CN109679979A - The production method of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid - Google Patents
The production method of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid Download PDFInfo
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- CN109679979A CN109679979A CN201910030458.4A CN201910030458A CN109679979A CN 109679979 A CN109679979 A CN 109679979A CN 201910030458 A CN201910030458 A CN 201910030458A CN 109679979 A CN109679979 A CN 109679979A
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- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 108010053835 Catalase Proteins 0.000 title claims abstract description 56
- 241000894006 Bacteria Species 0.000 title claims abstract description 46
- 102000016938 Catalase Human genes 0.000 title claims abstract description 38
- 230000014509 gene expression Effects 0.000 title claims abstract description 35
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 title claims abstract description 34
- 239000013598 vector Substances 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 230000001276 controlling effect Effects 0.000 claims abstract description 18
- 230000001105 regulatory effect Effects 0.000 claims abstract description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 36
- 239000002609 medium Substances 0.000 claims description 28
- 238000000855 fermentation Methods 0.000 claims description 26
- 230000004151 fermentation Effects 0.000 claims description 26
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 17
- 229910052760 oxygen Inorganic materials 0.000 claims description 17
- 239000001301 oxygen Substances 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 241001052560 Thallis Species 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 230000004069 differentiation Effects 0.000 claims description 9
- 239000000835 fiber Substances 0.000 claims description 9
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 239000006143 cell culture medium Substances 0.000 claims description 5
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 claims description 5
- 229960002413 ferric citrate Drugs 0.000 claims description 5
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 238000012549 training Methods 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 239000013589 supplement Substances 0.000 claims description 4
- 108090000854 Oxidoreductases Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 102000004316 Oxidoreductases Human genes 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 12
- 229910052708 sodium Inorganic materials 0.000 description 9
- 239000011734 sodium Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 7
- 235000013923 monosodium glutamate Nutrition 0.000 description 7
- 239000004223 monosodium glutamate Substances 0.000 description 6
- 229940049906 glutamate Drugs 0.000 description 5
- 229930195712 glutamate Natural products 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 3
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 3
- 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 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000006052 feed supplement Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 241000194103 Bacillus pumilus Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000936835 Streptomyces afghaniensis Species 0.000 description 1
- 241000521072 Streptomyces afghaniensis 772 Species 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000235015 Yarrowia lipolytica Species 0.000 description 1
- 241000222126 [Candida] glabrata Species 0.000 description 1
- -1 acyl group nitrile compound Chemical class 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 238000004176 ammonification Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- WDRWZVWLVBXVOI-QTNFYWBSSA-L dipotassium;(2s)-2-aminopentanedioate Chemical compound [K+].[K+].[O-]C(=O)[C@@H](N)CCC([O-])=O WDRWZVWLVBXVOI-QTNFYWBSSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 235000013919 monopotassium glutamate Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
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- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01006—Catalase (1.11.1.6)
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Abstract
The present invention provides the production methods of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid, belong to molecular biotechnology and enzyme engineering field.The recombinant vector of expression L-GLOD and catalase provided by the invention, by the regulating and controlling sequence SD for being inserted into the L-GLOD gene LGOX and catalase gene CAT expression in the carrier, it improves L-GLOD and catalase conversion of substrate generates the ability of α-ketoglutaric acid, there is preferable actual use value.
Description
Technical field
The present invention relates to molecular biotechnology and enzyme engineering field, in particular to expression L-GLOD and
The production method of the recombinant vector of catalase, engineering bacteria and application and α-ketoglutaric acid.
Background technique
α-ketoglutaric acid is the key node of organism tricarboxylic acid cycle and amino acid metabolism, can pass through glutamte dehydrogenase
Ammonification or transaminase generate glutamic acid, are widely used in the fields such as diet, medical treatment, cosmetics, fine chemistry industry or even sports
Field is also as the assisted class tonic for mitigating ammonia accumulation injury, and the market demand is greater than supply, and price is higher.Synthesis α-at present
There are mainly three types of methods for ketoglutaric acid: organic chemical synthesis, fermentation accumulation and enzymatic conversion method.
The traditional method of organic synthesis has acyl group nitrile compound Hydrolyze method, oxalic acid class ethyl ester Hydrolyze method, and several steps is needed to get to,
It is relatively complicated, it is seriously polluted；Fermentation accumulation method has been achieved with compared with quantum jump, and glucose culture torulopsis glabrata accumulates α -one penta
Diacid, 64h reach 43.7g/L；With glycerol culture Yarrowia lipolytica, 117h reaches 186g/L, although yield is very high,
Other heteroacid are more, it is big to isolate and purify difficulty, and fermentation period is too long, not yet realization industrialized production.
Summary of the invention
The embodiment of the present application provides a kind of recombinant vector, engineering bacteria for expressing L-GLOD and catalase
And the production method of application and α-ketoglutaric acid facilitates production by constructing the recombinant vector of high efficient expression；Pass through recombinant vector
Engineering bacteria is obtained, facilitates carry out whole-cell catalytic, the yield of α-ketoglutaric acid can be improved by the method for engineering bacteria catalyzed conversion.
