CN114606275A - Method for producing L-isoleucine through fermentation - Google Patents
Method for producing L-isoleucine through fermentation Download PDFInfo
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- CN114606275A CN114606275A CN202210128772.8A CN202210128772A CN114606275A CN 114606275 A CN114606275 A CN 114606275A CN 202210128772 A CN202210128772 A CN 202210128772A CN 114606275 A CN114606275 A CN 114606275A
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- isoleucine
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- 238000000855 fermentation Methods 0.000 title claims abstract description 198
- 230000004151 fermentation Effects 0.000 title claims abstract description 198
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 title claims abstract description 89
- 229960000310 isoleucine Drugs 0.000 title claims abstract description 89
- 229930182844 L-isoleucine Natural products 0.000 title claims abstract description 83
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 25
- 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 abstract description 64
- 239000008103 glucose Substances 0.000 claims abstract description 64
- 239000000463 material Substances 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 50
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 37
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 37
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 37
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000001963 growth medium Substances 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 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 abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 45
- 239000001301 oxygen Substances 0.000 claims description 45
- 229910052760 oxygen Inorganic materials 0.000 claims description 45
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 44
- 239000002609 medium Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 23
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 23
- 239000002904 solvent Substances 0.000 claims description 23
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 22
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 22
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 22
- 238000011218 seed culture Methods 0.000 claims description 21
- 238000005273 aeration Methods 0.000 claims description 20
- 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 18
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 18
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 18
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 14
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 14
- 239000004472 Lysine Substances 0.000 claims description 14
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 14
- 239000011790 ferrous sulphate Substances 0.000 claims description 14
- 229960002989 glutamic acid Drugs 0.000 claims description 14
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 14
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 14
- 229940099596 manganese sulfate Drugs 0.000 claims description 14
- 235000007079 manganese sulphate Nutrition 0.000 claims description 14
- 239000011702 manganese sulphate Substances 0.000 claims description 14
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 14
- 229930182817 methionine Natural products 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- 238000011081 inoculation Methods 0.000 claims description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 12
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 229960002685 biotin Drugs 0.000 claims description 9
- 235000020958 biotin Nutrition 0.000 claims description 9
- 239000011616 biotin Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000012262 fermentative production Methods 0.000 claims description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 9
- 229960001763 zinc sulfate Drugs 0.000 claims description 9
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 9
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 20
- 239000000243 solution Substances 0.000 description 25
- 239000002253 acid Substances 0.000 description 18
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 7
- 230000012010 growth Effects 0.000 description 6
- 241001052560 Thallis Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 230000002503 metabolic effect Effects 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 230000004060 metabolic process Effects 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 150000005693 branched-chain amino acids Chemical class 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- OCUSNPIJIZCRSZ-ZTZWCFDHSA-N (2s)-2-amino-3-methylbutanoic acid;(2s)-2-amino-4-methylpentanoic acid;(2s,3s)-2-amino-3-methylpentanoic acid Chemical compound CC(C)[C@H](N)C(O)=O.CC[C@H](C)[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O OCUSNPIJIZCRSZ-ZTZWCFDHSA-N 0.000 description 1
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 125000002061 L-isoleucyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](C([H])([H])[H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000001596 intra-abdominal fat Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Classifications
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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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for producing L-isoleucine by fermentation. The method comprises the following steps: inoculating the seed liquid of the L-isoleucine producing strain into a fermentation culture medium for fermentation to produce L-isoleucine; in the fermentation process, when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, feeding glucose, ammonium sulfate and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.5 wt%, the content of ammonium nitrogen to be 0.15-0.2 wt% and the mass ratio of the glucose to the small materials to be 8-9:1 within 0-20 h; after 20 hours, the content of residual reducing sugar in the fermentation liquor is controlled to be 0.5-1 wt%, the content of ammonium nitrogen is controlled to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials is 10-12: 1. The method provided by the invention can effectively shorten the fermentation period and improve the yield and the conversion rate of the L-isoleucine at the fermentation end point.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for producing L-isoleucine through fermentation.
Background
Isoleucine is also called as "isoleucine" and is systematically named as "alpha-amino-beta-methyl valeric acid" with the chemical formula C6H13NO2Is one of essential amino acids for human body, belongs to aliphatic neutral amino acid, is soluble in water and is slightly soluble in ethanol. The effects of isoleucine include the repair of muscle, the control of blood glucose, and the supply of energy to body tissues in cooperation with isoleucine and valine. It also increases the production of growth hormone and helps burn visceral fats, which are difficult to effectively act on by diet and exercise alone because they are inside the body. Leucine, isoleucine and valine are all branched chain amino acids that help promote muscle recovery after exercise. Among them, isoleucine is the most effective one of branched chain amino acids, and is effective for preventing muscle loss because it can be decomposed and converted into glucose more rapidly, and increasing glucose can prevent muscle tissue from being damaged, so that it is particularly suitable for body-building athletes. Since isoleucine is readily converted to glucose, it helps to regulate blood glucose levels. People with isoleucine deficiency can develop symptoms like hypoglycemia, such as headache, dizziness, fatigue, depression, confusion, irritability, and the like.
