CN113355371A - Method for preparing 4-hydroxyisoleucine through whole-cell catalysis - Google Patents
Method for preparing 4-hydroxyisoleucine through whole-cell catalysis Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 14
- OSCCDBFHNMXNME-WDCZJNDASA-N (2s,3s,4r)-2-amino-4-hydroxy-3-methylpentanoic acid Chemical compound C[C@@H](O)[C@@H](C)[C@H](N)C(O)=O OSCCDBFHNMXNME-WDCZJNDASA-N 0.000 title abstract description 5
- OSCCDBFHNMXNME-UHFFFAOYSA-N gamma-hydroxyisoleucine Natural products CC(O)C(C)C(N)C(O)=O OSCCDBFHNMXNME-UHFFFAOYSA-N 0.000 title abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 229960000310 isoleucine Drugs 0.000 claims abstract description 25
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 23
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 19
- 238000005342 ion exchange Methods 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 102000016680 Dioxygenases Human genes 0.000 claims abstract description 6
- 108010028143 Dioxygenases Proteins 0.000 claims abstract description 6
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 22
- 239000003480 eluent Substances 0.000 claims description 21
- 239000008399 tap water Substances 0.000 claims description 21
- 235000020679 tap water Nutrition 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 13
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 12
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims description 11
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 238000004090 dissolution Methods 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 7
- 238000005273 aeration Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 239000003729 cation exchange resin Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 210000001082 somatic cell Anatomy 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 238000004042 decolorization Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 238000000967 suction filtration Methods 0.000 claims 1
- 239000000047 product Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 229930182844 L-isoleucine Natural products 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 2
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241001312519 Trigonella Species 0.000 description 2
- 244000250129 Trigonella foenum graecum Species 0.000 description 2
- 235000001484 Trigonella foenum graecum Nutrition 0.000 description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 235000001019 trigonella foenum-graecum Nutrition 0.000 description 2
- OSCCDBFHNMXNME-DSDZBIDZSA-N 4-Hydroxy-L-isoleucine Chemical compound CC(O)[C@H](C)[C@H](N)C(O)=O OSCCDBFHNMXNME-DSDZBIDZSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000005171 Dysmenorrhea Diseases 0.000 description 1
- 206010013935 Dysmenorrhoea Diseases 0.000 description 1
- 201000005569 Gout Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007443 Neurasthenia Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 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 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 229930182852 proteinogenic amino acid Natural products 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
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- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for preparing 4-hydroxyisoleucine through whole-cell catalysis, and belongs to the technical field of biology. The invention utilizes a whole cell catalysis method to prepare 4-HIL, wherein the whole cell takes escherichia coli BL21(DE3) as a host, pET28a as a carrier to express isoleucine dioxygenase, isoleucine as a substrate to construct a whole cell catalysis system for conversion reaction, and a 4-HIL product is obtained by carrying out ultrafiltration and ion exchange on a conversion solution, decoloring, concentrating and drying. The conversion rate of the substrate can reach 98%, and the purity of the 4-HIL in the product can reach 98% by combining ultrafiltration and ion exchange means.
Description
Technical Field
The invention relates to a method for preparing 4-hydroxyisoleucine through whole-cell catalysis, and belongs to the technical field of biology.
Background
4-hydroxyisoleucine ((2S,3R,4S) -4-hydroxyisoeucine, 4-HIL) is a novel insulin secretion promoter, and can be used for treating type II diabetes. L-4-hydroxyisoleucine is a non-proteinogenic amino acid present in Trigonella species, and is predominantly present in the seed of Trigonella, accounting for about 80% of the total free amino acid content of the seed. The traditional Chinese medicine fenugreek is applied to treating late diabetes, dyspepsia, gastric ulcer, digestive disorder, tumor, dysmenorrhea, weakness, allergy, neurasthenia, gout, arthritis and the like.
Besides extraction and separation from fenugreek seeds, the synthesis methods of 4-HIL mainly include a chemical-enzymatic method and an enzymatic method. L-isoleucine hydroxylase (IDO) can specifically catalyze isoleucine to generate (2S,3R,4S) -4-HIL. The chemical-enzymatic synthesis of 4-HIL has complex steps, many intermediate byproducts and low conversion efficiency. The gene engineering bacteria expressing L-isoleucine hydroxylase are used for catalyzing isoleucine to synthesize 4-HIL, the conversion rate is low, and the subsequent separation and purification efficiency is low.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problems of complex steps and low conversion efficiency in the existing chemical-enzymatic synthesis of 4-HIL.
[ solution ]
The invention provides a method for preparing 4-HIL by using a whole cell catalysis method, wherein the whole cell takes escherichia coli BL21(DE3) as a host, pET28a as a vector to express isoleucine dioxygenase, isoleucine as a substrate to construct a whole cell catalysis system for conversion reaction, and a 4-HIL product is obtained by performing ultrafiltration and ion exchange on a conversion solution, decoloring, concentrating and drying.
