CN113233991A - Method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid - Google Patents
Method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000003197 catalytic effect Effects 0.000 title claims abstract description 22
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- OSCCDBFHNMXNME-UHFFFAOYSA-N gamma-hydroxyisoleucine Natural products CC(O)C(C)C(N)C(O)=O OSCCDBFHNMXNME-UHFFFAOYSA-N 0.000 title claims abstract description 14
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- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 6
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- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 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
- 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
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 235000011187 glycerol Nutrition 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
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- 238000009423 ventilation Methods 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
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- 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
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- 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
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- 201000005917 gastric ulcer Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011081 inoculation 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
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- 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
-
- 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
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Abstract
The invention discloses a method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid, and belongs to the technical field of biology. The method takes recombinant escherichia coli as a catalyst and isoleucine as a substrate to carry out whole-cell catalytic reaction to obtain a mixture containing 4-hydroxyisoleucine; ultrafiltering whole cell catalytic liquid to remove thallus, purifying 4-HIL by ion exchange method, determining the end point of the collected liquid by using sodium hydroxide solution as eluent and conductivity when adopting the ion exchange method, and obtaining the 4-HIL product by decoloring, concentrating and crystallizing, secondary decoloring and concentrating and crystallizing. The conversion rate of the substrate can reach 98 percent, and the purity of the 4-HIL in the product can reach 99.5 percent by combining ultrafiltration and ion exchange means.
Description
Technical Field
The invention relates to a method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid, 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. The chemical-enzymatic synthesis of 4-HIL has complex steps, many intermediate byproducts and low conversion efficiency. And, in the process of separating and extracting the 4-HIL, the 4-HIL needs to be eluted from the ion exchange column by using ammonia water, which causes the generation of waste water containing ammonia nitrogen.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problems that the existing chemical-enzymatic method for synthesizing 4-HIL has complex steps and low conversion efficiency, and generates ammonia nitrogen wastewater during separation and extraction.
[ solution ]
The invention provides a method for extracting 4-hydroxyisoleucine from whole-cell catalytic solution, wherein the whole-cell catalytic solution is obtained by mixing recombinant escherichia coli cells, a substrate and a buffer solution, and converting for 20-24 hours at 28-32 ℃; ultrafiltering whole cell catalytic liquid to remove thallus, purifying 4-HIL by ion exchange method, determining the end point of the collected liquid by using sodium hydroxide solution as eluent and conductivity when adopting the ion exchange method, and obtaining the 4-HIL product by decoloring, concentrating and crystallizing, secondary decoloring and concentrating and crystallizing.
The whole cell is recombinant escherichia coli, and escherichia coli BL21(DE3) is used as a host, pET28a is used as a vector to express isoleucine dioxygenase, wherein the isoleucine dioxygenase is derived from bacillus subtilis.
The whole-cell catalytic solution is obtained according to the following method: mixing the somatic cells, a substrate and a buffer solution, and converting for 20-24h at the temperature of 28-32 ℃ to obtain a whole-cell catalytic solution; in the transformation system, the concentration of the bacterial cells is 120-180g/L, the concentration of isoleucine is 80-100g/L, the concentration of alpha-ketoglutaric acid is 80-120g/L, Vc (used as a reducing agent) is 8-12g/L, and the concentration of ferrous sulfate heptahydrate (used as a cofactor) is 0.3-1.0 g/L.
The method for extracting 4-hydroxyisoleucine from whole-cell catalytic liquid comprises the following steps:
(1) carrying out ultrafiltration treatment on the whole-cell catalytic liquid (conversion liquid) by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear liquid;
(2) carrying out ion exchange treatment on the clear liquid, wherein an adopted ion exchange column is 732 cation exchange resin, after adsorption saturation, washing with water for 4-8 column volumes, then eluting with 1-2% sodium hydroxide solution, and collecting eluent with refraction not less than 0.1 and conductivity not more than 1000 mus/cm;
(3) adding 12-15% active carbon into the eluate, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL crude product;
(4) adding 10-15 times of water into the crude product, adding 12-15% of active carbon, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL 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 99.5 percent by combining ultrafiltration and ion exchange means.