At present, three methods of producing L-isoleucine include an extraction method, a chemical synthesis method and a fermentation method. The extraction method and the chemical synthesis method are not suitable for industrial production because the produced L-isoleucine is difficult to separate from its isomer, the raw material source is limited, the production cost is high, the environment is polluted and the like. The fermentation method has mild conditions, environment friendliness and stable product quality, and becomes the most main method for producing L-isoleucine.
In the fermentation production process of L-isoleucine, factors such as strains, culture conditions and the like all influence the yield of L-isoleucine. The existing method for producing L-isoleucine by fermentation generally has the problems of long fermentation period and low acid production rate. Therefore, the optimization of the fermentation culture process to improve the yield of the L-isoleucine has important significance for the development of the L-isoleucine industry.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for producing L-isoleucine by fermentation. Compared with the prior art, the method can effectively shorten the fermentation period and improve the yield and the conversion rate of the L-isoleucine at the fermentation end point.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for the fermentative production of L-isoleucine comprising the steps of:
inoculating the seed liquid of the L-isoleucine producing strain into a fermentation culture medium for fermentation to produce L-isoleucine;
in the fermentation process, when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, feeding glucose, ammonium sulfate and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after feeding, wherein the mass ratio of the fed glucose to the small materials is 8-9: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-1.0 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and controlling the mass ratio of fed-batch glucose to small materials to be 10-12: 1;
the small material comprises the following components: 0.6-0.7g/L of zinc sulfate, 0.4-0.6g/L of magnesium sulfate, 0.4-0.6g/L of monopotassium phosphate, 0.4-0.6g/L of dipotassium phosphate and VB310-12mg/L, and the solvent is water.
In the invention, after the glucose, the ammonium sulfate and the small materials are fed in, the content of residual reducing sugar has the same endpoint value of 1 wt% within the first 20h and the content of ammonium nitrogen has the same endpoint value of 0.15 wt% within the first 20h, and the technical scheme that the content of residual reducing sugar is maintained to be 1 wt% in the whole process and/or the content of ammonium nitrogen is maintained to be 0.15 wt% in the whole process after the glucose, the ammonium sulfate and the small materials are fed in the beginning is not understood by the technical scheme in the invention, because the concentration of the materials in the actual fermentation process is dynamically changed, and can only fluctuate within a certain range and can not be always maintained at a determined value.
According to the invention, the residual reducing sugar concentration, the ammonium nitrogen concentration and the small material addition amount in different fermentation stages are controlled, so that the thalli have respective suitable external environments in the logarithmic growth phase and the stable metabolism phase, the growth, the proliferation and the metabolic acid production are balanced, and other process steps are combined, so that the fermentation period is shortened, and the yield and the conversion rate of the L-isoleucine at the fermentation end point are improved.
In some embodiments of the present invention, the method for preparing the seed liquid comprises the following steps:
inoculating the L-isoleucine producing strain into a primary seed culture medium, culturing for 16-18h under the conditions that the culture temperature is 30 +/-0.5 ℃, the rotation speed of a shaking table is 250-300rpm, and inoculating the strain into a secondary seed culture medium when the OD value of the strain at the 620nm position is more than or equal to 1.0, wherein the inoculation amount is 8-10% of the volume of the secondary seed culture medium, and culturing for 10-12h under the conditions that the temperature is 30 +/-0.5 ℃, the pressure is 0.05-0.08Mpa, the stirring rotation speed is 300-500rpm, the aeration ratio is 1:0.5-0.8, the pH is 7.2 +/-0.5, and the DO is more than or equal to 20 wt% to obtain the seed solution.
In some embodiments of the invention, the primary seed medium is LB liquid medium.
Preferably, the secondary seed culture medium comprises the following components: 38-40g/L of glucose, 0.8-0.9g/L of magnesium sulfate, 1.5-1.6g/L of monopotassium phosphate, 2.8-3.0g/L of ammonium sulfate, 0.1-0.12g/L of ferrous sulfate, 8-10mg/L of manganese sulfate, 2.8-3.0g/L of yeast extract powder, 0.6-0.7g/mL of methionine, 10-12g/L of glutamic acid, 0.75-0.8g/L of lysine and 10-12g/L of corn steep liquor, and the solvent is water.
In some embodiments of the invention, the L-isoleucine producing bacterium is Corynebacterium glutamicum.
In some embodiments of the invention, the inoculum size of the seed solution is 8-12% of the fermentation medium volume.