The isoleucine dioxygenase may be selected from isoleucine dioxygenase derived from Bacillus subtilis.
The method comprises the following steps:
(1) carrying out fermentation culture on the recombinant escherichia coli, and collecting thalli;
(2) mixing the bacterial cells, a substrate and a buffer solution, and converting for 20-24h at the temperature of 28-32 ℃ to obtain a conversion solution; in the transformation system, the concentration of the somatic cells is 120-180g/L, the concentration of isoleucine is 80-100g/L, the concentration of alpha-ketoglutaric acid is 80-120g/L, Vc is 8-12g/L, and the concentration of ferrous sulfate heptahydrate is 0.3-1.0 g/L; vc is used as a reducing agent, and ferrous sulfate heptahydrate is used as a cofactor;
(3) carrying out ultrafiltration treatment on the conversion solution by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear solution;
(4) performing ion exchange treatment on the clear liquid, wherein the adopted ion exchange column is 732 cation exchange resin and is eluted by 1-2% sodium hydroxide solution; collecting the eluent when the refraction of the eluent is more than or equal to 0.1, and collecting the eluent with the conductivity of less than or equal to 1000 us/cm;
(6) adding 12-15% active carbon into the eluate, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, and drying to obtain 4-HIL product.
In one embodiment of the invention, in the step (1), isoleucine 90g, alpha-ketoglutaric acid 100.5g, Vc10.6g, and ferrous sulfate heptahydrate 0.45g are weighed, dissolved by adding tap water, poured into a 5L fermentation tank, the pH of the feed liquid is adjusted to 7.0, the centrifuged thallus dosage is 150g, the tap water is supplemented to 3L, 0.5mL of natural enemy is added, and the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 24 h.
In one embodiment of the invention, in the step (1), 85g of isoleucine, 100g of alpha-ketoglutaric acid, 9g of Vc, and 0.4g of ferrous sulfate heptahydrate are weighed, tap water is added for dissolution, the mixture is poured into a 5L fermentation tank, the pH of the feed liquid is adjusted to 7.0, the centrifuged bacterial cell amount is added to 130g, the tap water is supplemented to 3L, 0.5ml of natural enemy is added, and the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 20 h.
In one embodiment of the invention, in the step (1), isoleucine 100g, alpha-ketoglutaric acid 110g, Vc12g, and ferrous sulfate heptahydrate 0.8g are weighed, tap water is added for dissolution, the mixture is poured into a 5L fermentation tank, the pH of the feed liquid is adjusted to 7.0, the centrifuged thallus dosage is 180g, the tap water is supplemented to 3L, and the natural plant is added with the natural plant, wherein the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 24 h.
In one embodiment of the invention, in the step (4), 15% of activated carbon is added into the eluent, decolorized for 30min at 60 ℃, filtered, concentrated and dried to obtain the product.
[ advantageous effects ]
The invention takes the whole cells as the catalyst and the isoleucine as the substrate, the substrate conversion rate can reach 98 percent, and the purity of the 4-HIL in the product can reach 98 percent by combining ultrafiltration and ion exchange means.
The invention uses the conductivity to accurately judge the collection end point of the eluent.
When the target substance is eluted from the ion exchange column, the sodium hydroxide solution is used as the eluent, and no waste water containing ammonia nitrogen is generated.
Detailed Description
The present invention will be further illustrated below with reference to specific examples and comparative examples.
4-detection method of HIL: HPLC; column: c18, wavelength 254nm, flow rate 0.8ml/min, mobile phase methanol: water: phosphoric acid (0.55: 0.45: 0.001) was added in an amount of 10. mu.l.
EXAMPLE 1 cultivation of recombinant E.coli cells
Test tube seed culture medium: 10g/L peptone (Oxoid), 5g/L yeast extract (Oxoid), 10g/L sodium chloride, dissolving with tap water, packaging into test tubes (4 mL/tube) after constant volume, and sterilizing at 121 deg.C for 20 min. Kanamycin was added to a final concentration of 50. mu.g/mL prior to use.
Shake flask seed culture medium: 10g/L peptone (Oxoid), 10g/L, NaCl 10g/L yeast extract (Oxoid), dissolved in tap water, and pH adjusted to 7.0 with NaOH. Subpackaging with 500mL shake flask, 100mL each, sterilizing at 121 deg.C for 20 min. Kanamycin (50. mu.g/mL) was added before use.
