CN110714043A - Method for preparing guanosine triphosphate by immobilized enzyme method - Google Patents
Method for preparing guanosine triphosphate by immobilized enzyme method Download PDFInfo
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- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 title claims abstract description 140
- 238000000034 method Methods 0.000 title claims abstract description 32
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 88
- 108090000790 Enzymes Proteins 0.000 claims abstract description 88
- 238000004519 manufacturing process Methods 0.000 claims abstract description 57
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 102100040468 Guanylate kinase Human genes 0.000 claims abstract description 17
- 101100453821 Homo sapiens GUK1 gene Proteins 0.000 claims abstract description 17
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 8
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 7
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 6
- 239000000047 product Substances 0.000 claims description 24
- 239000012295 chemical reaction liquid Substances 0.000 claims description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 108020004202 Guanylate Kinase Proteins 0.000 claims description 10
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 102000006638 guanylate kinase Human genes 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000013375 chromatographic separation Methods 0.000 claims description 9
- 238000002425 crystallisation Methods 0.000 claims description 9
- 230000008025 crystallization Effects 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 5
- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 108020000161 polyphosphate kinase Proteins 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims description 5
- 235000019982 sodium hexametaphosphate Nutrition 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims description 5
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 8
- 239000012535 impurity Substances 0.000 abstract description 3
- 239000002773 nucleotide Substances 0.000 abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 abstract description 3
- 239000000049 pigment Substances 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 239000008363 phosphate buffer Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 235000013928 guanylic acid Nutrition 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 239000004226 guanylic acid Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/305—Pyrimidine nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04001—Polyphosphate kinase (2.7.4.1)
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- General Chemical & Material Sciences (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a method for preparing guanosine triphosphate by an immobilized enzyme method, which comprises the following steps: (1) preparing GTP production enzyme, (2) immobilizing GTP production enzyme, and (3) separating a product; the method adopts two GTP production enzymes of PPK and GMPK, can synthesize GTP only by two-step enzymatic reaction, and has the advantages of simpler reaction process, easier control of the reaction and more stable product quality compared with the traditional process for producing GTP by using beer yeast; the GTP is prepared by adopting an immobilized enzyme catalysis method, and the immobilized enzyme can be continuously and repeatedly used for many times, so that the production cost is greatly reduced; meanwhile, pigment and other types of nucleotide and other impurities introduced by using yeast are avoided, and the purification is easier; the method is suitable for large-scale production of GTP.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing guanosine triphosphate by an immobilized enzyme method.
Background
The glycolysis pathway of yeast is utilized, guanylic acid is taken as a substrate, and the synthesis of GTP through matrix level phosphorylation is the most common method and is also the method generally adopted in the industrial production of Guanosine Triphosphate (GTP) at present. However, the reaction process of catalytically synthesizing GTP by a yeast cell enzyme system is complex, a plurality of enzyme systems participating in catalytic reaction are provided, the reaction process is not easy to control, and the quality difference among product batches is large. Meanwhile, the quality of yeast enzyme systems is greatly different due to different suppliers, different batches and even different seasons. The yeast bacterial enzyme system has unstable quality, fast enzyme activity reduction and short service life, and is generally used for one time. In the reaction process, a large amount of yeast cell enzyme liquid is required to be added, and a large amount of pigment and other impurities such as nucleotide existing in the yeast are introduced, so that great difficulty is brought to later purification. At present, the GTP production level in China is generally low, and the cost control, the product quality and the like of the product are also deficient. In view of the above technical problems, there is an urgent need to develop a novel and stable reaction process, simplify the reaction process and improve the product quality.
In addition, in practical application, the cost of the enzyme is high, the GTP is produced by using free enzyme, the enzyme activity is reduced rapidly, the GTP cannot be effectively recycled, the production cost is high, and the practical application value is influenced.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a method for preparing guanosine triphosphate by an immobilized enzyme method.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing guanosine triphosphate by an immobilized enzyme method specifically comprises the following steps:
(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;
(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;
(3) and (3) separating a product: directly separating the immobilized GTP production enzyme in a reaction tank, recovering the immobilized GTP production enzyme from the separated reaction liquid through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Preferably, said preparing a GTP-producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing GMPK, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the GTP production enzyme.