In some embodiments of the invention, the fermentation medium comprises the following components: 58-60g/L of glucose, 1.8-2.0g/L of magnesium sulfate, 2.0-2.2g/L of monopotassium phosphate, 5.0-5.2g/L of ammonium sulfate, 8-10mg/L of ferrous sulfate, 6.0-8.0mg/L of manganese sulfate, 1.0-1.2g/L of dipotassium phosphate, 3.0-3.5g/L of yeast extract powder, 0.3-0.4g/mL of methionine, 10-12g/L of glutamic acid, 1.0-1.2g/L of lysine, 15-18g/L of corn steep liquor and 0.08-0.1g/L of biotin, wherein the solvent is water, and the pH value is 7.0 +/-0.5.
In some embodiments of the invention, the initial pressure of the fermentation is 0.05-0.1MPa, the initial stirring speed is 300-.
In some embodiments of the invention, the temperature is increased from 30. + -. 0.5 ℃ to 33. + -. 0.5 ℃ within 0-12h after the start of the fermentation and is maintained at 33. + -. 0.5 ℃ after 12 h. Preferably, the temperature is raised by 1 ℃ every 4h after the start of the fermentation.
The temperature is gradually increased, so that the thalli can be logarithmically proliferated at a proper temperature, the metabolism is accelerated at 33 +/-0.5 ℃, and the target product is accumulated.
In some embodiments of the invention, during the fermentation process, when the dissolved oxygen in the fermentation broth reaches below 20% of the initial dissolved oxygen, the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen in the fermentation broth at 15-25% of the initial dissolved oxygen.
In some embodiments of the present invention, ammonia is fed during the fermentation process, so that the pH of the fermentation liquid is maintained at 7.0 ± 0.5.
In some embodiments of the invention, when the production rate of L-isoleucine is slowed or no longer increased, the feeding of ammonium sulfate and the small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is terminated when the dissolved oxygen amount begins to rise.
In some embodiments of the invention, the method comprises the steps of:
(1) inoculating corynebacterium glutamicum to a first-level seed culture medium, culturing for 16-18h under the conditions that the culture temperature is 30 +/-0.5 ℃, the shaking table rotating speed is 250-300rpm, and inoculating corynebacterium glutamicum to a second-level seed culture medium when the OD value of the strain at the 620nm is more than or equal to 1.0, wherein the inoculation amount is 8-10% of the volume of the second-level seed culture medium, and culturing for 10-12h under the conditions that the temperature is 30 +/-0.5 ℃, the pressure is 0.05-0.08MPa, the stirring rotating speed is 300-500rpm, the aeration ratio is 1:0.5-0.8, the pH is 7.2 +/-0.5, and the DO is more than or equal to 20 wt%, so as to obtain a seed solution;
wherein the primary seed culture medium is an LB liquid culture medium;
the secondary seed culture medium comprises the following components: 38-40g/L of glucose, 0.8-0.9g/L of magnesium sulfate, 1.5-1.6g/L of monopotassium phosphate, 2.8-3.0g/L of ammonium sulfate, 0.1-0.12g/L of ferrous sulfate, 8-10mg/L of manganese sulfate, 2.8-3.0g/L of yeast extract powder, 0.6-0.7g/mL of methionine, 10-12g/L of glutamic acid, 0.75-0.8g/L of lysine and 10-12g/L of corn steep liquor, and the solvent is water;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 8-12% of the volume of the fermentation culture medium;
the fermentation medium comprises the following components: 58-60g/L glucose, 1.8-2.0g/L magnesium sulfate, 2.0-2.2g/L potassium dihydrogen phosphate, 5.0-5.2g/L ammonium sulfate, 8-10mg/L ferrous sulfate, 6.0-8.0mg/L manganese sulfate, 1.0-1.2g/L dipotassium hydrogen phosphate, 3.0-3.5g/L yeast extract powder, 0.3-0.4g/mL methionine, 10-12g/L glutamic acid, 1.0-1.2g/L lysine, 15-18g/L corn steep liquor and 0.08-0.1g/L biotin, wherein the solvent is water;
adjusting the initial temperature to be 30 +/-0.5 ℃, the initial pressure to be 0.05-0.1Mpa, the initial stirring rotation speed to be 300-650rpm, the initial aeration ratio to be 1:0.5-0.8, and adjusting the pH value to be 7.0 +/-0.5 by using ammonia water;
the temperature is raised by 1 ℃ every 4h after the fermentation is started, the temperature reaches 33 +/-0.5 ℃ at 12h, and then the temperature is maintained at 33 +/-0.5 ℃;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting aeration ratio, stirring speed and pressure to maintain the dissolved oxygen in the fermentation liquor at 15-25% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, feeding glucose, ammonium sulfate and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after feeding, wherein the mass ratio of the fed-batch glucose to the small materials is 8-9: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-1.0 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and controlling the mass ratio of fed-batch glucose to small materials to be 10-12: 1; the small material comprises the following components: 0.6-0.7g/L of zinc sulfate and sulfurMagnesium 0.4-0.6g/L, potassium dihydrogen phosphate 0.4-0.6g/L, dipotassium hydrogen phosphate 0.4-0.6g/L and VB310-12mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the residual reducing sugar concentration, the ammonium nitrogen concentration and the small material addition amount in different fermentation stages are controlled, so that the growth, proliferation and metabolic acid production of the thalli are balanced in different growth stages, and other process steps are combined, so that the fermentation period is shortened, the yield and the conversion rate of L-isoleucine at the fermentation end point are improved, the fermentation period is shortened to be below 46h, the L-isoleucine content at the fermentation end point is above 40g/L, and the sugar-acid conversion rate is above 20%.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the embodiment of the invention, the preparation method of the adopted seed liquid comprises the following steps:
inoculating corynebacterium glutamicum (Shanghai Xuan Yam biotechnology limited company, trademark XY-WSW-1755) into a primary seed culture medium, culturing for 18h under the conditions that the temperature is 30 +/-0.5 ℃ and the rotating speed of a shaking table is 250rpm, and when the OD value of a thallus at 620nm is not less than 1.0, inoculating the strain into a secondary seed culture medium, wherein the inoculation amount is 10% of the volume of the secondary seed culture medium, the temperature is 30 +/-0.5 ℃, the pressure is 0.06MPa, the stirring rotating speed is 400rpm, the aeration ratio is 1:0.5, the pH is 7.2 +/-0.5 (controlled by ammonia water), and culturing for 12h under the conditions that the DO is not less than 30% to obtain a seed solution;
wherein the primary seed culture medium is an LB liquid culture medium;
the secondary seed culture medium comprises the following components: 40g/L glucose, 0.8g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate, 3g/L ammonium sulfate, 0.1g/L ferrous sulfate, 8mg/L manganese sulfate, 3g/L yeast extract powder, 0.6g/mL methionine, 10g/L glutamic acid, 0.75g/L lysine and 10g/L corn steep liquor, and the solvent is water.
In the examples of the present invention, the measurement method of each index is as follows:
OD value: measuring with a 7230G visible spectrophotometer at 620nm wavelength;
pH value: measured with a bench-top pH meter for METTER OLEDO;
content of residual reducing sugar: measuring with SBA-40C enzyme membrane instrument;
ammonium nitrogen content: using methyl red-methylene blue as an indicator, and titrating by using a hydrochloric acid standard solution;
content of L-isoleucine: the measurement is carried out by chromatography on paper.
The saccharic acid conversion rate is L-isoleucine yield/total sugar consumption x 100%.
Example 1
This example provides a method for fermentative production of L-isoleucine comprising the steps of:
inoculating the seed solution into a 50L fermentation medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 10% of the volume of the fermentation medium;
the fermentation medium comprises the following components: 60g/L glucose, 2g/L magnesium sulfate, 2g/L potassium dihydrogen phosphate, 5g/L ammonium sulfate, 10mg/L ferrous sulfate, 8mg/L manganese sulfate, 1.0g/L dipotassium hydrogen phosphate, 3g/L yeast extract powder, 0.3g/mL methionine, 10g/L glutamic acid, 1g/L lysine, 18g/L corn steep liquor and 0.1g/L biotin, wherein the solvent is water;
the initial temperature was adjusted to 30. + -. 0.5 ℃ and the initial pressure to 0.05MPa, the initial stirring speed was 300rpm, and the initial aeration ratio was 1: 0.5.
After the fermentation is started, raising the temperature by 1 ℃ every 4h, and keeping the temperature at 33 +/-0.5 ℃ after reaching 33 ℃;
in the fermentation process, ammonia water with the concentration of 25 wt% is fed-batch in the whole process, so that the pH value of the fermentation liquor is maintained to be 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen in the fermentation liquor to be 25% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, beginning to feed glucose solution with the concentration of 700g/L, ammonium sulfate solution with the concentration of 45 wt% and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.3 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after beginning to feed, and controlling the mass ratio of the glucose to the small materials to be 8: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-0.8 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials to be 10: 1; the small material comprises the following components: 0.6g/L zinc sulfate, 0.6g/L magnesium sulfate, 0.4g/L potassium dihydrogen phosphate, 0.6g/L dipotassium hydrogen phosphate and VB310mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
The fermentation period of the embodiment is 45.1h, the content of L-isoleucine in the fermentation liquor at the end of the fermentation is 41.095g/L, the yield of L-isoleucine is 1643.8g, the total sugar consumption is 7122g, and the sugar-acid conversion rate is 23.08%.
Example 2
This example provides a method for fermentative production of L-isoleucine comprising the steps of:
inoculating the seed solution to 500L of fermentation medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 10% of the volume of the fermentation medium;
the fermentation medium comprises the following components: 58g/L glucose, 1.8g/L magnesium sulfate, 2.2g/L potassium dihydrogen phosphate, 5.2g/L ammonium sulfate, 8mg/L ferrous sulfate, 6mg/L manganese sulfate, 1.2g/L dipotassium hydrogen phosphate, 3.5g/L yeast extract powder, 0.4g/mL methionine, 12g/L glutamic acid, 1.2g/L lysine, 15g/L corn steep liquor and 0.08g/L biotin, and the solvent is water;
the initial temperature was adjusted to 30. + -. 0.5 ℃ and the initial pressure to 0.1MPa, the initial stirring speed was 650rpm, and the initial aeration ratio was 1: 0.8.
