CN116904537A - Method for preparing adenosine diphosphate pure product by using biological enzyme - Google Patents
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- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 title claims abstract description 87
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 81
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000000047 product Substances 0.000 claims abstract description 46
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- 238000001728 nano-filtration Methods 0.000 claims abstract description 28
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 18
- 238000000746 purification Methods 0.000 claims abstract description 11
- 238000001035 drying Methods 0.000 claims abstract description 10
- 108010021582 Glucokinase Proteins 0.000 claims abstract description 8
- 239000006227 byproduct Substances 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 6
- 239000008103 glucose Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 61
- 238000000855 fermentation Methods 0.000 claims description 37
- 230000004151 fermentation Effects 0.000 claims description 37
- 238000003756 stirring Methods 0.000 claims description 28
- 238000002425 crystallisation Methods 0.000 claims description 22
- 230000008025 crystallization Effects 0.000 claims description 22
- 239000008213 purified water Substances 0.000 claims description 18
- 239000011347 resin Substances 0.000 claims description 18
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- 238000001179 sorption measurement Methods 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 15
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- 239000012295 chemical reaction liquid Substances 0.000 claims description 14
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000013078 crystal Substances 0.000 claims description 12
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- 241000588724 Escherichia coli Species 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical class [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000004042 decolorization Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
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- 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 6
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
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- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 3
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- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 241000235058 Komagataella pastoris Species 0.000 claims description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
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- 238000010369 molecular cloning Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
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- 238000000643 oven drying Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
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- 230000009465 prokaryotic expression Effects 0.000 claims description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 abstract description 14
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 abstract description 14
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- 102100023038 WD and tetratricopeptide repeats protein 1 Human genes 0.000 description 46
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 7
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 5
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 4
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229960005305 adenosine Drugs 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 241000672609 Escherichia coli BL21 Species 0.000 description 2
- 102000009097 Phosphorylases Human genes 0.000 description 2
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- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 102000013009 Pyruvate Kinase Human genes 0.000 description 2
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- 239000010452 phosphate Substances 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 description 1
- 108010092060 Acetate kinase Proteins 0.000 description 1
- 229930183912 Cytidylic acid Natural products 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 description 1
- 102000007466 Purinergic P2 Receptors Human genes 0.000 description 1
- 108010085249 Purinergic P2 Receptors Proteins 0.000 description 1
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 1
- FOGRQMPFHUHIGU-UHFFFAOYSA-N Uridylic acid Natural products OC1C(OP(O)(O)=O)C(CO)OC1N1C(=O)NC(=O)C=C1 FOGRQMPFHUHIGU-UHFFFAOYSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 1
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
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- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 description 1
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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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
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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
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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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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01002—Glucokinase (2.7.1.2)
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Abstract
The application discloses a method for preparing an adenosine diphosphate pure product by using biological enzyme, which comprises the following steps: s1, preparing an enzyme protein a and constructing a GLK enzyme protein engineering strain; b. fermenting GLK recombinant enzyme protein; c. preparing GLK enzyme liquid; s2, performing enzyme catalytic reaction; s3, decoloring the reaction solution; s4, separating by a chromatographic column; s5, nanofiltration, salting and concentrating; s6, crystallizing; s7, recrystallizing; s8, drying the finished product. According to the application, glucokinase (GLK, EC: 2.7.1.2) obtained by screening is used as a catalyst, adenosine Triphosphate (ATP) and glucose which are low in cost and easy to obtain are used as raw materials, biosynthesis of ADP products is carried out in vitro, and ADP raw materials with higher purity are obtained through a simple separation and purification process, the whole biosynthesis process is carried out in a water phase, the reaction is mild and easy to control, few byproducts are generated, the difficulty of the separation and purification process is greatly reduced, the industrialized production cost and environmental protection pressure are reduced, and the final ADP purity is more than 99%.
Description
Technical Field
The application relates to the technical field of adenosine diphosphate preparation, in particular to a method for preparing an adenosine diphosphate pure product by using biological enzyme.
Background
Adenosine diphosphate (Adenosine Diphosphate, abbreviated as ADP) is a compound composed of one molecule of adenosine with two linked phosphates and has the molecular formula C10H15N5O10P2. In vivo, adenosine Triphosphate (ATP) is usually hydrolyzed to lose a phosphate, i.e., to break a high energy phosphate bond, and to release energy.
