH7.0 in the fermentation process, and controlling the concentration of glucose in the fermentation liquid at about 1g/L in the fermentation process.
The glucose content was measured using an SBA biosensor, the free ammonium content was measured using a Kjeldahl determination apparatus, and the L-threonine content was measured using HPLC.
As shown in FIG. 2, the fermentation time was 36 hours, the L-threonine concentration was 110g/L, the conversion was 54%, the maximum OD value was 30, and the threonine production was 2540 g.
Example 3
This example uses threonine producing strains: MHZ-0216-5, and carrying out single fermentation. The lysine fermentation formulation of example 1 was used with the inoculum sizes of 1L, 0.5L, 0.25L, and 0.125L, respectively.
Seed culture medium: 20g/L of glucose, 15g/L of corn steep liquor, 5g/L of soybean meal hydrolysate and MgSO 4 ·7H 2 O0.5g/L，KH 2 PO 4 2g/L。
Fermentation medium: glucose 20g/L, H 3 PO 4 1.5g/L，KCl 0.5g/L，MgSO 4 0.7g/L of molasses, 10g/L of corn steep liquor, 60g/L of soybean meal hydrolysate, 30g/L of MnSO 4 0.002g/L，FeSO 4 0.002g/L，VB 1 100 mu g/L and biotin 200 mu g/L. 600g/L of glucose and 500g/L of ammonium sulfate are fed in, and threonine is not fed in.
The sterilized L-threonine seed culture medium is added into a sterile seed tank 10L, water is added to adjust the initial volume of the seed culture medium to 6L, and 200mL of seed solution is inoculated. The culture conditions are as follows: aeration quantity is 0.8vvm, rotation speed is 300rpm, culture pH is 7.0, temperature is 37 ℃, and dissolved oxygen is 20%. And stopping culturing when the seeds grow to the OD value of 10.
Inoculating the sterilized L-threonine fermentation medium into a sterile fermentation tank 50L, adding sterile water to a constant volume of 15L, and inoculating the seed solution. Controlling conditions in the fermentation process: 0.5-0.8vvm, 300-700rpm, 0.07MPa of tank pressure, 7.0 of pH value, 37 ℃ of temperature, 20 percent of dissolved oxygen and 36 hours of fermentation period.
When the residual sugar content in the fermentation solution is 0.1g/L, feeding carbon source, controlling pH to 7.0 with ammonia water as pH regulator, preparing 600g/L glucose solution as feeding carbon source, controlling pH to 7.0 with 25% ammonia water, controlling glucose concentration in the fermentation solution to about 1g/L, and controlling fermented ammonia nitrogen (inorganic free ammonia NH) with ammonium sulfate of 500g/L 4 + ) The concentration is 0.8-1.2 g/L.
The fermentation results are shown in FIG. 3, wherein Thr-1 and OD-1 are the test results of the inoculum size of 1L, Thr-2 and OD-2 are the test results of the inoculum size of 0.5L, Thr-3 and OD-3 are the test results of the inoculum size of 0.25L, and Thr-4 and OD-4 are the test results of the inoculum size of 0.125L, respectively, and it can be seen that the fermentation time is 36h, the threonine concentration of the inoculum size of 1L is 7.2g/L, the fermentation liquid size is 18.88L, and the yield is 136 g; inoculating 0.5L of threonine with the concentration of 5g/L, the volume of fermentation liquid of 16L and the yield of 80 g; inoculating 0.25L threonine with concentration of 4.4g/L, fermentation broth volume of 15.9L, and yield of 70 g; the threonine concentration of 0.125L of the inoculum is 3.1g/L, the volume of the fermentation liquid is 15.48L, and the yield is 48 g.
Example 4
This example uses threonine producing strains: MHZ-0216-5, and carrying out single fermentation. The lysine fermentation formulation of example 1 was used with an inoculum size of 0.25L.
Seed culture medium: 20g/L of glucose, 15g/L of corn steep liquor, 5g/L of soybean meal hydrolysate and MgSO 4 ·7H 2 O0.5g/L，KH 2 PO 4 2g/L。
Fermentation medium: glucose 20g/L, H 3 PO 4 1.5g/L，KCl 0.5g/L，MgSO 4 0.7g/L of molasses, 10g/L of corn steep liquor and 6g/L of corn steep liquor0g/L, 30g/L of soybean meal hydrolysate and MnSO 4 0.002g/L，FeSO 4 0.002g/L，VB 1 100 mu g/L and biotin 200 mu g/L. Adding glucose 600g/L, adding threonine, adding ammonium sulfate (concentration is 500g/L) to control ammonia nitrogen (inorganic free ammonia NH) in fermentation process 4 + ) The concentrations were 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, respectively.