After the fermentation is started, raising the temperature by 1 ℃ every 4h, and keeping the temperature at 33 +/-0.5 ℃ after the temperature reaches 33 ℃;
in the fermentation process, ammonia water with the concentration of 25 wt% is fed-batch in the whole process, so that the pH value of the fermentation liquor is maintained to be 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen in the fermentation liquor at 15% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, beginning to feed glucose solution with the concentration of 700g/L, ammonium sulfate solution with the concentration of 45 wt% and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1.2-1.4 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after beginning to feed, and controlling the mass ratio of the glucose to the small materials to be 9: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.7-1.0 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials to be 12: 1; the small material comprises the following components: 0.7g/L of zinc sulfate, 0.4g/L of magnesium sulfate, 0.6g/L of monopotassium phosphate, 0.4g/L of dipotassium phosphate and VB312mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
The fermentation period of the example is 45.8h, the content of L-isoleucine in the fermentation broth at the end of the fermentation is 40.59g/L, the yield of L-isoleucine is 15018.3g, the total sugar consumption is 65040.6g, and the sugar-acid conversion rate is 23.09%.
Example 3
This example provides a method for fermentative production of L-isoleucine comprising the steps of:
inoculating the seed solution into a 50L fermentation medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 8% of the volume of the fermentation medium;
the fermentation medium comprises the following components: 60g/L glucose, 2g/L magnesium sulfate, 2g/L potassium dihydrogen phosphate, 5g/L ammonium sulfate, 10mg/L ferrous sulfate, 8mg/L manganese sulfate, 1.0g/L dipotassium hydrogen phosphate, 3g/L yeast extract powder, 0.3g/mL methionine, 10g/L glutamic acid, 1g/L lysine, 18g/L corn steep liquor and 0.1g/L biotin, wherein the solvent is water;
the initial temperature was adjusted to 30. + -. 0.5 ℃ and the initial pressure to 0.08MPa, the initial stirring speed was 450rpm, and the initial aeration ratio was 1: 0.6.
After the fermentation is started, raising the temperature by 1 ℃ every 4h, and keeping the temperature at 33 +/-0.5 ℃ after the temperature reaches 33 ℃;
in the fermentation process, ammonia water with the concentration of 25 wt% is fed-batch in the whole process, so that the pH value of the fermentation liquor is maintained to be 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen in the fermentation liquor at 20% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, beginning to feed glucose solution with the concentration of 700g/L, ammonium sulfate solution with the concentration of 45 wt% and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.3 wt%, the content of ammonium nitrogen to be 0.15-0.2 wt% and the mass ratio of the glucose to the small materials to be 8.5:1 within 0-20 h; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.7-1 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials to be 11: 1; the small material comprises the following components: 0.6g/L zinc sulfate, 0.6g/L magnesium sulfate, 0.4g/L potassium dihydrogen phosphate, 0.6g/L dipotassium hydrogen phosphate and VB310mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
The fermentation period of the example is 46h, the content of L-isoleucine in the fermentation broth at the end of the fermentation is 40.02g/L, the yield of L-isoleucine is 1640.8g, the total sugar consumption is 7193.4g, and the sugar-acid conversion rate is 22.81%.
Example 4
This example provides a method for fermentative production of L-isoleucine comprising the steps of:
inoculating the seed solution into a 50L fermentation medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 10% of the volume of the fermentation medium;
the fermentation medium comprises the following components: 58g/L glucose, 1.8g/L magnesium sulfate, 2.2g/L potassium dihydrogen phosphate, 5.2g/L ammonium sulfate, 8mg/L ferrous sulfate, 6mg/L manganese sulfate, 1.2g/L dipotassium hydrogen phosphate, 3.5g/L yeast extract powder, 0.4g/mL methionine, 12g/L glutamic acid, 1.2g/L lysine, 15g/L corn steep liquor and 0.08g/L biotin, and the solvent is water;
the initial temperature was adjusted to 30. + -. 0.5 ℃ and the initial pressure to 0.05MPa, the initial stirring speed was 300rpm, and the initial aeration ratio was 1: 0.7.