ADP is the earliest and most important substance found in the body for inducing platelet aggregation, exists in high-density particles in platelet cells, is released when the platelet undergoes a coagulation reaction, and influences the shape and biological behavior of the platelet through an ADP receptor on the platelet, thereby further accelerating the coagulation process of the platelet. ADP is currently used as an important biological raw material in biochemical research, and is applied to the field of preparing polynucleotides by measuring the activity of pyruvate kinase.
The current methods for preparing ADP mainly comprise a chemical synthesis method and a biological fermentation method. The chemical method mainly uses adenosine or adenosine monophosphate as a substrate, and phosphorylates by using phosphorus oxychloride organophosphorus reagent to obtain the corresponding ADP raw material. The biological fermentation is to carry out gene modification on the metabolic pathway of ADP in microbial cells through metabolic engineering, so that the purpose of enriching the target product ADP is achieved, but due to the fact that the fermentation synthesis concentration of the ADP is limited, the components of fermentation liquor are complex, products of other series of nucleotides (such as adenosine, cytidylic acid and uridylic acid) are easy to generate in the microbial metabolic process, the physical and chemical properties of the substances are similar, the purification difficulty of later products is greatly increased, and the industrial production of the products is not facilitated. At present, phosphorylase or kinase is often used as cyclic regeneration between ADP-ATP (adenosine monophosphate) in research, such as nucleoside phosphorylase, polyphosphate kinase, acetate kinase and pyruvate kinase, but if the phosphorylase or kinase is used for producing ADP raw materials, the enzymatic reaction of the cyclic regeneration systems is always in an equilibrium state, the substrate conversion is incomplete, ADP is easy to be continuously hydrolyzed into Adenosine Monophosphate (AMP) by the enzyme, the types of byproducts in the reaction process are increased, the industrialization difficulty is high, and the cost is high. The synthesis of ADP using adenosine or adenylate as a substrate has been reported to have the problems of the reaction balance and the byproducts, and the purity of the commercial ADP raw material is about 95% due to the difficulty of separation and purification. Therefore, there is a need to devise a method for preparing pure adenosine diphosphate using biological enzymes to solve the above problems.
Disclosure of Invention
The application aims to provide a method for preparing an adenosine diphosphate pure product by using biological enzyme, which aims to solve the defects in the prior art.
In order to achieve the above object, the present application provides the following technical solutions:
a method for preparing an adenosine diphosphate pure product by using biological enzyme, which comprises the following steps:
s1, enzyme protein preparation
a. Construction of GLK enzyme protein engineering strains:
molecular cloning of the glucokinase Gene (GLK) from E.coli K12 strain;
b. GLK recombinase protein fermentation:
performing enzyme protein fermentation by using a common escherichia coli fermentation medium, and performing protein induction expression by using isopropyl thiogalactoside (IPTG) or lactose;
c. preparation of GLK enzyme solution: suspending the collected GLK recombinant bacteria fermentation bacteria by using 2mM phosphate buffer solution with pH of 7.0, wherein the volume of the collected GLK recombinant bacteria fermentation bacteria is 0.2-0.5 times of that of the fermentation liquid, crushing cells by using an ultrasonic cell crusher or a high-pressure homogenizer, and centrifugally collecting supernatant to obtain enzyme liquid containing GLK recombinant enzyme protein;
s2, enzyme catalytic reaction
d. The raw material ATP is weighed so that the final concentration is between 100 and 300mM, preferably 200mM, and the concentration ratio of glucose to ADP is 1: 1-2, weighing raw materials into a reaction kettle, adding purified water to reach a target concentration, and stirring to completely dissolve the raw materials;
e. adjusting the pH value of the raw material liquid to 5-9 by using a saturated sodium hydroxide solution, or ammonia water, sodium carbonate or an organic alkali alkaline solution;
f. according to the mass of the raw material ATP of which the volume is 1:5-10 times, preferably 1:8, measuring GLK enzyme liquid, and slowly adding the GLK enzyme liquid into the reaction liquid;