The sterilized L-threonine seed culture medium is added into a sterile seed tank 10L, water is added to adjust the initial volume of the seed culture medium to 6L, and 200mL of seed solution is inoculated. The culture conditions are as follows: aeration quantity is 0.8vvm, rotation speed is 300rpm, culture pH is 7.0, temperature is 37 ℃, and dissolved oxygen is 20%. And stopping culturing when the seeds grow to the OD value of 10.
Inoculating the sterilized L-threonine fermentation medium into a sterile fermentation tank 50L, adding sterile water to a constant volume of 15L, and inoculating the seed solution. Controlling conditions in the fermentation process: 0.5-0.8vvm at 300-700rpm under 0.07MPa and pH7.0 at 37 deg.C, dissolved oxygen of 20%, and fermentation period of 36 h.
When the residual sugar content in the fermentation solution is 0.1g/L, feeding carbon source, using ammonia water as pH regulator to control pH to 7.0, preparing 600g/L glucose solution as feeding carbon source, using 25% ammonia water to control pH to 7.0, controlling the concentration of glucose in the fermentation solution to about 1g/L, and fermenting ammonia nitrogen (inorganic free ammonia NH) 4 + ) The concentration is controlled by feeding ammonium sulfate and is respectively 0.5g/L, 1.0g/L, 1.5g/L and 2.0 g/L.
The fermentation result is shown in figure 4, in the figure, Thr-1 and OD-1 are respectively the test result of controlling the ammonia nitrogen concentration to be 0.5g/L, Thr-2 and OD-2 are respectively the test result of controlling the ammonia nitrogen concentration to be 1g/L, Thr-3 and OD-3 are respectively the test result of controlling the ammonia nitrogen concentration to be 1.5g/L, and Thr-4 and OD-4 are respectively the test result of controlling the ammonia nitrogen concentration to be 2g/L, so that the fermentation time is 36h, the threonine concentration of controlling the ammonia nitrogen concentration to be 0.5g/L is 4g/L, the volume of the fermentation liquid is 18.0L, and the yield is 72 g; the threonine concentration of 1g/L ammonia nitrogen concentration is controlled to be 2.7g/L, the volume of the fermentation liquor is 17.3L, and the yield is 46.7 g; controlling the ammonia nitrogen concentration to be 1.5g/L, the threonine concentration to be 1.8g/L, the volume of the fermentation liquor to be 16.6L and the yield to be 30 g; threonine concentration of ammonia nitrogen concentration controlled to be 2.0g/L is 1g/L, volume of fermentation liquor is 15.5L, and yield is 15.5 g.
Example 5
The lysine MHZ-0914-producing strain and the threonine MHZ-0216-5-producing strain are respectively subjected to seed culture according to the records of examples 1 and 2, and then seed liquids of the two strains are inoculated into 15L of lysine fermentation medium in example 1, wherein the lysine inoculation amount is 20 percent, and the threonine inoculation amount is 1.6 percent.
Specifically, when threonine seeds and lysine seeds reach the OD of a lower tank, the threonine seeds and the lysine seeds are simultaneously inoculated into a lysine fermentation medium, the initial fermentation glucose concentration is 20g/L, the fermentation temperature is 36-38 ℃, the fermentation pressure is controlled at 0.08MPa, the aeration rate is 1vvm, the culture is carried out for 36h, and 600g/L of high-concentration glucose solution and ammonium sulfate (the concentration is 500g/L) are continuously fed into a fermentation tank. In the fermentation process, residual sugar is zero sugar, the concentration of fermentation ammonia nitrogen is 0.8-1.2g/L, 25% ammonia water is used for regulating and controlling pH, the fermentation pH value is 6.7-7.1, fermentation culture is carried out, when the volume of a culture solution in a fermentation tank is 70% of the volume of the fermentation tank, the culture solution is discharged, the discharge volume is 5% of the volume of a culture medium, and the fermentation time is 36 h.
The fermentation result is shown in figure 5, and the results show that the fermentation time is 36h, the lysine final acid content is 192g/L, the total sugar content in the fermentation is 10105g, the conversion rate is 65.3%, the theoretical acid content is 6598g, the threonine content in the fermentation tank is 0g, and the ammonia nitrogen content in the fermentation process is controlled to be 0.8-1.2 g/L.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for producing lysine is characterized in that fermentation bacteria for producing lysine and fermentation bacteria for producing threonine are subjected to co-fermentation culture in the same fermentation medium.
2. The method as claimed in claim 1, wherein the lysine producing fermented bacteria are MHZ-0914, and the preservation number of MHZ-0914 is CGMCC No. 22648;
the fermentation bacteria for producing threonine is MHZ-0216-5.