The application's in a first aspect, providing the recombinant vector of expression L-GLOD and catalase, weight
Group carrier includes the L-GLOD gene LGOX for expressing L-GLOD, the hydrogen peroxide for expressing catalase
The enzyme gene CAT and regulating and controlling sequence SD of regulation L-GLOD gene LGOX and catalase gene CAT expression；L-
The base sequence of glucose oxidation enzyme gene LGOX is as shown in SEQ ID No.1, and the base sequence of catalase gene CAT is such as
Shown in SEQ ID No.2, the base sequence of regulating and controlling sequence SD is as shown in SEQ ID No.3.
The principle of α-ketoglutaric acid is produced by coexpression L-GLOD and hydrogen peroxide enzymatic conversion glutamate
It is as follows:
Converting glutamate by L-GLOD is α-ketoglutaric acid, while generating hydrogen peroxide, with reaction
The concentration of progress hydrogen peroxide will increase, the progress of reaction will be inhibited, so degrading by the effect of catalase
Hydrogen oxide simultaneously generates water and oxygen, guarantees that the continuous and effective of reaction carries out.
By regulating and controlling L-GLOD gene LGOX and catalase gene in expression vector in the present embodiment
The regulating and controlling sequence SD of CAT expression, regulating and controlling sequence SD have the site of ribosomes combination and regulate and control L-GLOD gene
LGOX and catalase gene CAT expression, reaches the complex equilibrium of the expression quantity of L-GLOD and catalase,
It realizes preferable reaction rate, improves the yield of α-ketoglutaric acid.
The second aspect of the application provides the recombinant vector of above-mentioned expression L-GLOD and catalase
Application in preparation α-ketoglutaric acid.
The third aspect of the application provides the engineering bacteria of expression L-GLOD and catalase, engineering bacteria
Including expressing the L-GLOD gene LGOX of L-GLOD, expressing the catalase gene of catalase
The CAT and regulating and controlling sequence SD of regulation L-GLOD gene LGOX and catalase gene CAT expression；Pidolidone
The base sequence of oxidase gene LGOX is as shown in SEQ ID No.1, the base sequence of catalase gene CAT such as SEQ ID
Shown in No.2, the base sequence of regulating and controlling sequence SD is as shown in SEQ ID No.3.
The fourth aspect of the application, the engineering bacteria for providing above-mentioned expression L-GLOD and catalase exist
Prepare the application in α-ketoglutaric acid.
The application the 5th aspect, provide a kind of production method of α-ketoglutaric acid, production method the following steps are included:
The recombinant vector of building expression L-GLOD and catalase is thin by recombinant vector transformed competence colibacillus
Born of the same parents obtain engineering bacteria；
Engineering bacteria obtains fermentation thalli by primary culture；
Fermentation thalli is inoculated in the reaction solution of bioreactor and is reacted, production obtains α-ketoglutaric acid.
In embodiment, the recombinant vector (weight of expression L-GLOD and catalase is co-expressed by building
The skeleton carrier of group carrier can be there are many selection, and the present invention is not specifically limited), and the operation carrier is transformed into competence
Cell (competent cell can there are many selections, are not specifically limited in the application), which obtains capable of co-expressing, expresses Pidolidone oxygen
Change the engineering bacteria of enzyme and catalase, then obtain thallus by expanding culture, thallus is used for whole-cell catalytic reaction, just
It can generate and obtain α-ketoglutaric acid.
In some embodiments of aforementioned 5th aspect, primary culture includes level-one culture and second level culture, level-one culture
Under the conditions of the temperature that condition is 37 DEG C, 220-250rpm cultivates 8-12h；Level-one culture is that 3.8-4.1 switching carries out two to OD600
Grade culture, under the conditions of the condition of second level culture is 37 DEG C of temperature, 200-235rpm cultivates 4-5h.
In the present embodiment, by expanding culture step by step, enough thallus can be quickly obtained and carry out catalysis reaction.
It further include supplement culture after second level culture, supplement culture is by second level in some embodiments of aforementioned 5th aspect
The bacterium solution of culture is forwarded to fermentation medium and ferments, and the pH and/or dissolved oxygen of fermentation medium rise, and adds feed-batch culture
Base starts to be cooled to 29-30 DEG C, the IPTG Fiber differentiation 11- of 0.1-0.2mM is added when culture reaches 22-28 to OD600
14h obtains fermentation thalli.
It, can be in bioreactor by the way that after second level culture, continuation Additional nutrient solution is simultaneously cultivated, and the thallus of high concentration is obtained
The middle concentration for keeping thallus certain, improves reaction efficiency；And by addition IPTG induction, the expression and production of a large amount of albumen are obtained
Raw, thallus, which can be accumulated, generates a large amount of L-GLOD and catalase, generates to obtain α -one by enzymic catalytic reaction
Glutaric acid.