Initial culture medium of fermentation tank: 5g/L of glycerol, 5g/L of peptone (Oxoid), and 5g/L, Na of yeast extract (Oxoid)2HPO4·12 H2O 5g/L、Na2SO4 0.7g/L、KH2PO4 3.4g/L、MgSO4 0.25g/L、NH4Cl 2.7g/L, weighed according to 3L fermentation broth. Adding the weighed culture medium into a 5L fermentation tank, adding 2.8L of tap water, stirring until the mixture is completely dissolved, adding 0.5mL of a defoaming agent, adding sodium hydroxide to adjust the pH value to 7.2-7.3, and sterilizing at 121 ℃ for 20 min.
a. Picking recombinant Escherichia coli single colony with sterilized toothpick, inoculating into test tube containing 4mL seed culture medium, shake culturing at 37 deg.C and 200rpm in shaking table for 10 hr, OD600Reaching above 0.6 (actually measured 1.0), transferring into 500mL Erlenmeyer flask containing 100mL seed culture medium with inoculum size of 2mL, and continuously culturing in shaker at 37 deg.C and 200rpm to OD600Up to about 0.6.
b. B, inoculating 7% of the seed liquid obtained in the step aThe amount of the solution was measured into a fermenter medium, and the temperature and the stirring speed of the fermenter were set to 37 ℃ and 400rpm, respectively, and the aeration rate was adjusted to 1vvm (3L/min). After each parameter of the fermentation tank is stable, 200mL of seed liquid is inoculated into the fermentation tank under the protection of flame, and the fermentation is started. Dissolved Oxygen (DO) decreased as the cells grew, and the agitation speed was increased when the DO dropped below 30% to a speed increase of 500 rpm. Acid and alkali are fed during the fermentation process to adjust the pH value to about 7.0. Sampling every 1h after fermentation for 2h, and detecting cell concentration (OD) in the fermentation liquid600). After fermentation culture for 4h, carbon-nitrogen source (supplemented carbon-nitrogen source: 250g/L glycerol, 60g/L peptone, 60g/L yeast extract, 400 mL/tank, pH was not adjusted) was supplemented at a flow rate of 35 mL/h. During fermentation culture for 5h, the temperature of the feed solution in the fermentation tank is adjusted to 25 ℃, and then IPTG aqueous solution (the concentration of the mother solution is 1M, and the final concentration is 0.2mM) is added to perform induction expression on the target protein, and the feeding rate is reduced to 27 mL/h. Sampling every 2h after induction expression, determining OD600After 12h of induction, the fermentation was terminated.
c. And (5) putting the strain into a tank, and centrifugally collecting the strain.
Example 2 method for synthesizing 4-HIL Using recombinant E.coli Whole cell
(1) Weighing isoleucine 90g, alpha-ketoglutaric acid 100.5g, Vc10.6g and ferrous sulfate heptahydrate 0.45g, adding tap water for dissolution, pouring into a 5L fermentation tank, adjusting the pH of the feed liquid to 7.0, adding centrifuged thallus dosage 150g, supplementing tap water to 3L, adding 0.5ml of natural enemy, and converting conditions: reacting at 30 ℃ and 250rpm with the ventilation of 1vvm for 24 h;
(2) carrying out ultrafiltration treatment on the conversion solution by adopting an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear solution;
(3) adsorbing 732 cation exchange resin (columns of type 2.5L x 2 are connected in series, and WA-2 is used as filler) on the clear liquid of the ultrafiltration membrane, eluting with 1% sodium hydroxide solution, collecting the eluent when the refractive index of the eluent is more than or equal to 0.1, and collecting the eluent with the conductivity of less than or equal to 1000 us/cm;
(4) adding 15% active carbon into the eluent, decolorizing at 60 deg.C for 30min, vacuum filtering, concentrating, and drying to obtain 120g product with purity of 98%.
Example 3 method for synthesizing 4-HIL Using recombinant E.coli Whole cell
(1) Weighing 85g of isoleucine, 100g of alpha-ketoglutaric acid, 9g of Vc and 0.4g of ferrous sulfate heptahydrate, adding tap water for dissolution, pouring into a 5L fermentation tank, adjusting the pH of the feed liquid to 7.0, adding 130g of centrifuged thallus, supplementing the tap water to 3L, adding 0.5ml of natural enemy, and converting: reacting at 30 ℃ and 250rpm with the ventilation of 1vvm for 20 h;
(2) carrying out ultrafiltration treatment on the conversion solution by adopting an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear solution;
(3) adsorbing 732 cation exchange resin (columns of type 2.5L x 2 are connected in series, and WA-2 is used as filler) on the clear liquid of the ultrafiltration membrane, eluting with 2% sodium hydroxide solution, collecting the eluent when the refractive index of the eluent is more than or equal to 0.1, and collecting the eluent with the conductivity of less than or equal to 1000 mus/cm;
(4) adding 15% active carbon into the eluent, decolorizing at 60 deg.C for 50min, vacuum filtering, concentrating, and drying to obtain 107g product with purity of 98.2%.