3. The method of claim 1 or 2, wherein the ratio of the inoculum size of the lysine producing yeast to the inoculum size of the threonine producing yeast is 1: (0.04-0.16), preferably 1: 0.08.
4. The method according to any one of claims 1 to 3, wherein in the co-fermentation culture, the initial fermentation glucose concentration is 19 to 21g/L, the fermentation temperature is 36 to 38 ℃, the fermentation pressure is controlled at 0.07 to 0.09MPa, and the aeration rate is 0.9 to 1.1 vvm; continuously feeding glucose and ammonium sulfate in a flowing manner in the fermentation process, controlling the concentration of residual sugar in the fermentation process to be 0-1g/L and the concentration of fermentation ammonia nitrogen to be 0.8-1.2g/L, regulating and controlling the pH value by ammonia water, wherein the fermentation pH value is 6.7-7.1, and the fermentation time is 35-37 h; when the volume of the culture solution in the fermentation tank is 68-72% of the total volume of the fermentation tank, discharging the culture solution, wherein the discharge volume is 4.8-5.2% of the volume of the culture medium.
5. The method of any one of claims 1 to 4, wherein the carbon source of the fermentation medium comprises glucose and sucrose and the nitrogen source comprises ammonium sulfate and ammonia.
6. The method of any one of claims 1-5, wherein the fermentation medium comprises:
glucose 19-21g/L, H 3 PO 4 1.2-1.7g/L，KCl 0.4-0.6g/L，MgSO 4 0.6-0.8g/L of molasses, 9-11g/L of corn steep liquor, 58-62g/L of soybean meal hydrolysate, 29-31g/L of MnSO 4 0.0018-0.0022g/L，FeSO 4 0.0018-0.0022g/L，VB 1 95-105 μ g/L, biotin 190-210 μ g/L; glucose and ammonium sulfate were fed.
7. The method of claim 6, wherein the fermentation medium comprises: glucose 20g/L, H 3 PO 4 1.5g/L，KCl 0.5g/L，MgSO 4 0.7g/L of molasses, 10g/L of corn steep liquor, 60g/L of soybean meal hydrolysate, 30g/L of MnSO 4 0.002g/L，FeSO 4 0.002g/L，VB 1 100 mu g/L and biotin 200 mu g/L.
8. The method of any one of claims 1-7, wherein the seed culture medium of lysine producing fermentors comprises: 39-41g/L of glucose, KH 2 PO 4 1.4-1.6g/L，MgSO 4 1.4-1.6g/L of molasses, 11-13g/L of corn steep liquor, 24-26g/L of ammonium sulfate and MnSO 4 0.0015-0.0025g/L，FeSO 4 0.0015-0.0025g/L。
9. The method of any one of claims 1-8, wherein the seed culture medium of the threonine-producing fermenting bacteria comprises: 18-22g/L of glucose, 10-20g/L of corn steep liquor, 3-7g/L of soybean meal hydrolysate and MgSO 4 ·7H 2 O 0.4-0.6g/L，KH 2 PO 4 1.8-2.2g/L。
10. Use of the method of any one of claims 1-9 to reduce the cost of producing lysine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1118719A (en) * | 1966-03-14 | 1968-07-03 | Kyowa Hakko Kogyo Kk | Process for producing l-lysine by fermentation |
| RU2347807C1 (en) * | 2007-12-13 | 2009-02-27 | Общество с ограниченной ответственностью "БИОРЕАКТОР" | Escherichia coli-lysine producer strain, method of making feed additive, containing this strain, composition, obtained using this method and method of monogastric animals and birds |
| CN113846132A (en) * | 2020-06-28 | 2021-12-28 | 廊坊梅花生物技术开发有限公司 | Construction of threonine producing strain and method for producing threonine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1118719A (en) * | 1966-03-14 | 1968-07-03 | Kyowa Hakko Kogyo Kk | Process for producing l-lysine by fermentation |
| RU2347807C1 (en) * | 2007-12-13 | 2009-02-27 | Общество с ограниченной ответственностью "БИОРЕАКТОР" | Escherichia coli-lysine producer strain, method of making feed additive, containing this strain, composition, obtained using this method and method of monogastric animals and birds |
| CN113846132A (en) * | 2020-06-28 | 2021-12-28 | 廊坊梅花生物技术开发有限公司 | Construction of threonine producing strain and method for producing threonine |
Non-Patent Citations (1)
| Title |
|---|
| ALISSA KERNER等: "A Programmable Escherichia coli Consortium via Tunable Symbiosis", PLOS ONE, vol. 7, no. 3, pages 1 - 10 * |
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