g. controlling the temperature of the reaction solution to be 20-45 ℃, preferably 37 ℃, stirring to perform biocatalysis reaction, and adjusting the pH of the reaction solution to be 5-9, preferably pH7.5 by using saturated NaOH solution in the reaction process;
h. sampling every 1 hour in the reaction process, performing HPLC detection, observing the progress of the reaction liquid, and finally reacting for 3-8 hours, wherein the ATP conversion rate of the substrate is more than 98%, and stopping the reaction;
i. the liquid phase purity of the product ADP in the enzyme catalysis reaction liquid is more than 90 percent, the by-product AMP accounts for less than 5 percent, and the enzyme catalysis reaction is finished;
s3, decoloring the reaction solution
j. After the enzyme catalytic reaction is finished, heating the reaction solution to 60-80 ℃, and stirring for 20-60 min, preferably 30min;
k. adding 0.5-5 mill active carbon, preferably 1 mill active carbon, into the heated enzyme catalytic reaction liquid, stirring for 1-4 h at 30-60 ℃ for adsorption decolorization, preferably 45 ℃ for 2h, collecting supernatant as decolorization liquid, and detecting and analyzing the content of decolorization liquid products by HPLC, wherein the loss rate is less than 2%;
s4, separating by chromatographic column
Separating and purifying the decolorized solution by using strong-alkali anion exchange resin to remove saccharide substances in the decolorized solution;
pretreatment of m, LKA20 type anion exchange resin
Preprocessing LKA20 resin according to the instruction of a resin manufacturer;
n, sample adsorption
The pH of the decolorized solution is controlled to 2.0 to 11.0, preferably pH7.0, and the ratio of 1: 5-20 mass percent, weighing pretreated LKA20 resin, adding the pretreated LKA20 resin into the decolorized solution, slowly stirring the solution for 6-12h at the temperature of 25-40 ℃, carrying out ADP sample adsorption, and enabling the adsorption and mounting rate of the final resin to the product ADP to be up to 95%;
o, sample elution:
filling the LKA20 resin after the sample adsorption into a chromatographic column, flushing the chromatographic column with purified water of which the volume is 1-5 times, preferably 2 times, and eluting residual reaction solvent and saccharide impurities;
washing the non-adsorbed impurities and solvent with 1-3 column volumes, preferably 2 volumes of purified water;
eluting the nucleotide impurities by using 2-6 times of column volume, preferably 4 times of 0.05M-0.1M NaCl solution, and detecting ADP residual amount <5% in the impurity eluent;
eluting the target product ADP by using 0.2M NaCl solution with the volume of 3-8 times of the column volume, detecting the purity of the ADP eluent, and collecting the eluent with the combined purity of more than 95 percent;
s5, nanofiltration, salt removal and concentration
p, adjusting the pH value of the product eluent collected by column chromatography to 4-7, desalting and concentrating by using a nanofiltration membrane system, wherein the filtration molecular weight of the nanofiltration membrane is 150-500 Da, and controlling the temperature of a sample to be 10-35 ℃ in the nanofiltration process;
q, when the eluent volume of the nanofiltration concentrated product reaches 1/2 of the original volume, adding 1/2 of pre-cooling purified water at the temperature of 4 ℃ and repeating for 3-8 times, preferably 5 times;
r, continuing nanofiltration and concentration to 1/8-1/3, preferably 1/5, of the original volume, and stopping nanofiltration; collecting a product concentrated solution, and detecting the purity of a sample to be more than 97%;
s6, crystallization
s, quantifying the concentrated product solution after nanofiltration, slowly adding 1-3 times of absolute ethyl alcohol, preferably 2.5 times of volume, stirring while adding, crystallizing at 0-20 ℃ after stirring and mixing uniformly, preferably 10 ℃ for 10-24 hours, wherein the crystallization time is more than 97% and the ADP crystallization rate is not changed, and stopping crystallization;
s7, recrystallizing
t, collecting crystallized crystals, adding purified water with the volume of 0.1-1 times of the reaction liquid, preferably 0.3 times, and redissolving the crystals at the temperature of 30-60 ℃;
u, adding 1-3 times of absolute ethyl alcohol, preferably 2.5 times of volume into the complex solution, stirring while adding, crystallizing at 0-20 ℃ after stirring and mixing uniformly, preferably 10 ℃, wherein the crystallization time is 2-10 h, the crystallization rate of ADP is more than 97% and is not changed, and then stopping crystallization and collecting crystals;
s8, drying the finished product
v, drying the collected ADP crystals to obtain a final ADP finished product;
w, the HPLC purity of the final ADP product is more than 99%, the content is more than 97%, and the purification yield is 90% -96%.
