CN111635917A - Preparation method of beta-nicotinamide ribodinucleotide - Google Patents
Preparation method of beta-nicotinamide ribodinucleotide Download PDFInfo
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- CN111635917A CN111635917A CN202010529554.6A CN202010529554A CN111635917A CN 111635917 A CN111635917 A CN 111635917A CN 202010529554 A CN202010529554 A CN 202010529554A CN 111635917 A CN111635917 A CN 111635917A
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- nicotinamide
- transferase
- ribokinase
- beta
- mononucleotide
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- 229960003966 nicotinamide Drugs 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 52
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- 108090000992 Transferases Proteins 0.000 claims abstract description 30
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- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 25
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 claims abstract description 24
- 102000046755 Ribokinases Human genes 0.000 claims abstract description 24
- 235000020956 nicotinamide riboside Nutrition 0.000 claims abstract description 21
- 241000588724 Escherichia coli Species 0.000 claims abstract description 20
- 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 abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000002994 raw material Substances 0.000 claims abstract description 8
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 12
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- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
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- 108010021066 nicotinamide riboside kinase Proteins 0.000 claims description 5
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- 230000006698 induction Effects 0.000 claims description 2
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- 230000000694 effects Effects 0.000 abstract description 10
- 239000000411 inducer Substances 0.000 abstract description 4
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 10
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 9
- 229950006238 nadide Drugs 0.000 description 9
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 239000012466 permeate Substances 0.000 description 6
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 5
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- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
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- 229930195729 fatty acid Natural products 0.000 description 1
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- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01022—Ribosylnicotinamide kinase (2.7.1.22)
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07001—Nicotinamide-nucleotide adenylyltransferase (2.7.7.1)
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Abstract
The invention relates to the field of bio-enzyme catalytic preparation, and in particular relates to a preparation method of beta-nicotinamide ribodinucleotide. The method comprises the steps of firstly preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, and then producing beta-nicotinamide ribodinucleotide by nicotinamide ribose and ATP under the action of the nicotinamide ribokinase and the nicotinamide mononucleotide transferase. The invention provides a high-density fermentation method for preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase, which uses a fermentation culture medium containing essential elements suitable for growth of escherichia coli, enables the escherichia coli to grow well in the fermentation culture medium under the action of an inducer IPTG, can produce high-yield nicotinamide ribokinase and nicotinamide mononucleotide transferase, and has high enzyme activity. The preparation method of the beta-nicotinamide riboside provided by the invention has the advantages of high efficiency, simple operation and high yield, and the used nicotinamide riboside and ATP are common raw materials, so that the cost is low, and the preparation method is suitable for industrial production.
Description
Technical Field
The invention relates to the field of bio-enzyme catalytic preparation, and in particular relates to a preparation method of beta-nicotinamide ribodinucleotide.
Background
Of all the enzymes currently found, about 30-35% are oxidoreductases. In industrial biotechnology, especially in biopharmaceutical and fine chemical industries, oxidoreductases are important enzymes. In the case of the reaction catalyzed by the oxidoreductase, the production of the product is accompanied by the consumption of a certain coenzyme for the transfer of electrons and protons. According to statistics, about 80% of reactions need nicotinamide ribodinucleotide, namely oxidized coenzyme I (NAD +) as coenzyme, and the phosphorylation derivative of 2' -position of a ribose ring system connected with adenine in the NAD + is nicotinamide adenine dinucleotide phosphate, also called oxidized coenzyme II, (NADP +) is an important coenzyme. The oxidized coenzyme II transfers protons, electrons and energy in a redox reaction mode, participates in a plurality of metabolic reactions of cells, synthesizes lipids, fatty acid and nucleotide, can activate a multienzyme system, promotes the synthesis and metabolism of nucleic acid, protein and polysaccharide, improves the material transfer and regulation control, and improves the metabolic function. Research shows that the oxidized coenzyme II can promote metabolism, energy metabolism, resist cell senility and resist oxidation.
NAD + is widely present in living organisms, but is present in very low amounts, with NAD + extracted from living organisms being at a market price of tens of thousands of dollars per kilogram. NAD + is used in industrial production, but the production method is less. Current chemical and biological enzymatic methods of NAD +. The chemical method uses nicotinamide ribose as a raw material, and also uses expensive reagents and explosive reagents, so that accidents can occur due to high cost. Biological enzyme catalysis is gradually the mainstream method for industrially preparing nicotinamide ribodinucleotide (NAD +) because of the inherent advantages of high efficiency, environmental protection and the like, but the existing biological enzyme method has complex operation and low yield.