The invention uses sodium hydroxide to replace ammonia water for elution, so that waste water containing ammonia nitrogen cannot be discharged.
The invention uses the conductivity to accurately judge the collection end point of the eluent.
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
The recombinant Escherichia coli cell expresses isoleucine dioxygenase derived from Bacillus subtilis by using Escherichia coli BL21(DE3) as a host and pET28a as a vector.
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)2HPO4·12H2O 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. And c, inoculating the seed solution obtained in the step a into a fermentation tank culture medium according to the inoculation amount of 7%, setting the temperature of the fermentation tank and the stirring speed to be 37 ℃ and 400rpm respectively, and adjusting the ventilation quantity to be 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 (feed carbon-nitrogen source: 250g/L glycerin, 60g/L egg) is supplementedWhite peptone, 60g/L yeast extract, 400 mL/jar, pH not adjusted), flow rate 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 value of the feed liquid to 7.0, adding centrifuged thallus dosage 150g, supplementing tap water to 3L, adding foam enemy 0.5ml, and converting conditions: the reaction was carried out at 30 ℃ and 250rpm with a ventilation of 1vvm for 24 hours, and the resulting conversion solution was collected.
Example 34 extraction of HIL
(1) Carrying out ultrafiltration treatment on the conversion solution obtained in the embodiment 2 by using an ultrafiltration membrane to remove protein and thalli, and obtaining ultrafiltration membrane clear solution;
(2) performing ion exchange treatment on 7L of the clear solution, wherein the adopted ion exchange column is 2L of 732 cation exchange resin (column model 2.5L x 2 in series and WA-2 filler), and the flow rate of sample loading is 1.2L/h; after adsorption saturation, washing with 15L of water, then eluting with 1% sodium hydroxide solution, and collecting 6L of eluent with refraction of not less than 0.1 and conductivity of not more than 1000us/cm, wherein the content of 4-HIL is 3%;
(3) adding 15% of active carbon into the eluent, decoloring for 30min at 60 ℃, filtering, concentrating, crystallizing and drying to obtain 120g of a 4-HIL crude product;
(4) adding 1L water into 100g crude product (water content 20%), adding 15% active carbon, decolorizing at 60 deg.C for 30min, filtering, concentrating, crystallizing, and drying to obtain 40g 4-HIL product. The purification yield was 40g/100g × 80% ═ 50%, and the purity was 99.5%.
Example 44 extraction of HIL
(1) Carrying out ultrafiltration treatment on the conversion solution obtained in the embodiment 2 by using an ultrafiltration membrane to remove protein and thalli, and obtaining ultrafiltration membrane clear solution;
(2) performing ion exchange treatment on 7L of the clear solution, wherein the adopted ion exchange column is 2L of 732 cation exchange resin (column model 2.5L x 2 in series and WA-2 filler), and the flow rate of sample loading is 1.2L/h; after adsorption saturation, washing with 10L of water, then eluting with 2% sodium hydroxide solution, and collecting the eluate with refraction not less than 0.1 and conductivity not more than 1000 us/cm;
(3) adding 12% active carbon into the eluate, decolorizing at 55 deg.C for 50min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL crude product;
(4) adding 12 times of water by mass into the crude product, adding 12% of activated carbon, decolorizing at 55 deg.C for 50min, filtering, concentrating, crystallizing, and drying to obtain 4-HIL product.
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 extracting 4-hydroxyisoleucine from whole-cell catalytic liquid is characterized by ultrafiltering whole-cell catalytic liquid to remove thallus, purifying 4-HIL by an ion exchange method, judging the end point of a collected liquid by using sodium hydroxide solution as an eluent and utilizing electric conductivity when the ion exchange method is adopted, and then carrying out decoloration, concentrated crystallization, secondary decoloration and concentrated crystallization to obtain a 4-HIL product.