In some embodiments of aforementioned 5th aspect, first cell culture medium is LB culture medium, and secondary medium is TB culture medium,
Fermentation medium contains yeast extract 28g/L, peptone 15g/L, glycerol 10g/L, dipotassium hydrogen phosphate trihydrate 16.4g/L, phosphoric acid
Potassium dihydrogen 2.3g/L, bitter salt 0.3g/L, ferric citrate 0.1g/L；Supplemented medium contains yeast extract 100g/L,
Peptone 30g/L, glycerol 400g/L.
In some embodiments of aforementioned 5th aspect, the solid-liquid ratio of fermentation thalli and reaction solution is 20-35g/L.
Contain a large amount of L-GLOD and catalase in the thallus that fermentation generates, by fermentation thalli and instead
Liquid is answered suitably to match, L-GLOD and hydrogen peroxide enzymatic reaction can improve the speed of reaction, avoid too low
Fermentation thalli causes to ferment for a long time, influences production efficiency.
In some embodiments of aforementioned 5th aspect, reaction solution includes glutamate, the reaction solution reaction of bioreactor
Temperature be 30 DEG C of temperature, 260-320rpm, and controlling pH is 6.5-7.0.
Glutamate (including sodium glutamate, potassium glutamate etc.) is added in fermentation liquid and is used as reaction substrate, can react generation
α-ketoglutaric acid.
Compared with prior art, the beneficial effect comprise that expression L-GLOD provided by the invention and
The recombinant vector of catalase, by being inserted into regulation L-GLOD gene LGOX and catalase base in the carrier
Because of the regulating and controlling sequence SD of CAT expression, improves L-GLOD and catalase conversion of substrate generates α-ketoglutaric acid
Ability has preferable actual use value.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described.It should be appreciated that the following drawings illustrates only certain embodiments of the present invention, therefore it is not construed as pair
The restriction of range.It for those of ordinary skill in the art, without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 provides 1 recombinant vector structure chart for experimental example of the present invention；
Fig. 2 is the SDS-PAGE egg of L-GLOD and catalase expression quantity that experimental example 1 of the present invention provides
White matter electrophoretic analysis detection figure.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of recombinant vectors and engineering bacteria for expressing L-GLOD and catalase, this is heavy
Group carrier includes the L-GLOD gene LGOX for expressing L-GLOD, the hydrogen peroxide for expressing catalase
The enzyme gene CAT and regulating and controlling sequence SD of regulation L-GLOD gene LGOX and catalase gene CAT expression；L-
The base sequence of glucose oxidation enzyme gene LGOX is as shown in SEQ ID No.1, and the base sequence of catalase gene CAT is such as
Shown in SEQ ID No.2, the base sequence of regulating and controlling sequence SD is as shown in SEQ ID No.3.
It expresses L-GLOD and the recombinant vector of catalase and the acquisition process of engineering bacteria is as follows:
1.1 according to the L-GLOD gene of Streptomyces afghaniensis 772 go full length sequence into
Row optimizes and removes the signal peptide of sequence, obtains L-GLOD gene, and designs amplimer amplification Pidolidone oxygen
Change enzyme gene, and L-GLOD gene is connected to carrier T, building obtains T-LGOX carrier；
1.2 optimize to obtain catalase gene CAT according to bacillus pumilus catalase gene, and design is drawn
Regulating and controlling sequence SD is simultaneously designed on primer by object sequence, by amplification, is obtained SD sequence-catalase gene CAT and is connected
To pUC57 carrier, pUC57-SD-CAT carrier is obtained；
1.3 select suitable enzymes double zyme cutting T-LGOX carrier, pUC57-SD-CAT carrier and pET32a carrier framework
System；Endonuclease bamhi is recycled, and the segment of the connection double digestion of digestion is obtained into expression L-GLOD with T4 ligase
With the pET32a-LGOX-SD-CAT recombinant vector of catalase, carrier structure figure is shown in Fig. 1；
1.4 convert obtained pET32a-LGOX-SD-CAT recombinant vector in DH5 α competent cell, extract plasmid simultaneously
It goes in e. coli bl21 (DE3), obtains the engineering bacteria pET32a- of expression L-GLOD and catalase
LGOX-SD-CAT/BL21(DE3)。
By the change of recombinant vector conversion into e. coli bl21 (DE3) competent cell, coated plate culture 15h chooses single bacterium
It falls in LB test tube and cultivates 9h, be transferred to Fiber differentiation 16h in TB culture medium, expression quantity is detected by SDS-PAGE, as a result such as
Shown in Fig. 2, the bacterium of the equal normal expression of double enzymes is selected, that is, successfully obtains engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3),
It is stand-by in ultra low temperature freezer to save several permanent bacterium glycerol tubes.
It therefore, can be by pET32a-LGOX-SD-CAT recombinant vector or engineering bacteria pET32a-LGOX-SD-CAT/BL21
(DE3) for co-expressing L-GLOD and catalase.