Example 4 method for synthesizing 4-HIL Using recombinant E.coli Whole cell
(1) Weighing 100g of isoleucine, 110g of alpha-ketoglutaric acid, 12g of Vc and 0.8g of ferrous sulfate heptahydrate, adding tap water for dissolution, pouring into a 5L fermentation tank, adjusting the pH value of the feed liquid to 7.0, adding 180g of centrifuged thallus, supplementing the tap water to 3L, adding 0.5ml of natural enemy, and converting: reacting at 30 ℃ and 250rpm with the ventilation of 1vvm for 24 h;
(2) carrying out ultrafiltration treatment on the conversion solution by adopting an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear solution;
(3) adsorbing 732 cation exchange resin (columns of type 2.5L x 2 are connected in series, and WA-2 is used as filler) on the clear liquid of the ultrafiltration membrane, eluting with 1% sodium hydroxide solution, collecting the eluent when the refractive index of the eluent is more than or equal to 0.1, and collecting the eluent with the conductivity of less than or equal to 1000 us/cm;
(4) adding 10% active carbon into the eluent, decolorizing at 60 deg.C for 50min, vacuum filtering, concentrating, and drying to obtain 123g product with purity of 98.2%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A method for preparing 4-HIL by using a whole-cell catalysis method is characterized in that the whole cell takes Escherichia coli BL21(DE3) as a host, pET28a as a carrier to express isoleucine dioxygenase, isoleucine as a substrate to construct a whole-cell catalysis system for conversion reaction, and a 4-HIL product is obtained by carrying out ultrafiltration and ion exchange on a conversion solution, decoloring, concentrating and drying;
the method comprises the following steps:
(1) carrying out fermentation culture on the recombinant escherichia coli, and collecting thalli;
(2) mixing the bacterial cells, a substrate and a buffer solution, and converting for 20-24h at the temperature of 28-32 ℃ to obtain a conversion solution; in the transformation system, the concentration of the somatic cells is 120-180g/L, the concentration of isoleucine is 80-100g/L, the concentration of alpha-ketoglutaric acid is 80-120g/L, Vc is 8-12g/L, and the concentration of ferrous sulfate heptahydrate is 0.3-1.0 g/L;
(3) carrying out ultrafiltration treatment on the conversion solution by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear solution;
(4) performing ion exchange treatment on the clear liquid, wherein the adopted ion exchange column is 732 cation exchange resin and is eluted by 1-2% sodium hydroxide solution; collecting the eluent when the refraction of the eluent is more than or equal to 0.1, and collecting the eluent with the conductivity of less than or equal to 1000 us/cm;
(6) adding 12-15% active carbon into the eluate, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, and drying to obtain 4-HIL product.
2. The method for preparing 4-HIL according to claim 1, wherein said isoleucine dioxygenase is derived from Bacillus subtilis.
3. The method for preparing 4-HIL by using the whole-cell catalysis method according to claim 1, wherein in the step (1), isoleucine 90g, alpha-ketoglutaric acid 100.5g, Vc10.6g and ferrous sulfate heptahydrate 0.45g are weighed, tap water is added for dissolution, the mixture is poured into a 5L fermentation tank, the pH value of the feed liquid is adjusted to 7.0, the centrifuged thallus dosage is 150g, the tap water is supplemented to 3L, 0.5mL of natural enemy is added, and the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 24 h.
4. The method for preparing 4-HIL by whole cell catalysis according to claim 1, wherein in step (4), 15% of activated carbon is added into the eluate, decolorization is carried out at 60 ℃ for 30min, and the product is obtained by suction filtration, concentration and drying.
5. The method for preparing 4-HIL by using the whole-cell catalysis method according to claim 1, wherein in the step (1), 85g of isoleucine, 100g of alpha-ketoglutaric acid, 9g of Vc, and 0.4g of ferrous sulfate heptahydrate are weighed, tap water is added for dissolution, the mixture is poured into a 5L fermentation tank, the pH value of the material liquid is adjusted to 7.0, 130g of centrifuged thallus is added, the tap water is added for supplementing to 3L, 0.5ml of natural enemy is added, and the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 20 h.
6. The method for preparing 4-HIL by using the whole-cell catalysis method according to claim 1, wherein in the step (1), isoleucine 100g, alpha-ketoglutaric acid 110g, Vc12g and ferrous sulfate heptahydrate 0.8g are weighed, tap water is added for dissolution, the mixture is poured into a 5L fermentation tank, the pH value of the feed liquid is adjusted to 7.0, the centrifuged thallus dosage 180g is added, the tap water is added for supplementing to 3L, the natural killer 0.5ml is added, and the conversion conditions are as follows: the reaction was carried out at 30 ℃ and 250rpm with a 1vvm aeration for 24 h.
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