After the fermentation is started, raising the temperature by 1 ℃ every 4h, and keeping the temperature at 33 +/-0.5 ℃ after the temperature reaches 33 ℃;
in the fermentation process, ammonia water with the concentration of 25 wt% is fed-batch in the whole process, so that the pH value of the fermentation liquor is maintained to be 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen in the fermentation liquor at 20% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, beginning to feed glucose solution with the concentration of 700g/L, ammonium sulfate solution with the concentration of 45 wt% and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1.2-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after beginning to feed, and controlling the mass ratio of the glucose to the small materials to be 8: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-0.8 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials to be 10: 1; the small material comprises the following components: 0.7g/L of zinc sulfate, 0.4g/L of magnesium sulfate, 0.6g/L of monopotassium phosphate, 0.4g/L of dipotassium phosphate and VB312mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
The fermentation period of the example is 44.9h, the content of L-isoleucine in the fermentation broth at the end of the fermentation is 40.99g/L, the yield of L-isoleucine is 1599.6g, the total sugar consumption is 6988.2g, and the sugar-acid conversion rate is 22.89%.
Example 5
This example provides a method for fermentative production of L-isoleucine comprising the steps of:
inoculating the seed solution into a 50L fermentation medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 12% of the volume of the fermentation medium;
the fermentation medium comprises the following components: 60g/L glucose, 2g/L magnesium sulfate, 2g/L potassium dihydrogen phosphate, 5g/L ammonium sulfate, 10mg/L ferrous sulfate, 8mg/L manganese sulfate, 1.0g/L dipotassium hydrogen phosphate, 3g/L yeast extract powder, 0.3g/mL methionine, 10g/L glutamic acid, 1g/L lysine, 18g/L corn steep liquor and 0.1g/L biotin, wherein the solvent is water;
adjusting the initial temperature to be 30 +/-0.5 ℃, the initial pressure to be 0.05Mpa, the initial stirring rotation speed to be 300rpm, and the initial aeration ratio to be 1: 0.5;
after the fermentation is started, raising the temperature by 1 ℃ every 4h, and keeping the temperature at 33 +/-0.5 ℃ after the temperature reaches 33 ℃;
in the fermentation process, ammonia water with the concentration of 25 wt% is fed-batch in the whole process, so that the pH value of the fermentation liquor is maintained to be 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio and the stirring speed to maintain the dissolved oxygen in the fermentation liquor at 20% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, beginning to feed glucose solution with the concentration of 700g/L, ammonium sulfate solution with the concentration of 45 wt% and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1.2-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after beginning to feed, and controlling the mass ratio of the glucose to the small materials to be 8: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.6-0.9 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and the mass ratio of glucose to small materials to be 10: 1; the small material comprises the following components: 0.6g/L zinc sulfate, 0.6g/L magnesium sulfate, 0.4g/L potassium dihydrogen phosphate, 0.6g/L dipotassium hydrogen phosphate and VB310mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
The fermentation period of the example is 45.5h, the content of L-isoleucine in the fermentation broth at the end of the fermentation is 42.09g/L, the yield of L-isoleucine is 1692.1g, the total sugar consumption is 8061.5g, and the sugar-acid conversion rate is 20.99%.
Example 6
This example provides a method for fermentative production of L-isoleucine, differing from example 1 only in that: in the fermentation process, the temperature is controlled to be 30 +/-0.5 ℃ in the whole process.
The fermentation period of the embodiment is 50.3h, the content of L-isoleucine in the fermentation liquor at the end of the fermentation is 38.88g/L, the yield of L-isoleucine is 1664.1g, the total sugar consumption is 8837.5g, and the sugar-acid conversion rate is 18.83%.
Example 7
This example provides a method for fermentative production of L-isoleucine, differing from example 1 only in that: in the fermentation process, the temperature is controlled to be 33 +/-0.5 ℃ in the whole process.
The fermentation period of the embodiment is 48.6h, the content of L-isoleucine in the fermentation broth at the end of the fermentation is 40g/L, the yield of L-isoleucine is 1520g, the total sugar consumption is 8000g, and the sugar-acid conversion rate is 19%.
Comparative example 1
A process for the fermentative preparation of L-isoleucine is provided which differs from example 1 only in that: after the glucose solution, the ammonium sulfate solution and the small materials are fed in, the content of residual reducing sugar in the fermentation liquor is controlled to be 1.0-1.5 wt% and the content of ammonium nitrogen is controlled to be 0.15-0.2 wt% in the whole process, and the mass ratio of the glucose to the small materials is 8: 1.
The fermentation period of the comparative example 1 was 52.2h, the L-isoleucine content in the fermentation broth at the end of the fermentation was 38.3g/L, the L-isoleucine yield was 1646.9g, the total sugar consumption was 9098.9g, and the sugar-acid conversion rate was 18.1%.
Comparative example 2
A process for the fermentative preparation of L-isoleucine is provided which differs from example 1 only in that: after the glucose solution, the ammonium sulfate solution and the small materials are fed in, the content of residual reducing sugar in the fermentation liquor is controlled to be 0.5-0.8 wt% and the content of ammonium nitrogen is controlled to be 0.1-0.15 wt% in the whole process, and the mass ratio of glucose to the small materials is 10: 1.