Embodiment 2
The present embodiment provides a kind of production method of α-ketoglutaric acid, the step of production method, is as follows:
The method building pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a- of 1.1 reference implementation examples 1
LGOX-SD-CAT/BL21(DE3)；
1.2 are inoculated into engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) in first cell culture medium LB, carry out primary
The level-one cultivation stage of culture, under the conditions of the condition of level-one culture is 37 DEG C of temperature, 220rpm cultivates 12h；It is to OD600
3.8；
1.3 are transferred to the bacterium solution of level-one culture the second level cultivation stage training for continuing primary culture in secondary medium TB
It supports, under the conditions of the temperature that the condition of second level culture is 37 DEG C, 200rpm cultivates 4h；
1.4 by the thallus of second level culture be inoculated into added with fermentation medium (fermentation medium contains yeast extract 28g/L,
Peptone 15g/L, glycerol 10g/L, dipotassium hydrogen phosphate trihydrate 16.4g/L, potassium dihydrogen phosphate 2.3g/L, bitter salt
0.3g/L, ferric citrate 0.1g/L) fermentor in 37 DEG C of culture 4h-5h；
1.5 when pH in fermentor and dissolved oxygen are begun to ramp up, and (supplemented medium contains yeast to addition supplemented medium
Cream 100g/L, peptone 30g/L, glycerol 400g/L), when cell concentration OD600 reaches 28, cooling processing, and use 0.1mM
IPTG Fiber differentiation 14h, collect thallus it is spare；
The thallus collected in step 1.5 is taken 150g thallus to be added in 7.5L reaction solution with the solid-liquid ratio of 20g/L by 1.6,
Monosodium glutamate (L-sodium) in reaction solution containing 1370.4g, in 30 DEG C of temperature, 260rpm is simultaneously passed through filtrated air conversion training
It supports；
1.7 with the sulfuric acid of 4mol/L control pH be 6.5-7.0, detects and is free of L-sodium in reaction solution, dissolved oxygen is greater than
50%, end of reaction.
Embodiment 3
The present embodiment provides a kind of production method of α-ketoglutaric acid, the step of production method, is as follows:
The method building pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a- of 1.1 reference implementation examples 1
LGOX-SD-CAT/BL21(DE3)；
1.2 are inoculated into engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) in first cell culture medium LB, carry out primary
The level-one cultivation stage of culture, under the conditions of the condition of level-one culture is 37 DEG C of temperature, 250rpm cultivates 8h；It is to OD600
4.1；
1.3 are transferred to the bacterium solution of level-one culture the second level cultivation stage training for continuing primary culture in secondary medium TB
It supports, under the conditions of the temperature that the condition of second level culture is 37 DEG C, 235rpm cultivates 5h；
1.4 by the thallus of second level culture be inoculated into added with fermentation medium (fermentation medium contains yeast extract 28g/L,
Peptone 15g/L, glycerol 10g/L, dipotassium hydrogen phosphate trihydrate 16.4g/L, potassium dihydrogen phosphate 2.3g/L, bitter salt
0.3g/L, ferric citrate 0.1g/L) fermentor in 37 DEG C of culture 4h-5h；
1.5 when pH in fermentor and dissolved oxygen are begun to ramp up, and (supplemented medium contains yeast to addition supplemented medium
Cream 100g/L, peptone 30g/L, glycerol 400g/L), when cell concentration OD600 reaches 22, cooling processing, and use 0.2mM
IPTG Fiber differentiation 11h, collect thallus it is spare；
The thallus collected in step 1.5 is taken 262.5g thallus to be added to 7.5L reaction solution with the solid-liquid ratio of 35g/L by 1.6
In, the monosodium glutamate (L-sodium) in reaction solution containing 1370.4g, in 30 DEG C of temperature, 320rpm is simultaneously passed through filtrated air turn
Change culture；
1.7 with the sulfuric acid of 4mol/L control pH be 6.5-7.0, detects and is free of L-sodium in reaction solution, dissolved oxygen is greater than
50%, end of reaction.
Embodiment 4
The present embodiment provides a kind of production method of α-ketoglutaric acid, the step of production method, is as follows:
The method building pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a- of 1.1 reference implementation examples 1
LGOX-SD-CAT/BL21(DE3)；
1.2 are inoculated into engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) in first cell culture medium LB, carry out primary
The level-one cultivation stage of culture, under the conditions of the condition of level-one culture is 37 DEG C of temperature, 240rpm cultivates 9h；It is 4 to OD600；
1.3 are transferred to the bacterium solution of level-one culture the second level cultivation stage training for continuing primary culture in secondary medium TB
It supports, under the conditions of the temperature that the condition of second level culture is 37 DEG C, 220rpm cultivates 5h；
1.4 by the thallus of second level culture be inoculated into added with fermentation medium (fermentation medium contains yeast extract 28g/L,
Peptone 15g/L, glycerol 10g/L, dipotassium hydrogen phosphate trihydrate 16.4g/L, potassium dihydrogen phosphate 2.3g/L, bitter salt
0.3g/L, ferric citrate 0.1g/L) fermentor in 37 DEG C of culture 4h-5h；
1.5 when pH in fermentor and dissolved oxygen are begun to ramp up, and (supplemented medium contains yeast to addition supplemented medium
Cream 100g/L, peptone 30g/L, glycerol 400g/L), when cell concentration OD600 reaches 22, cooling processing, and use 0.2mM
IPTG Fiber differentiation 11h, collect thallus it is spare；
The thallus collected in step 1.5 is taken 200g thallus to be added to 7.5L reaction solution with the solid-liquid ratio of 26.67g/L by 1.6
In, the monosodium glutamate (L-sodium) in reaction solution containing 1370.4g, in 30 DEG C of temperature, 300rpm is simultaneously passed through filtrated air turn
Change culture；
1.7 with the sulfuric acid of 4mol/L control pH be 6.5-7.0, detects and is free of L-sodium in reaction solution, dissolved oxygen is greater than
50%, end of reaction.