Further, in the step a in the step S1, the recombinant expression engineering strain of the glucokinase can be constructed by cloning the recombinant expression strain into eukaryotic expression hosts such as saccharomyces cerevisiae and pichia pastoris, and expressing the target protein in prokaryotic expression systems such as escherichia coli and bacillus subtilis hosts, preferably using escherichia coli BL21 as a recombinant protein expression host.
Further, in the step b in the step S1, the fermentation medium is a TB liquid medium, and the TB liquid medium contains: 12g/L peptone, 24g/L yeast powder, 4g/L glycerol, 2.31g/L KH2PO4, 16.43g/L K2 HPO4.3H2O; the culture temperature of the induced expression of the protein is 20-40 ℃, the culture is continued for 16-24 hours, the fermentation can be stopped when the OD600 of the fermentation broth is more than 60-80, and the thalli of the fermentation broth are collected; the GLK recombinase protein can be colibacillus fermentation bacteria containing GLK recombinant genes, cell disruption enzyme liquid (GLK enzyme liquid for short) containing the GLK recombinase protein, or freeze-dried powder containing the GLK recombinase protein, and the GLK recombinase protein can be combined with a corresponding carrier material to become immobilized enzyme of the GLK recombinase protein.
Further, in the step d in the step S2, the temperature of the solution after adding the purified water is controlled to be 20-45 ℃, preferably 37 ℃; in step e of S2, the pH is preferably 7.5; in the step g in S2, the saturated NaOH solution may be any one of aqueous ammonia, sodium carbonate, and an organic alkaline solution.
Further, in the step S4, the anion exchange resin commonly used includes 201×7 type, AM207 type, XRK209 type, LKA20 type, preferably LKA20 type anion exchange resin as a chromatographic separation medium.
Further, in the step p in the step S5, the pH is preferably 5; the molecular weight of nanofiltration membrane filtration is preferably 170Da, and the temperature of the sample is preferably 15 ℃.
Further, in the step v in S8, the drying mode includes one of vacuum oven drying and freeze-drying.
In the technical scheme, the method for preparing the adenosine diphosphate pure product by using the biological enzyme takes the glucokinase (GLK, EC: 2.7.1.2) obtained by screening as a catalyst, uses the low-cost and easily-obtained Adenosine Triphosphate (ATP) and glucose as raw materials to carry out the biosynthesis of the ADP product in vitro, and obtains the ADP raw material with higher purity through a simple separation and purification process, the whole biosynthesis process is carried out in a water phase, the reaction is regulated to be mild and easy to control, the concentration of a catalytic substrate is up to 300mM, the substrate conversion is thorough, the highest concentration can be 99%, the produced ADP is stable and does not continue to hydrolyze, the byproducts are fewer, the difficulty of the separation and purification process is greatly reduced, the industrialized production cost and the environmental protection pressure are reduced, and the final ADP purity is more than 99%.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings for a person having ordinary skill in the art.
FIG. 1 is a flow chart of a production process provided by an embodiment of a method for preparing pure adenosine diphosphate by using biological enzymes.
FIG. 2 is a diagram showing the reaction scheme of the enzyme-catalyzed synthesis provided in the example of the method for preparing pure adenosine diphosphate by using biological enzyme.
FIG. 3 is a graph showing the variation of the components of an enzyme-catalyzed reaction according to an example of a method for preparing pure adenosine diphosphate using biological enzymes according to the present application.
FIG. 4 is a graph showing adsorption rates of LKA20 filler to ADP under different pH conditions according to an embodiment of a method for preparing pure adenosine diphosphate using biological enzyme according to the present application.
FIG. 5 is a graph showing the change of ADP crystallization tendency under different ethanol ratios provided by the embodiment of the method for preparing pure adenosine diphosphate by using biological enzyme.
FIG. 6 is a chart showing HPLC detection at different times of enzyme-catalyzed reaction provided by an example of a method for preparing pure adenosine diphosphate by using biological enzyme.
Detailed Description
In order to make the technical scheme of the present application better understood by those skilled in the art, the present application will be further described in detail with reference to the accompanying drawings.