The fermentation period of the comparative example 2 was 51.8h, the L-isoleucine content in the fermentation broth at the end of the fermentation was 39.2g/L, the L-isoleucine yield was 1540.6g, the total sugar consumption was 8151.3g, and the sugar-acid conversion rate was 18.9%.
The embodiment shows that the residual reducing sugar concentration, the ammonium nitrogen concentration and the small material addition amount in different fermentation stages are controlled, so that the growth, proliferation and metabolic acid production of the thalli are balanced in different growth stages, the fermentation period is shortened, the yield and the conversion rate of L-isoleucine at the fermentation end point are improved, the fermentation period is less than 46h, the content of the L-isoleucine at the fermentation end point is more than 40g/L, and the sugar-acid conversion rate is more than 20%.
Compared with the example 1, the temperature is controlled to be about 30 ℃ in the whole process of the example 6, so that the thallus can proliferate too fast, the bacterial quantity is large, the time for L-isoleucine metabolism accumulation is prolonged, the final fermentation period is longer, and the yield and the conversion rate of L-isoleucine at the fermentation end point are reduced; in example 7, the total temperature was controlled to about 33 ℃ to decrease the amount of cell growth, and accumulation of L-isoleucine via metabolism was rapidly started, but the increase in the amount of acid gradually slowed down, the final fermentation period was prolonged, and the yield and conversion of L-isoleucine at the end of the fermentation were reduced.
Compared with the example 1, the comparative examples 1 and 2 do not adjust different reducing sugar concentrations, ammonium nitrogen concentrations and small material addition amounts aiming at different fermentation stages, so that thalli are affected in logarithmic proliferation stages and metabolic acid production stages, the growth, proliferation and metabolic acid production are unbalanced, the final fermentation period is longer, and the yield and the conversion rate of L-isoleucine at the fermentation end point are remarkably reduced.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can 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 the fermentative production of L-isoleucine, said method comprising the steps of:
inoculating the seed liquid of the L-isoleucine producing strain into a fermentation culture medium for fermentation to produce L-isoleucine;
in the fermentation process, when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, feeding glucose, ammonium sulfate and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after feeding, wherein the mass ratio of the fed glucose to the small materials is 8-9: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-1.0 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and controlling the mass ratio of fed-batch glucose to small materials to be 10-12: 1;
the small material comprises the following components: 0.6-0.7g/L of zinc sulfate, 0.4-0.6g/L of magnesium sulfate, 0.4-0.6g/L of monopotassium phosphate, 0.4-0.6g/L of dipotassium phosphate and VB310-12mg/L, and the solvent is water.
2. The method of claim 1, wherein the method of preparing the seed liquid comprises the steps of:
inoculating the L-isoleucine producing strain into a primary seed culture medium, culturing for 16-18h under the conditions that the culture temperature is 30 +/-0.5 ℃, the rotating speed of a shaking table is 250-.
3. The method of claim 2, wherein the primary seed medium is LB liquid medium;
preferably, the secondary seed culture medium comprises the following components: 38-40g/L of glucose, 0.8-0.9g/L of magnesium sulfate, 1.5-1.6g/L of monopotassium phosphate, 2.8-3.0g/L of ammonium sulfate, 0.1-0.12g/L of ferrous sulfate, 8-10mg/L of manganese sulfate, 2.8-3.0g/L of yeast extract powder, 0.6-0.7g/mL of methionine, 10-12g/L of glutamic acid, 0.75-0.8g/L of lysine and 10-12g/L of corn steep liquor, and the solvent is water.
4. The method according to any one of claims 1 to 3, wherein the L-isoleucine producing bacterium is Corynebacterium glutamicum;
preferably, the inoculation amount of the seed liquid is 8-12% of the volume of the fermentation medium;
and/or the fermentation medium comprises the following components: 58-60g/L glucose, 1.8-2.0g/L magnesium sulfate, 2.0-2.2g/L potassium dihydrogen phosphate, 5.0-5.2g/L ammonium sulfate, 8-10mg/L ferrous sulfate, 6.0-8.0mg/L manganese sulfate, 1.0-1.2g/L dipotassium hydrogen phosphate, 3.0-3.5g/L yeast extract powder, 0.3-0.4g/mL methionine, 10-12g/L glutamic acid, 1.0-1.2g/L lysine, 15-18g/L corn steep liquor and 0.08-0.1g/L biotin, wherein the solvent is water, and the pH value is 7.0 +/-0.5.
5. The method as claimed in any one of claims 1 to 4, wherein the initial pressure of the fermentation is 0.05 to 0.1MPa, the initial stirring rotation speed is 300 and 650rpm, and the initial aeration ratio is 1:0.5 to 0.8.
6. The method according to any one of claims 1 to 5, wherein the temperature is raised from 30 ± 0.5 ℃ to 33 ± 0.5 ℃ within 0 to 12 hours after the start of the fermentation, and the temperature is maintained at 33 ± 0.5 ℃ after 12 hours.