Experimental example 1
The method building pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a-LGOX- of reference implementation example 1
SD-CAT/BL21(DE3)。
Recombinant: culture transferring engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) is trained in level-one in an aseptic environment
It supports in base, cultivates 9h under the conditions of 37 DEG C of temperature, revolving speed 240rpm, OD600 is inoculated into secondary medium after reaching 3~4 and (connects
10%) kind amount, cultivates 4~5h at 37 DEG C of temperature, revolving speed 220rpm；Inoculate 37 DEG C of culture (inoculum concentrations in fermentor
10%) feed supplement is opened when, pH and dissolved oxygen rise, when bacteria concentration OD600 reaches 22~28, starts to be cooled to 30 DEG C, 0.1mM is added
Inducer IPTG Fiber differentiation put within 12 hours tank, it is stand-by to collect thallus.
Transformation experiment: it weighs 200g wet thallus and is suspended in 7.5L water, pour into 10L bioreactor, separately weigh 1370.4g
Monosodium glutamate is gradually put into, and 30 DEG C, 300rpm, logical filtrated air conversion control pH6.5~7.0 with 4mol/L sulfuric acid.Conversion 24~
28h, TLC put tank after detecting no L-sodium, and dissolved oxygen is greater than 50%, and conversion finishes.It is detected through HPLC, α-ketoglutaric acid contains
Amount is 123.37g/L, conversion ratio 93.25%.
Experimental example 2
Recombinant: culture transferring engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) is trained in level-one in an aseptic environment
It supports in base, cultivates 9h under the conditions of 37 DEG C of temperature, revolving speed 240rpm, OD600 is inoculated into secondary medium after reaching 3~4 and (connects
10%) kind amount, cultivates 4~5h at 37 DEG C of temperature, revolving speed 220rpm；Inoculate 37 DEG C of culture (inoculum concentrations in fermentor
10%) feed supplement is opened when, pH and dissolved oxygen rise, when bacteria concentration OD600 reaches 22~28, starts to be cooled to 30 DEG C, be added
The inducer IPTG Fiber differentiation of 0.15mM puts tank for 12 hours, and it is stand-by to collect thallus.
Transformation experiment: measuring 1.5L zymocyte liquid, and deionized water constant volume 7.5L is added, pours into 10L bioreactor, another to claim
1370.4g monosodium glutamate is taken gradually to put into, 30 DEG C, 300rpm, logical filtrated air conversion control pH6.5~7.0 with 4mol/L sulfuric acid.
24~28h is converted, TLC puts tank after detecting no L-sodium, and dissolved oxygen is greater than 50%, and conversion finishes.It is detected through HPLC, α -one
Glutaric acid content is 120.4g/L, conversion ratio 91.03%.
Experimental example 3
Recombinant: culture transferring engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE3) is trained in level-one in an aseptic environment
It supports in base, cultivates 9h under the conditions of 37 DEG C of temperature, revolving speed 240rpm, OD600 is inoculated into secondary medium after reaching 3~4 and (connects
10%) kind amount, cultivates 4~5h at 37 DEG C of temperature, revolving speed 220rpm；Inoculate 37 DEG C of culture (inoculum concentrations in fermentor
10%) feed supplement is opened when, pH and dissolved oxygen rise, when bacteria concentration OD600 reaches 22~28, starts to be cooled to 30 DEG C, be added
The inducer IPTG Fiber differentiation of 0.20mM puts tank for 12 hours, and it is stand-by to collect thallus.
Transformation experiment: measuring 1.7L zymocyte liquid, and deionized water constant volume 7.5L is added, pours into 10L bioreactor, another to claim
1370.4g monosodium glutamate is taken gradually to put into, 30 DEG C, 300rpm, logical filtrated air conversion control pH6.5~7.0 with 4mol/L sulfuric acid.
24~28h is converted, TLC puts tank after detecting no L-sodium, and dissolved oxygen is greater than 50%, and conversion finishes.It is detected through HPLC, α -one
Glutaric acid content 126.3g/L, conversion ratio 95.46%.