Disclosure of Invention
Aiming at the problems of complex operation and low yield of the preparation method of beta-nicotinamide ribodinucleotide in the prior art, the invention aims to provide a preparation method of beta-nicotinamide ribodinucleotide.
In order to achieve the purpose, the invention adopts the following technical scheme.
A preparation method of beta-nicotinamide ribodinucleotide comprises the following steps.
Step one, inoculating escherichia coli to a solid culture medium of LB, and culturing for 12-18 h at 37 ℃ to obtain a solid inclined plane containing the escherichia coli.
And step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotation speed of a shaking table of 180-200 r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle grows well.
Step three, preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, wherein a fermentation medium consists of the following raw materials in percentage by mass: peptone 1.0-2.0%, yeast extract 0.2-1.0%, potassium dihydrogen phosphate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.10%, glycerol 0.5-1.0%, magnesium sulfate 0.05-0.2%, defoaming agent 0.05-0.10%, and drinking water in balance; sterilizing the culture medium by using steam, respectively inoculating escherichia coli containing nicotinamide ribokinase and nicotinamide mononucleotide transferase genes, culturing at the temperature of 33-37 ℃, the ventilation volume of 3-10L/min and the stirring speed of 300-600rpm in a fermentation tank for 5-6 hours, adding 0.5g of isopropyl thiogalactoside IPTG for induction, and continuously culturing for 15-22 hours to obtain a culture solution.
Step four, respectively centrifuging the two culture solutions in the step three to obtain thalli, homogenizing and crushing the thalli by using the pressure of 0.2-0.6MPa, filtering to obtain filtrate, and filtering and concentrating the filtrate by using a hollow membrane to obtain an enzyme concentrate of the amide ribokinase and the nicotinamide mononucleotide transferase.
Step five, dissolving 34g of nicotinamide riboside, 100g of ATP and 8-16g of magnesium chloride into 2L of purified water, respectively adding 20-80g of enzyme concentrated solution of nicotinamide riboside kinase and nicotinamide mononucleotide transferase in the step four, controlling the temperature at 30-37 ℃, controlling the pH value to be 6.40-6.60 by using sodium hydroxide solution in the process, detecting the residue of nicotinamide riboside and the generated beta-nicotinamide riboside, and under the action of enzyme, the nicotinamide riboside is remained below 0.02g/L within 4 hours, so that the generated beta-nicotinamide riboside reaches the highest concentration.
And step six, carrying out post-treatment ultrafiltration, nanofiltration, resin adsorption elution and crystallization on the reaction liquid to obtain a product.
Further, the fermentation medium in the third step is composed of the following raw materials by mass percent: peptone 1.0-1.5%, yeast extract 0.5-0.8%, potassium dihydrogen phosphate 0.05-0.10%, dipotassium hydrogen phosphate 0.05-0.08%, glycerol 0.5-0.8%, magnesium sulfate 0.10-0.15%, defoaming agent 0.05-0.08%, and water in balance.
Further, the amount of magnesium chloride added in step five is 10-15 g.
Further, the amount of the enzyme concentrate of nicotinamide ribokinase and nicotinamide mononucleotide transferase added in step five is 30-60 g.
Further, the temperature in the fifth step is controlled to be 35-37 ℃.
Further, the pH value is controlled to be 6.45-6.55 in the fifth step.
Compared with the prior art, the invention has the following beneficial effects.
The enzymatic activities of nicotinamide ribokinase and nicotinamide mononucleotide transferase in the preparation method for preparing beta-nicotinamide ribodinucleotide provided by the invention are high, nicotinamide ribokinase and ATP can be rapidly converted into beta-nicotinamide ribodinucleotide, the conversion rate is high, the purity of the produced NAD + is high, the fermentation process is simple, the extraction is easy, the cost is low, and the industrial production is easy.
The fermentation medium contains essential elements suitable for growth of escherichia coli, the escherichia coli can grow well in the fermentation medium under the action of an inducer IPTG, high yield of nicotinamide ribokinase and nicotinamide mononucleotide transferase can be achieved, the enzyme activity is high, the medium is simple in component, low in cost and wide in application prospect, and the fermentation method is simple, practical and easy to operate.
The invention provides an enzymatic reaction method of nicotinamide riboside, which has high efficiency, simple operation and high yield, and the used nicotinamide riboside and ATP are common raw materials, so the cost is low and the method can be industrialized.