Example 1
As shown in fig. 1-6, the method for preparing pure adenosine diphosphate by using biological enzyme provided by the embodiment of the application comprises the following steps:
s1, enzyme protein preparation
a. Construction of GLK enzyme protein engineering strains:
molecular cloning of the glucokinase Gene (GLK) from E.coli K12 strain;
b. GLK recombinase protein fermentation:
performing enzyme protein fermentation by using a common escherichia coli fermentation medium, and performing protein induction expression by using isopropyl thiogalactoside (IPTG) or lactose;
c. preparation of GLK enzyme solution: suspending the collected GLK recombinant bacteria fermentation bacteria by using 2mM phosphate buffer solution with pH of 7.0, wherein the volume of the collected GLK recombinant bacteria fermentation bacteria is 0.2-0.5 times of that of the fermentation liquid, crushing cells by using an ultrasonic cell crusher or a high-pressure homogenizer, and centrifugally collecting supernatant to obtain enzyme liquid containing GLK recombinant enzyme protein;
s2, enzyme catalytic reaction
d. The raw material ATP is weighed so that the final concentration is between 100 and 300mM, preferably 200mM, and the concentration ratio of glucose to ADP is 1: 1-2, weighing raw materials into a reaction kettle, adding purified water to reach a target concentration, and stirring to completely dissolve the raw materials;
e. adjusting the pH value of the raw material liquid to 5-9 by using a saturated sodium hydroxide solution, or ammonia water, sodium carbonate or an organic alkali alkaline solution;
f. according to the mass of the raw material ATP of which the volume is 1:5-10 times, preferably 1:8, measuring GLK enzyme liquid, and slowly adding the GLK enzyme liquid into the reaction liquid;
g. controlling the temperature of the reaction solution to be 20-45 ℃, preferably 37 ℃, stirring to perform biocatalysis reaction, and adjusting the pH of the reaction solution to be 5-9, preferably pH7.5 by using saturated NaOH solution in the reaction process;
h. sampling every 1 hour in the reaction process, performing HPLC detection, observing the progress of the reaction liquid, and finally reacting for 3-8 hours, wherein the ATP conversion rate of the substrate is more than 98%, and stopping the reaction;
i. the liquid phase purity of the product ADP in the enzyme catalysis reaction liquid is more than 90 percent, the by-product AMP accounts for less than 5 percent, and the enzyme catalysis reaction is finished;
s3, decoloring the reaction solution
j. After the enzyme catalytic reaction is finished, heating the reaction solution to 60-80 ℃, and stirring for 20-60 min, preferably 30min;
k. adding 0.5-5 mill active carbon, preferably 1 mill active carbon, into the heated enzyme catalytic reaction liquid, stirring for 1-4 h at 30-60 ℃ for adsorption decolorization, preferably 45 ℃ for 2h, collecting supernatant as decolorization liquid, and detecting and analyzing the content of decolorization liquid products by HPLC, wherein the loss rate is less than 2%;
s4, separating by chromatographic column
Separating and purifying the decolorized solution by using strong-alkali anion exchange resin to remove saccharide substances in the decolorized solution;
pretreatment of m, LKA20 type anion exchange resin
Preprocessing LKA20 resin according to the instruction of a resin manufacturer;
n, sample adsorption
The pH of the decolorized solution is controlled to 2.0 to 11.0, preferably pH7.0, and the ratio of 1: 5-20 mass percent, weighing pretreated LKA20 resin, adding the pretreated LKA20 resin into the decolorized solution, slowly stirring the solution for 6-12h at the temperature of 25-40 ℃, carrying out ADP sample adsorption, and enabling the adsorption and mounting rate of the final resin to the product ADP to be up to 95%;
o, sample elution:
filling the LKA20 resin after the sample adsorption into a chromatographic column, flushing the chromatographic column with purified water of which the volume is 1-5 times, preferably 2 times, and eluting residual reaction solvent and saccharide impurities;
washing the non-adsorbed impurities and solvent with 1-3 column volumes, preferably 2 volumes of purified water;
eluting the nucleotide impurities by using 2-6 times of column volume, preferably 4 times of 0.05M-0.1M NaCl solution, and detecting ADP residual amount <5% in the impurity eluent;
eluting the target product ADP by using 0.2M NaCl solution with the volume of 3-8 times of the column volume, detecting the purity of the ADP eluent, and collecting the eluent with the combined purity of more than 95 percent;
s5, nanofiltration, salt removal and concentration
p, adjusting the pH value of the product eluent collected by column chromatography to 4-7, desalting and concentrating by using a nanofiltration membrane system, wherein the filtration molecular weight of the nanofiltration membrane is 150-500 Da, and controlling the temperature of a sample to be 10-35 ℃ in the nanofiltration process;