Post-treated to obtain 98.5% α-ketoglutaric acid crystal, total recovery is 80~85%.
In conclusion the recombinant vector of expression L-GLOD provided in an embodiment of the present invention and catalase,
The production method of engineering bacteria and application and α-ketoglutaric acid obtains the recombinant vector of the efficient table of energy, by transformed cells, obtains high
Expression system is imitated, and links up everfermentation energy Efficient Conversion glutamate and is converted to α-ketoglutaric acid, and improve substrate glutamic acid
Salt is converted to the efficiency of α-ketoglutaric acid, improves the yield and the rate of recovery of product.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110>Sichuan Ji Sheng biological medicine Co., Ltd
<120>recombinant vector, engineering bacteria and the application and α -one penta of L-GLOD and catalase are expressed
The production method of diacid
<170> PatentIn version 3.5
<210> 1
<211> 1863
<212> DNA
<213> Streptomyces afghaniensis
<400> 1
atgccgtctg ctgaccgtgg tgctgacttc gaccgttgcc tggctgttgc tcgtgctctg 60
ctggttctgg acaccgacaa ccgtccgctg gttccgcgtt accagcgtgt tctggaaaaa 120
ggtctgccgg ctcagcgtcg tacccgtccg aaaaacgttc tggttatcgg tgctggtccg 180
gctggtctgg ttaccgcttg gctgctgaaa aaagctggtc accgtgttac cgttctggaa 240
gctaacggta accgtgctgg tggtcgtatc aaaaccttcc gtaacggtgg tcacgaacac 300
gctgaacagc cgttcgctga cccgcgtcag tacgctgaag ctggtgctat gcgtatcccg 360
ggttctcacc cgctggttat ggaactgatc gaccagttcg gtctgaaaaa acgtcgtttc 420
cactacgttg acgttgacaa cgaaggtcgt ccggctggtc gtacctggat ccacgttaac 480
ggtatccgta tgcgtcgtgc tgactacgct cgtgctccgc gtcgtatcaa ccgttctttc 540
ggtgttccgc gtgctcgttg ggacaccccg gctgctgcta tcctgcgttc tgttctggac 600
ccggttcgtg acgaattctc ttacgttaac ggtgacggta aacgtgttga caaaccgctg 660
ccggaacgtc tgcgtggttg ggctcgtgtt gttcagcgtt tcggtgactg gtctatgttc 720
cgtttcctga ccgaacacgc tggtctggac gaacgtacca tcgacctgat cggtaccctg 780
gaaaacctga cctctcgtct gccgctgtct ttcatccact ctttcaccgg ttcttctctg 840
atctctccgg acaccccgtt ctacgaactg gaaggtggta ccgctgttct gccggacgct 900
ctgctggaac gtgttcgtgg tgacgttcgt ttcgaccgtc gtgttacccg tatcgaatac 960
caccacccgg accgtccgtc tccggacacc gaacacgttc gtggtaaagg tccgcacgtt 1020
tgggttgaca ccgtttctga aggtcgtgac ggtccggttg ttcgtgaaca gttcaccgct 1080
gacgttgctg ttgttaccgt tccgttctct ggtctgcgtc acgttcagat cgctccgccg 1140
atgtcttacg gtaaacgtcg tgctgtttgc gaactgcact acgactctgc taccaaagtt 1200
ctgctggaat tctctcgtcg ttggtgggaa ttcgacgaag ctgactggaa acgtgaactg 1260
caggctatcg ctccgggtct gtacgacgct taccgtaccg gtcgtgctgc tggtgacggt 1320
tctctgctgg gtgctcaccc gtctgttccg ggtggtcaca tcaccgctgg tcagcgtacc 1380
cactacgctg ctaaccgtgc tatcgctcgt gaccagccgg aagctgttga cgttgttggt 1440
ggtggttctg tttctgacaa cgctaaccgt ttcatgttcc acccgtctca cccggttccg 1500
ggttctgctg gtggtgttgt tctggcttct tactcttggg ctgacgacgc tctgcgttgg 1560
gactctctgg acgacgaagc tcgttacccg cacgctctgt gcggtctgca gcaggtttac 1620
ggtcagcgta tcgaagtttt ctactctggt gctggtcgta cccagtcttg gctgcgtgac 1680
ccgtacgctt acggtgaagc ttctgttctg ctgccgggtc agcacaccga actgctgccg 1740
gctatcccgg ttcgtgaagg tccgctgcac ttcgctggtg accacacctc tgttaaaccg 1800
gcttggatcg aaggtgctgt tgaatctgct gttcgtgctg ctctggaaat ccacaccgct 1860
taa 1863
<210> 2
<211> 1476
<212> DNA
<213> Bacillus pumilus
<400> 2
atgaccaact ctaaccacaa aaacctgacc accaaccagg gtgttccggt tggtgacaac 60
cagaactctc gtaccgctgg tcaccgtggt ccgaccttcc tggacgacta ccacctgatc 120
gaaaaactgg ctcacttcga ccgtgaacgt atcccggaac gtgttgttca cgctcgtggt 180
gctggtgctt acggtgtttt cgaagttgaa aactctatgg aaaaacacac caaagctgct 240
ttcctgtctg aagaaggtaa acagaccgac gttttcgttc gtttctctac cgttatccac 300
ccgaaaggtt ctccggaaac cctgcgtgac ccgcgtggtt tcgctgttaa attctacacc 360
gaagaaggta actacgacct ggttggtaac aacctgccga tcttcttcat ccgtgacgct 420
ctgaaattcc cggacatggt tcactctctg aaaccggacc cggttaccaa catccaggac 480