Detailed Description
For better understanding of the present invention, the following examples are further illustrated, but the following examples do not limit the scope of the present invention. In the following examples, experimental procedures and methods not described in detail are conventional means well known in the art.
Example 1 fermentation Process for amidoribokinase and nicotinamide mononucleotide transferase.
The fermentation method of the amide ribokinase and the nicotinamide mononucleotide transferase comprises the following specific steps.
Step one, inoculating escherichia coli into a solid culture medium of LB, and culturing for 14h at 37 ℃ to obtain a solid slant containing the escherichia coli.
And step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle is good in length.
Step three, preparing 1.5 percent of fermentation medium peptone, 0.5 percent of yeast extract powder, 0.10 percent of potassium dihydrogen phosphate, 0.05 percent of dipotassium hydrogen phosphate, 0.5 percent of glycerol, 0.10 percent of magnesium sulfate, 0.05 percent of defoaming agent and the balance of drinking water into a 10L tank, metering and preparing according to 6.0L, sterilizing by using steam at the temperature of 123 ℃ for 30min, inoculating the grown shake flask seed liquid into a fermentation tank, inoculating 150ml of the seed liquid, the temperature of the tank is 33-37 ℃, the pressure of the tank is 0.03-0.06MPa, the ventilation volume is 3-10L/min, the rotating speed of 300 ℃ of 600rpm, adding 0.5g of inducer of isopropyl thiogalactoside IPTG when the culture is carried out for 6 hours, and the culture period is 22 hours. OD at can discharge600nm=56。
And step four, centrifuging the culture solution obtained in the step 3, discarding the supernatant, homogenizing and crushing the thalli by using a homogenizer at 0.4Mpa, filtering, washing the thalli for 2 times by using purified water, and combining the filtrate. Then, the mixture was concentrated through a hollow membrane having a molecular weight of 20000 to obtain 820ml of a crude enzyme solution of nicotinamide ribokinase, and 850ml of a crude enzyme solution of nicotinamide mononucleotide transferase was obtained in the same manner.
Two enzyme activity detection methods are as follows: using 2g of ATP and 0.68g of nicotinamide ribose, using 0.14g of magnesium sulfate, using 50ml of constant volume, adding 1ml of each of two enzyme solutions, controlling the process temperature to be 37 ℃ and the process pH to be 6.40-6.50 by using 0.4g/L of sodium hydroxide solution, detecting and cooling to be below 20 ℃ after 1 hour, using hydrochloric acid to adjust the pH to be 3.8-4.0, and detecting nicotinamide residue after centrifugation, wherein the enzyme activities of the two enzymes = the milligrams of the consumed nicotinamide ribose. The enzyme activities of the above two enzymes were 300.
Example 2 fermentation Process for amidoribokinase and nicotinamide mononucleotide transferase.
The fermentation method of the amide ribokinase and the nicotinamide mononucleotide transferase comprises the following specific steps.
Step one, inoculating escherichia coli into a solid culture medium of LB, and culturing for 14h at 37 ℃ to obtain a solid slant containing the escherichia coli.
And step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle is good in length.
Step three, preparing 1.0% of fermentation medium peptone, 0.8% of yeast extract powder, 0.10% of potassium dihydrogen phosphate, 0.05% of dipotassium hydrogen phosphate, 0.8% of glycerol, 0.10% of magnesium sulfate, 0.05% of defoaming agent and the balance of drinking water into a 10L tank, preparing the materials according to 6.0L, sterilizing the well-grown shake flask seed liquid by using steam at the temperature of 121-. OD at can discharge600nm=65。
And step four, centrifuging the culture solution obtained in the step 3, discarding the supernatant, homogenizing and crushing the thalli by using a homogenizer at 0.4Mpa, filtering, washing the thalli for 2 times by using purified water, and combining the filtrate. Then, the mixture was concentrated by passing through a hollow membrane having a molecular weight of 20000 to obtain 850ml of a crude enzyme solution of nicotinamide ribokinase. 900ml of crude enzyme solution of nicotinamide mononucleotide transferase was obtained in the same manner.
Two enzyme activity detection methods are as follows: using 2g of ATP and 0.68g of nicotinamide ribose, using 0.14g of magnesium sulfate, using 50ml of constant volume, adding 1ml of each of two enzyme solutions, controlling the process temperature to be 37 ℃ and the process pH to be 6.40-6.50 by using 0.4g/L of sodium hydroxide solution, detecting and cooling to be below 20 ℃ after 1 hour, using hydrochloric acid to adjust the pH to be 3.8-4.0, centrifuging, detecting nicotinamide residues, and using the enzyme activity of the two enzymes = the mg of the consumed nicotinamide ribose. The enzyme activity of the two enzymes is 360.