q, when the eluent volume of the nanofiltration concentrated product reaches 1/2 of the original volume, adding 1/2 of pre-cooling purified water at the temperature of 4 ℃ and repeating for 3-8 times, preferably 5 times;
r, continuing nanofiltration and concentration to 1/8-1/3, preferably 1/5, of the original volume, and stopping nanofiltration; collecting a product concentrated solution, and detecting the purity of a sample to be more than 97%;
s6, crystallization
s, quantifying the concentrated product solution after nanofiltration, slowly adding 1-3 times of absolute ethyl alcohol, preferably 2.5 times of volume, stirring while adding, crystallizing at 0-20 ℃ after stirring and mixing uniformly, preferably 10 ℃ for 10-24 hours, wherein the crystallization time is more than 97% and the ADP crystallization rate is not changed, and stopping crystallization;
s7, recrystallizing
t, collecting crystallized crystals, adding purified water with the volume of 0.1-1 times of the reaction liquid, preferably 0.3 times, and redissolving the crystals at the temperature of 30-60 ℃;
u, adding 1-3 times of absolute ethyl alcohol, preferably 2.5 times of volume into the complex solution, stirring while adding, crystallizing at 0-20 ℃ after stirring and mixing uniformly, preferably 10 ℃, wherein the crystallization time is 2-10 h, the crystallization rate of ADP is more than 97% and is not changed, and then stopping crystallization and collecting crystals;
s8, drying the finished product
v, drying the collected ADP crystals to obtain a final ADP finished product;
w, the HPLC purity of the final ADP product is more than 99%, the content is more than 97%, and the purification yield is 90% -96%.
The application provides a method for preparing adenosine diphosphate pure product by biological enzyme, which takes glucokinase (GLK, EC: 2.7.1.2) obtained by screening as catalyst, uses cheap and easily available Adenosine Triphosphate (ATP) and glucose as raw materials to carry out biological synthesis of ADP product in vitro, and obtains ADP raw material with higher purity by simple separation and purification process, the whole biological synthesis process is carried out in water phase, the reaction is regulated and controlled to be mild and easy, the concentration of catalytic substrate is up to 300mM, the substrate conversion is thorough, the highest can be 99%, the produced ADP is stable and does not continue hydrolysis, the by-product is less, the difficulty of separation and purification process is greatly reduced, the industrialized production cost and environmental protection pressure are reduced, and the final ADP purity is more than 99%.
Example two
In the step a in the S1, cloning into eukaryotic expression hosts such as Saccharomyces cerevisiae and Pichia pastoris, and expressing target proteins in prokaryotic expression systems such as escherichia coli and bacillus subtilis hosts, wherein escherichia coli BL21 is preferably used as a recombinant protein expression host to construct a glucokinase recombinant expression engineering strain; in the step b in the step S1, the fermentation medium is a TB liquid medium, and the TB liquid medium comprises: 12g/L peptone, 24g/L yeast powder, 4g/L glycerol, 2.31g/L KH2PO4, 16.43g/L K2 HPO4.3H2O; the culture temperature of the induced expression of the protein is 20-40 ℃, the culture is continued for 16-24 hours, the fermentation can be stopped when the OD600 of the fermentation broth is more than 60-80, and the thalli of the fermentation broth are collected; the GLK recombinant enzyme protein can be colibacillus fermentation bacteria containing GLK recombinant genes, cell disruption enzyme liquid (GLK enzyme liquid for short) containing the GLK recombinant enzyme protein, or freeze-dried powder containing the GLK recombinant enzyme protein, and the GLK recombinant enzyme protein can be combined with a corresponding carrier material to become immobilized enzyme of the GLK recombinant enzyme protein. In the step d in the step S2, the temperature of the solution after adding the purified water is controlled to be 20-45 ℃, and preferably 37 ℃; in step e in S2, the pH is preferably 7.5; in the step g in S2, the saturated NaOH solution can be any one of ammonia water, sodium carbonate or organic alkali alkaline solution. In step S4, the usual anion exchange resins include 201X 7 type, AM207 type, XRK209 type, LKA20 type, preferably LKA20 type anion exchange resins as the chromatographic separation medium. In step p in S5, the pH is preferably 5; the molecular weight of nanofiltration membrane filtration is preferably 170Da, and the temperature of the sample is preferably 15 ℃. In step v in S8, the drying mode includes one of vacuum oven drying and freeze-drying.