ccggaccgtt actgggactt catgaccctg accccggaat ctacccacat gctgacctgg 540
ctgttctctg acgaaggtat cccggctaac tacgctgaaa tgcgtggttc tggtgttcac 600
accttccgtt gggttaacaa atacggtgaa accaaatacg ttaaatacca ctggcgtccg 660
tctgaaggta tccgtaacct gtctatggaa gaagctgctg aaatccaggc taacgacttc 720
cagcacgcta cccgtgacct gtacgaccgt atcgaaaacg gtaactaccc ggcttgggac 780
ctgtacgttc agctgatgcc gctgtctgac tacgacgacc tggactacga cccgtgcgac 840
ccgaccaaaa cctggtctga agaagactac ccgctgcaga aagttggtcg tatgaccctg 900
aaccgtaacc cggaaaactt cttcgctgaa accgaacagt ctgctttcac cccgtctgct 960
ctggttccgg gtatcgaagc ttctgaagac aaactgctgc agggtcgtct gttctcttac 1020
ccggacaccc agcgtcaccg tctgggtgct aactacatgc gtatcccggt taactgcccg 1080
tacgctccgg ttcacaacaa ccagcaggac ggtttcatga ccaccacccg tccgtctggt 1140
cacatcaact acgaaccgaa ccgttacgac gaccagccga aagaaaaccc gcactacaaa 1200
gaatctgaac aggttctgca cggtgaccgt atggttcgtc agaaaatcga aaaaccgaac 1260
gacttcaaac aggctggtga aaaataccgt tcttactctg aagaagaaaa acaggctctg 1320
atcaaaaacc tgaccgctga cctgaaagac gttaacgaca aaaccaaact gctggctatc 1380
tgcaacttct accgtgctga cgaagactac ggtcagcgtc tggctgactc tctgggtgtt 1440
gacatccgtt cttacctgca gggttctatg aaataa 1476
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized
<400> 3
agatctggaa ggaatacaaa 20
Claims (10)
1. expressing the recombinant vector of L-GLOD and catalase, which is characterized in that the recombinant vector includes table
Up to L-GLOD L-GLOD gene LGOX, express catalase catalase gene CAT with
And regulate and control the regulating and controlling sequence SD of the L-GLOD gene LGOX and catalase gene CAT expression；It is described
The base sequence of L-GLOD gene LGOX is as shown in SEQ ID No.1, the base of the catalase gene CAT
Sequence is as shown in SEQ ID No.2, and the base sequence of the regulating and controlling sequence SD is as shown in SEQ ID No.3.
2. the recombinant vector of expression L-GLOD and catalase is in preparation α -one penta 2 as described in claim 1
Application in acid.
3. expressing the engineering bacteria of L-GLOD and catalase, which is characterized in that the engineering bacteria includes expression L-
The L-GLOD gene LGOX of dglutamic oxidase, the catalase gene CAT and tune for expressing catalase
Control the regulating and controlling sequence SD of the L-GLOD gene LGOX and catalase gene CAT expression；The L- paddy
The base sequence of amino acid oxidase gene LGOX is as shown in SEQ ID No.1, the base sequence of the catalase gene CAT
As shown in SEQ ID No.2, the base sequence of the regulating and controlling sequence SD is as shown in SEQ ID No.3.
4. the engineering bacteria of expression L-GLOD and catalase is in preparation α-ketoglutaric acid as claimed in claim 3
In application.
5. a kind of production method of α-ketoglutaric acid, which is characterized in that the production method the following steps are included:
The recombinant vector of building expression L-GLOD and catalase is thin by the recombinant vector transformed competence colibacillus
Born of the same parents obtain engineering bacteria；
The engineering bacteria obtains fermentation thalli by primary culture；
The fermentation thalli is inoculated in the reaction solution of bioreactor and is reacted, production obtains α-ketoglutaric acid.
6. the production method of α-ketoglutaric acid according to claim 5, which is characterized in that the primary culture includes level-one
Culture and second level culture, under the conditions of the condition of the level-one culture is 37 DEG C of temperature, 220-250rpm cultivates 8-12h；It is described
Level-one culture is that 3.8-4.1 switching carries out the second level culture, the temperature strip that the condition of the second level culture is 37 DEG C to OD600
Under part, 200-235rpm cultivates 4-5h.