Example 3 enzymatic reaction method of nicotinamide ribodinucleotide.
Nicotinamide ribose and ATP are used to produce beta-nicotinamide ribodinucleotide under the action of nicotinamide ribokinase and nicotinamide mononucleotide transferase.
Step one, dissolving 34g of nicotinamide riboside, 100g of ATP and 10-15g of magnesium chloride into 2L of purified water, controlling the temperature at 36.0-37.0 ℃, adjusting the pH to 6.50 by using a 4% sodium hydroxide solution, then adding 30g of nicotinamide riboside kinase concentrated solution and 30g of nicotinamide mononucleotide transferase concentrated solution, controlling the pH to 6.40-6.50 by using a 4% sodium hydroxide solution, detecting the residue of nicotinamide riboside and generated beta-nicotinamide riboside by using a high performance liquid phase, wherein the nicotinamide riboside is lower than 0.02g/L, the conversion rate is higher than 99.5%, finishing the reaction within 4 hours, cooling to below 20 ℃, adjusting the pH to 3.8-4.0 by using 10% hydrochloric acid, and terminating the reaction.
And step two, filtering the reaction solution to obtain a filtrate, passing the filtrate through an ultrafiltration membrane with the molecular weight of 5000 to obtain a permeate, and performing nanofiltration on the permeate with the molecular weight of 500 to obtain a concentrated solution of the beta-nicotinamide ribodinucleotide.
And step three, adsorbing and eluting the concentrated solution of the beta-nicotinamide ribodinucleotide by resin to obtain an eluent with the purity of more than 98.0%, and concentrating, crystallizing and drying to obtain a high-purity product with the purity of 98.5%.
Example 4 enzymatic reaction method of nicotinamide ribodinucleotide.
Nicotinamide ribose and ATP are used to produce beta-nicotinamide ribodinucleotide under the action of nicotinamide ribokinase and nicotinamide mononucleotide transferase.
Step one, dissolving 34g of nicotinamide riboside, 100g of ATP and 10-15g of magnesium chloride into 2L of purified water, controlling the temperature at 36.0-37.0 ℃, adjusting the pH to 6.50 by using a 4% sodium hydroxide solution, then adding 30g of nicotinamide riboside kinase concentrated solution and 40g of nicotinamide mononucleotide transferase concentrated solution, controlling the pH to 6.40-6.50 by using a 4% sodium hydroxide solution, detecting the residue of nicotinamide riboside and generated beta-nicotinamide riboside by using a high performance liquid phase, wherein the nicotinamide riboside is lower than 0.02g/L, the conversion rate is higher than 99%, finishing the reaction within 2 hours, cooling to below 20 ℃, adjusting the pH to 3.8-4.0 by using 10% hydrochloric acid, and terminating the reaction.
And step two, filtering the reaction solution to obtain a filtrate, passing the filtrate through an ultrafiltration membrane with the molecular weight of 5000 to obtain a permeate, and performing nanofiltration on the permeate with the molecular weight of 500 to obtain a concentrated solution of the beta-nicotinamide ribodinucleotide.
And step three, adsorbing and eluting the concentrated solution of the beta-nicotinamide ribodinucleotide by resin to obtain eluent with the purity of more than 98%, and concentrating, crystallizing and drying to obtain a high-purity product with the purity of 99.1%.
Example 5 enzymatic reaction method of nicotinamide ribodinucleotide.
Nicotinamide ribose and ATP are used to produce beta-nicotinamide ribodinucleotide under the action of nicotinamide ribokinase and nicotinamide mononucleotide transferase.
Step one, dissolving 34g of nicotinamide riboside, 100g of ATP and 10-15g of magnesium chloride into 2L of purified water, controlling the temperature at 36.0-37.0 ℃, adjusting the pH to 6.50 by using a 4% sodium hydroxide solution, then adding 30g of nicotinamide riboside kinase concentrated solution and 60g of nicotinamide mononucleotide transferase concentrated solution, controlling the pH to 6.40-6.50 by using a 4% sodium hydroxide solution, detecting the residue of nicotinamide riboside and generated beta-nicotinamide riboside by using a high performance liquid phase, wherein the nicotinamide riboside is lower than 0.02g/L, the conversion rate is higher than 99%, finishing the reaction within 3 hours, cooling to below 20 ℃, adjusting the pH to 3.8-4.0 by using 10% hydrochloric acid, and terminating the reaction.