7. The method according to any one of claims 1 to 6, wherein the aeration ratio, the stirring speed and the pressure are adjusted so that the dissolved oxygen in the fermentation broth is maintained at 15 to 25% of the initial dissolved oxygen when the dissolved oxygen in the fermentation broth reaches 20% or less of the initial dissolved oxygen during the fermentation.
8. The method according to any one of claims 1 to 7, wherein ammonia is fed in during the fermentation process to maintain the pH of the fermentation broth at 7.0 ± 0.5.
9. The method according to any one of claims 1 to 8, wherein the feeding of ammonium sulfate and the small material is stopped when the production rate of L-isoleucine is slowed or no longer increased, the feeding of glucose is stopped after 0.5h, and the fermentation is terminated when the dissolved oxygen amount starts to rise.
10. A method according to any of claims 1-9, characterized in that the method comprises the steps of:
(1) inoculating corynebacterium glutamicum into a primary seed culture medium, culturing for 16-18h under the conditions that the culture temperature is 30 +/-0.5 ℃, the rotating speed of a shaking table is 250-300rpm, and inoculating the corynebacterium glutamicum into a secondary seed culture medium when the OD value of the strain at 620nm is more than or equal to 1.0, wherein the inoculation amount is 8-10% of the volume of the secondary seed culture medium, and culturing for 10-12h under the conditions that the temperature is 30 +/-0.5 ℃, the pressure is 0.05-0.08MPa, the stirring rotating speed is 300-500rpm, the aeration ratio is 1:0.5-0.8, the pH is 7.2 +/-0.5, and the DO is more than or equal to 20 wt% to obtain a seed solution;
wherein the primary seed culture medium is an LB liquid culture medium;
the secondary seed culture medium comprises the following components: 38-40g/L of glucose, 0.8-0.9g/L of magnesium sulfate, 1.5-1.6g/L of monopotassium phosphate, 2.8-3.0g/L of ammonium sulfate, 0.1-0.12g/L of ferrous sulfate, 8-10mg/L of manganese sulfate, 2.8-3.0g/L of yeast extract powder, 0.6-0.7g/mL of methionine, 10-12g/L of glutamic acid, 0.75-0.8g/L of lysine and 10-12g/L of corn steep liquor, and the solvent is water;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium for fermentation to produce L-isoleucine, wherein the inoculation amount is 8-12% of the volume of the fermentation culture medium;
the fermentation medium comprises the following components: 58-60g/L of glucose, 1.8-2.0g/L of magnesium sulfate, 2.0-2.2g/L of monopotassium phosphate, 5.0-5.2g/L of ammonium sulfate, 8-10mg/L of ferrous sulfate, 6.0-8.0mg/L of manganese sulfate, 1.0-1.2g/L of dipotassium phosphate, 3.0-3.5g/L of yeast extract powder, 0.3-0.4g/mL of methionine, 10-12g/L of glutamic acid, 1.0-1.2g/L of lysine, 15-18g/L of corn steep liquor and 0.08-0.1g/L of biotin, wherein the solvent is water;
adjusting the initial temperature to 30 +/-0.5 ℃, the initial pressure to 0.05-0.1Mpa, the initial stirring rotation speed to 300-;
the temperature is increased by 1 ℃ every 4h after the fermentation is started, the temperature reaches 33 +/-0.5 ℃ in 12h, and then the temperature is maintained at 33 +/-0.5 ℃;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0 +/-0.5;
when the dissolved oxygen in the fermentation liquor reaches below 20% of the initial dissolved oxygen, adjusting the aeration ratio, the stirring speed and the pressure to maintain the dissolved oxygen in the fermentation liquor at 15-25% of the initial dissolved oxygen;
when the content of residual reducing sugar in the fermentation liquor is lower than 1 wt%, feeding glucose, ammonium sulfate and small materials, controlling the content of the residual reducing sugar in the fermentation liquor to be 1-1.5 wt% and the content of ammonium nitrogen to be 0.15-0.2 wt% within 0-20h after feeding, wherein the mass ratio of the fed-batch glucose to the small materials is 8-9: 1; after 20 hours, controlling the content of residual reducing sugar in the fermentation liquor to be 0.5-1.0 wt%, the content of ammonium nitrogen to be 0.1-0.15 wt%, and controlling the mass ratio of fed-batch glucose to small materials to be 10-12: 1; the small material comprises the following components: 0.6-0.7g/L of zinc sulfate, 0.4-0.6g/L of magnesium sulfate, 0.4-0.6g/L of monopotassium phosphate, 0.4-0.6g/L of dipotassium phosphate and VB310-12mg/L, and the solvent is water;
when the production rate of L-isoleucine is slowed down or no longer increased, the feeding of ammonium sulfate and small materials is stopped, the feeding of glucose is stopped after 0.5h, and the fermentation is ended when the dissolved oxygen amount begins to rise.
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