7. the production method of α-ketoglutaric acid according to claim 6, which is characterized in that further include supplement after second level culture
Culture, the supplement culture is that the bacterium solution of the second level culture is forwarded to fermentation medium to ferment, the fermented and cultured
The pH and/or dissolved oxygen of base rise, and add supplemented medium and start to be cooled to 29-30 DEG C when culture reaches 22-28 to OD600,
The IPTG Fiber differentiation 11-14h of 0.1~0.2mM is added, obtains fermentation thalli.
8. the production method of α-ketoglutaric acid according to claim 7, which is characterized in that the first cell culture medium is LB training
Base is supported, the secondary medium is TB culture medium, and the fermentation medium contains yeast extract 28g/L, peptone 15g/L, glycerol
10g/L, dipotassium hydrogen phosphate trihydrate 16.4g/L, potassium dihydrogen phosphate 2.3g/L, bitter salt 0.3g/L, ferric citrate
0.1g/L；The supplemented medium contains yeast extract 100g/L, peptone 30g/L, glycerol 400g/L.
9. the production method of α-ketoglutaric acid according to claim 5, which is characterized in that the fermentation thalli and described anti-
The solid-liquid ratio for answering liquid is 20-35g/L.
10. the production method of α-ketoglutaric acid according to claim 9, which is characterized in that the reaction solution includes paddy ammonia
Hydrochlorate, the temperature that the temperature of the reaction solution of the bioreactor is 30 DEG C, 260-320rpm, and controlling pH is 6.5-
7.0。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305927A (en) * | 2019-08-07 | 2019-10-08 | 郑州伊美诺生物技术有限公司 | Utilize the method for recombination bacillus coli fermentation preparation coating TP antigen |
| CN111676182A (en) * | 2020-07-02 | 2020-09-18 | 江南大学 | Method for producing refined ketone mixture by utilizing recombinant corynebacterium crenatum through fermentation |
| CN112625993A (en) * | 2021-01-05 | 2021-04-09 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821066A (en) * | 2016-05-26 | 2016-08-03 | 江南大学 | Dual-enzyme co-expression strain for producing alpha-oxoglutarate |
| CN106047913A (en) * | 2016-05-26 | 2016-10-26 | 江南大学 | Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate |
| CN106868030A (en) * | 2015-12-14 | 2017-06-20 | 中国科学院微生物研究所 | Recombinant vector, the engineering bacteria containing it and the application in product KG |
| CN107686850A (en) * | 2016-08-04 | 2018-02-13 | 中国科学院天津工业生物技术研究所 | It is a kind of to utilize the method for co-expressing recombinant bacterial strain conversion production alpha Ketoglutarate |
| CN108841878A (en) * | 2018-06-19 | 2018-11-20 | 四川同晟生物医药有限公司 | A method of coexpression Pfansteihl oxidizing ferment and catalase coupling production Sodium Pyruvate |
-
2019
- 2019-01-11 CN CN201910030458.4A patent/CN109679979B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868030A (en) * | 2015-12-14 | 2017-06-20 | 中国科学院微生物研究所 | Recombinant vector, the engineering bacteria containing it and the application in product KG |
| CN105821066A (en) * | 2016-05-26 | 2016-08-03 | 江南大学 | Dual-enzyme co-expression strain for producing alpha-oxoglutarate |
| CN106047913A (en) * | 2016-05-26 | 2016-10-26 | 江南大学 | Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate |
| CN107686850A (en) * | 2016-08-04 | 2018-02-13 | 中国科学院天津工业生物技术研究所 | It is a kind of to utilize the method for co-expressing recombinant bacterial strain conversion production alpha Ketoglutarate |
| CN108841878A (en) * | 2018-06-19 | 2018-11-20 | 四川同晟生物医药有限公司 | A method of coexpression Pfansteihl oxidizing ferment and catalase coupling production Sodium Pyruvate |
Non-Patent Citations (1)
| Title |
|---|
| GRUENING,B.A等: "GenBank: EPJ38564.1", 《GENBANK》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305927A (en) * | 2019-08-07 | 2019-10-08 | 郑州伊美诺生物技术有限公司 | Utilize the method for recombination bacillus coli fermentation preparation coating TP antigen |
| CN111676182A (en) * | 2020-07-02 | 2020-09-18 | 江南大学 | Method for producing refined ketone mixture by utilizing recombinant corynebacterium crenatum through fermentation |
| CN112625993A (en) * | 2021-01-05 | 2021-04-09 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
| CN112625993B (en) * | 2021-01-05 | 2023-01-20 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
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Denomination of invention: Recombinant vectors, engineering bacteria, and applications for expressing L-glutamic acid oxidase and catalase a- The production method of ketoglutaric acid Effective date of registration: 20231204 Granted publication date: 20230317 Pledgee: China Construction Bank Corporation Leshan branch Pledgor: SICHUAN JISHENG BIOPHARMACEUTICAL Co.,Ltd. Registration number: Y2023980068875 |
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