And step two, filtering the reaction solution to obtain a filtrate, passing the filtrate through an ultrafiltration membrane with the molecular weight of 5000 to obtain a permeate, and performing nanofiltration on the permeate with the molecular weight of 500 to obtain a concentrated solution of the beta-nicotinamide ribodinucleotide.
And step three, adsorbing and eluting the concentrated solution of the beta-nicotinamide ribodinucleotide by resin to obtain eluent with the purity of more than 98%, and concentrating, crystallizing and drying to obtain a high-purity product with the purity of 98.9%.
Embodiments one to two provide a fermentation medium and a method for fermenting nicotinamide ribokinase and nicotinamide mononucleotide transferase by using the fermentation medium, the fermentation medium contains essential elements suitable for growth of escherichia coli, under the action of an inducer IPTG, the escherichia coli can grow well in the fermentation medium and can produce high-yield nicotinamide ribokinase and nicotinamide mononucleotide transferase, the enzyme activity is high, the obtained enzyme activity is more than 200, and the reaction is completed within 4 hours during production, and the medium has simple components, low cost, wide application prospect, simple and practical fermentation method and easy operation.
Examples three to five provide enzymatic reaction methods of nicotinamide riboside, which have high efficiency, the nicotinamide riboside reaction is completed within 4 hours, the conversion rate exceeds 99.5%, the operation is simple, the conversion rate is high, the nicotinamide riboside and ATP used are common raw materials, the cost is low, and industrialization can be realized.
Claims (6)
1. A preparation method of beta-nicotinamide ribodinucleotide is characterized by comprising the following steps:
step one, inoculating escherichia coli to a solid culture medium of LB, and culturing for 12-18 h at 37 ℃ to obtain a solid inclined plane containing the escherichia coli;
step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotating speed of a shaking table of 180-200 r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle is good in length;
step three, preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, wherein a fermentation medium consists of the following raw materials in percentage by mass: peptone 1.0-2.0%, yeast extract 0.2-1.0%, potassium dihydrogen phosphate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.10%, glycerol 0.5-1.0%, magnesium sulfate 0.05-0.2%, defoaming agent 0.05-0.10%, and the balance of drinking water; sterilizing the culture medium by using steam, respectively inoculating escherichia coli containing nicotinamide ribokinase and nicotinamide mononucleotide transferase genes, culturing at the temperature of 33-37 ℃, the ventilation volume of 3-10L/min and the stirring speed of 300-600rpm in a fermentation tank for 5-6 hours, adding 0.5g of isopropyl thiogalactoside IPTG for induction, and continuously culturing for 15-22 hours to obtain a culture solution;
step four, respectively centrifuging the two culture solutions in the step three to obtain thalli, homogenizing and crushing the thalli by using the pressure of 0.2-0.6MPa, filtering to obtain filtrate, and filtering and concentrating the filtrate by using a hollow membrane to obtain an enzyme concentrate of the amide ribokinase and the nicotinamide mononucleotide transferase;
step five, dissolving 34g of nicotinamide riboside, 100g of ATP and 8-16g of magnesium chloride into 2L of purified water, respectively adding 20-80g of enzyme concentrated solution of nicotinamide riboside kinase and nicotinamide mononucleotide transferase in the step four, controlling the temperature at 30-37 ℃, controlling the pH value to be 6.40-6.60 by using sodium hydroxide solution in the process, detecting the residue of nicotinamide riboside and the generated beta-nicotinamide riboside, and under the action of enzyme, the nicotinamide riboside is remained below 0.02g/L within 4 hours, so that the generated beta-nicotinamide riboside reaches the highest concentration.
2. The method of claim 1, wherein the fermentation medium of step three comprises the following raw materials by weight: peptone 1.0-1.5%, yeast extract 0.5-0.8%, potassium dihydrogen phosphate 0.05-0.10%, dipotassium hydrogen phosphate 0.05-0.08%, glycerol 0.5-0.8%, magnesium sulfate 0.10-0.15%, defoaming agent 0.05-0.08%, and water in balance.
3. The method of claim 1, wherein the amount of magnesium chloride added in step five is 10-15 g.
4. The method of claim 1, wherein 30-60g of the enzyme concentrate of nicotinamide ribokinase and nicotinamide mononucleotide transferase is added in step five.
5. The method of claim 1, wherein the temperature in step five is controlled to 35-37 ℃.
6. The method of claim 1, wherein the pH is controlled to 6.45-6.55 in step five.
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