While certain exemplary embodiments of the present application have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that modifications may be made to the described embodiments in various different ways without departing from the spirit and scope of the application. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive of the scope of the application, which is defined by the appended claims.
Claims (7)
1. A method for preparing an adenosine diphosphate pure product by using biological enzyme, which is characterized by comprising the following steps:
s1, enzyme protein preparation
a. Construction of GLK enzyme protein engineering strains:
molecular cloning of the glucokinase Gene (GLK) from E.coli K12 strain;
b. GLK recombinase protein fermentation:
performing enzyme protein fermentation by using a common escherichia coli fermentation medium, and performing protein induction expression by using isopropyl thiogalactoside (IPTG) or lactose;
c. preparation of GLK enzyme solution: suspending the collected GLK recombinant bacteria fermentation bacteria by using 2mM phosphate buffer solution with pH of 7.0, wherein the volume of the collected GLK recombinant bacteria fermentation bacteria is 0.2-0.5 times of that of the fermentation liquid, crushing cells by using an ultrasonic cell crusher or a high-pressure homogenizer, and centrifugally collecting supernatant to obtain enzyme liquid containing GLK recombinant enzyme protein;
s2, enzyme catalytic reaction
d. Weighing raw material ATP to make the final concentration of the raw material ATP between 100 mM and 300mM, and the concentration ratio of glucose to ADP is 1: 1-2, weighing raw materials into a reaction kettle, adding purified water to reach a target concentration, and stirring to completely dissolve the raw materials;
e. adjusting the pH value of the raw material liquid to 5-9 by using a saturated sodium hydroxide solution, or ammonia water, sodium carbonate or an organic alkali alkaline solution;
f. according to the mass of the raw material ATP with the volume of 1:5-10 times, measuring GLK enzyme liquid, and slowly adding the GLK enzyme liquid into the reaction liquid;
g. controlling the temperature of the reaction solution to be 20-45 ℃, stirring to perform biocatalysis reaction, and adjusting the pH of the reaction solution to be 5-9 by using a saturated NaOH solution in the reaction process;
h. sampling every 1 hour in the reaction process, performing HPLC detection, observing the progress of the reaction liquid, and finally reacting for 3-8 hours, wherein the ATP conversion rate of the substrate is more than 98%, and stopping the reaction;
i. the liquid phase purity of the product ADP in the enzyme catalysis reaction liquid is more than 90 percent, the by-product AMP accounts for less than 5 percent, and the enzyme catalysis reaction is finished;
s3, decoloring the reaction solution
j. After the enzyme catalytic reaction is finished, heating the reaction solution to 60-80 ℃, and stirring for 20-60 min;
k. adding 0.5-5 mill active carbon into the enzyme catalytic reaction liquid after the heat treatment, stirring for 1-4 hours at 30-60 ℃ for adsorption decolorization, stirring for 2 hours, collecting supernatant as decolorization liquid, and detecting and analyzing the content of a decolorization liquid product by HPLC, wherein the loss rate is less than 2%;
s4, separating by chromatographic column
Separating and purifying the decolorized solution by using strong-alkali anion exchange resin to remove saccharide substances in the decolorized solution;
pretreatment of m, LKA20 type anion exchange resin
Preprocessing LKA20 resin according to the instruction of a resin manufacturer;
n, sample adsorption
The pH value of the decolorized solution is controlled to be 2.0-11.0 according to the following ratio of 1: 5-20 mass percent, weighing pretreated LKA20 resin, adding the pretreated LKA20 resin into the decolorized solution, slowly stirring the solution for 6-12h at the temperature of 25-40 ℃, carrying out ADP sample adsorption, and finally carrying out ADP adsorption mounting rate of the product by the resin by 95%;
o, sample elution:
filling LKA20 resin after sample adsorption into a chromatographic column, flushing the chromatographic column with purified water with the volume of 1-5 times of the column volume, and eluting residual reaction solvent and saccharide impurities;
washing the non-adsorbed impurities and the solvent with purified water in an amount of 1-3 times the column volume;
eluting the nucleotide impurities by using 0.05M-0.1M NaCl solution with the volume of 2-6 times of the column volume, and detecting ADP residual quantity of <5% in the impurity eluent;