2. The method for extracting 4-hydroxyisoleucine from the whole-cell catalytic solution according to claim 1, wherein the whole-cell catalytic solution is obtained by mixing recombinant escherichia coli cells, a substrate and a buffer solution, and converting the mixture at 28-32 ℃ for 20-24h to obtain a conversion solution; the whole cell is recombinant escherichia coli, and escherichia coli BL21(DE3) is used as a host, pET28a is used as a vector to express isoleucine dioxygenase, wherein the isoleucine dioxygenase is derived from bacillus subtilis.
3. The method for extracting 4-hydroxyisoleucine from the whole-cell catalytic solution according to claim 2, wherein the whole-cell catalytic solution is obtained by the following method: mixing 120-180g/L of thallus cells, 80-100g/L of isoleucine, 80-120g/L, Vc 8-12g/L of alpha-ketoglutaric acid and 0.3-1.0g/L of ferrous sulfate heptahydrate, and converting at 28-32 ℃ for 20-24 h.
4. The method for extracting 4-hydroxyisoleucine from a whole-cell catalytic solution according to any one of claims 1 to 3,
(1) carrying out ultrafiltration treatment on the whole-cell catalytic liquid by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear liquid;
(2) carrying out ion exchange treatment on the clear liquid, wherein an adopted ion exchange column is 732 cation exchange resin, after adsorption saturation, washing with water for 4-8 column volumes, then eluting with 1-2% sodium hydroxide solution, and collecting eluent with refraction not less than 0.1 and conductivity not more than 1000 us/cm;
(3) adding 12-15% active carbon into the eluate, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL crude product;
(4) adding 10-15 times of water into the crude product, adding 12-15% of active carbon, decolorizing at 55-60 deg.C for 30min-60min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL product.
5. The method of claim 4, wherein the extraction of 4-hydroxyisoleucine from the whole-cell catalyst solution,
(1) carrying out ultrafiltration treatment on the whole-cell catalytic liquid by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear liquid;
(2) carrying out ion exchange treatment on 7L of clear liquid, wherein the adopted ion exchange column is 2L of 732 cation exchange resin, and the sample loading flow rate is 1.2L/h; after adsorption saturation, washing with 15L of water, then eluting with 1% sodium hydroxide solution, and collecting the eluate with refraction not less than 0.1 and conductivity not more than 1000 us/cm;
(3) adding 15% active carbon into the eluate, decolorizing at 60 deg.C for 30min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL crude product;
(4) adding 10 times of water by mass into 100g of crude product, adding 15% of activated carbon, decolorizing at 60 ℃ for 30min, filtering, concentrating, crystallizing, and drying to obtain the 4-HIL product.
6. The method of claim 4, wherein the extraction of 4-hydroxyisoleucine from the whole-cell catalyst solution,
(1) carrying out ultrafiltration treatment on the whole-cell catalytic liquid by using an ultrafiltration membrane to remove protein and thalli to obtain ultrafiltration membrane clear liquid;
(2) carrying out ion exchange treatment on 7L of clear liquid, wherein the adopted ion exchange column is 2L of 732 cation exchange resin, and the sample loading flow rate is 1.2L/h; after adsorption saturation, washing with 10L of water, then eluting with 2% sodium hydroxide solution, and collecting the eluate with refraction not less than 0.1 and conductivity not more than 1000 us/cm;
(3) adding 12% active carbon into the eluate, decolorizing at 55 deg.C for 50min, vacuum filtering, concentrating, crystallizing, and drying to obtain 4-HIL crude product;
(4) adding 12 times of water by mass into the crude product, adding 12% of activated carbon, decolorizing at 55 deg.C for 50min, filtering, concentrating, crystallizing, and drying to obtain 4-HIL product.
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