eluting the target product ADP by using 0.2M NaCl solution with the volume of 3-8 times of the column volume, detecting the purity of the ADP eluent, and collecting the eluent with the combined purity of more than 95 percent;
s5, nanofiltration, salt removal and concentration
p, adjusting the pH value of the product eluent collected by column chromatography to 4-7, desalting and concentrating by using a nanofiltration membrane system, wherein the filtration molecular weight of the nanofiltration membrane is 150-500 Da, and controlling the temperature of a sample to be 10-35 ℃ in the nanofiltration process;
q, when the eluent volume of the nanofiltration concentrated product reaches 1/2 of the original volume, adding 1/2 of pre-cooling purified water at the temperature of 4 ℃ and repeating for 3-8 times;
r, continuing nanofiltration and concentration to 1/8-1/3 of the original volume, and stopping nanofiltration; collecting a product concentrated solution, and detecting the purity of a sample to be more than 97%;
s6, crystallization
s, quantifying the concentrated solution of the product after nanofiltration, slowly adding 1-3 times of absolute ethyl alcohol into the concentrated solution, stirring the mixture while adding, crystallizing the mixture at the temperature of 0-20 ℃ after stirring and mixing uniformly, wherein the crystallization time is 10-24 h, and the ADP crystallization rate is more than 97% and does not change any more, thus terminating the crystallization;
s7, recrystallizing
t, collecting crystallized crystals, adding purified water with the volume of 0.1-1 times of the volume of the reaction solution, and re-dissolving the crystals at the temperature of 30-60 ℃;
u, adding 1-3 times of absolute ethyl alcohol into the complex solution, stirring while adding, crystallizing at 0-20 ℃ after stirring and mixing uniformly, wherein the crystallization time is 2-10 h, the ADP crystallization rate is more than 97% and does not change any more, and then the crystallization can be stopped, and the crystals are collected;
s8, drying the finished product
v, drying the collected ADP crystals to obtain a final ADP finished product;
w, the HPLC purity of the final ADP product is more than 99%, the content is more than 97%, and the purification yield is 90% -96%.
2. The method for preparing pure adenosine diphosphate by using biological enzyme according to claim 1, wherein in step a in S1, the target protein is expressed by cloning into eukaryotic expression hosts such as Saccharomyces cerevisiae and Pichia pastoris, and prokaryotic expression systems such as E.coli and Bacillus subtilis.
3. The method for preparing pure adenosine diphosphate by biological enzyme according to claim 1, wherein in step b of S1, the fermentation medium is a TB liquid medium comprising: 12g/L peptone, 24g/L yeast powder, 4g/L glycerol, 2.31g/L KH2PO4, 16.43g/L K2 HPO4.3H2O; the culture temperature of the induced expression of the protein is 20-40 ℃, the culture is continued for 16-24 hours, the fermentation can be stopped when the OD600 of the fermentation broth is more than 60-80, and the thalli of the fermentation broth are collected; the GLK recombinant enzyme protein can be colibacillus fermentation thalli containing GLK recombinant genes, cell disruption enzyme liquid containing the GLK recombinant enzyme protein, freeze-dried powder containing the GLK recombinant enzyme protein, and the enzyme protein can be combined with a corresponding carrier material to become immobilized enzyme of the GLK recombinant enzyme protein.
4. The method for preparing pure adenosine diphosphate by biological enzyme according to claim 1, wherein in step d of S2, the temperature of the solution after adding purified water is controlled to be 20-45 ℃; in step e of S2, the pH is preferably 7.5; in the step g in S2, the saturated NaOH solution may be any one of aqueous ammonia, sodium carbonate, and an organic alkaline solution.
5. The method for preparing pure adenosine diphosphate by using biological enzyme according to claim 1, wherein the commonly used anion exchange resin in step S4 comprises 201×7 type, AM207 type, XRK209 type and LKA20 type.
6. The method for preparing pure adenosine diphosphate by biological enzyme according to claim 1, wherein in step p of S5, the pH is preferably 5; the molecular weight of nanofiltration membrane filtration is preferably 170Da, and the temperature of the sample is preferably 15 ℃.
7. The method for preparing pure adenosine diphosphate by biological enzyme according to claim 1, wherein in step v in S8, the drying mode comprises one of vacuum oven drying and freeze-drying.
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