Preferably, the immobilized GTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with 10L GTP production enzyme, and stirring at 150rpm for 4-6h at room temperature; filtering and collecting the carrier, and washing for 2-4 times by using 0.1MpH7.0 phosphate buffer solution (containing 1mol/L sodium chloride) to obtain the immobilized GTP production enzyme.
Preferably, the isolated product: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 200-300g of GMP, 200g of sodium hexametaphosphate, 130g of magnesium chloride 110-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.4-0.6kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 4-6h, and detecting the generation amount of GTP by high performance liquid chromatography; recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
The invention has the following beneficial effects:
1. the invention adopts two GTP production enzymes of PPK and GMPK, can synthesize GTP only by two-step enzymatic reaction, and has simpler reaction process, easier control of the reaction and more stable product quality compared with the traditional process for producing GTP by beer yeast.
2. The GTP is prepared by adopting an immobilized enzyme catalysis method, and the immobilized enzyme can be continuously and repeatedly used for many times, so that the production cost is greatly reduced; meanwhile, pigment and other types of nucleotide and other impurities introduced by using yeast are avoided, and the purification is easier; the method is suitable for large-scale production of GTP.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
The first embodiment is as follows:
a method for preparing guanosine triphosphate by an immobilized enzyme method specifically comprises the following steps:
(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;
(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;
(3) and (3) separating a product: directly separating the immobilized GTP production enzyme in a reaction tank, recovering the immobilized GTP production enzyme from the separated reaction liquid through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Specifically, the preparation of the GTP-producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.0kg of thallus containing PPK and 1.0kg of thallus containing GMPK, mixing and suspending with 10L of 0.1M phosphate buffer solution with the pH value of 7.0, crushing the bacteria by a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the GTP producing enzyme.
Specifically, the immobilized GTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the GTP production enzyme 10L, and stirring at 150rpm for 4h at room temperature; the carrier was collected by filtration and washed 2 times with 0.1M phosphate buffer (containing 1mol/L sodium chloride) pH7.0 to obtain immobilized GTP-producing enzyme.
Specifically, the separation product: preparing 10L reaction liquid containing GMP200g, sodium hexametaphosphate 100g, magnesium chloride 110g, GTP 4g and water in a 20L reaction tank; adjusting the pH of the reaction solution to 7.0 by NaOH, adding 0.4kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 4 hours, and detecting the generation amount of GTP by high performance liquid chromatography; and recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining 200g of guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Example two:
a method for preparing guanosine triphosphate by an immobilized enzyme method specifically comprises the following steps:
(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;
(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;
(3) and (3) separating a product: directly separating the immobilized GTP production enzyme in a reaction tank, recovering the immobilized GTP production enzyme from the separated reaction liquid through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Preferably, said preparing a GTP-producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 2.0kg of thallus containing PPK and 2.0kg of thallus containing GMPK, mixing and suspending with 10L of 0.1M phosphate buffer solution with the pH value of 7.0, crushing the bacteria by a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the GTP producing enzyme.
Preferably, the immobilized GTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with 10L GTP production enzyme, and stirring at 150rpm for 6h at room temperature; the carrier was collected by filtration and washed 4 times with 0.1M phosphate buffer (containing 1mol/L sodium chloride) pH7.0 to obtain immobilized GTP-producing enzyme.
Preferably, the isolated product: preparing 10L reaction liquid containing GMP300g, sodium hexametaphosphate 200g, magnesium chloride 130g, GTP 6g and water in a 20L reaction tank; adjusting the pH of the reaction solution to 7.0 by NaOH, adding 0.6kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 6 hours, and detecting the generation amount of GTP by high performance liquid chromatography; and recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining 250g of guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Example three:
a method for preparing guanosine triphosphate by an immobilized enzyme method specifically comprises the following steps:
(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;
(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;
(3) and (3) separating a product: directly separating the immobilized GTP production enzyme in a reaction tank, recovering the immobilized GTP production enzyme from the separated reaction liquid through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
Preferably, said preparing a GTP-producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.5kg of thallus containing PPK and 1.5kg of thallus containing GMPK, mixing and suspending with 10L of 0.1M phosphate buffer solution with the pH value of 7.0, crushing the bacteria by a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the GTP producing enzyme.
Preferably, the immobilized GTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the GTP production enzyme 10L, and stirring at 150rpm for 5h at room temperature; the carrier was collected by filtration and washed 3 times with 0.1M phosphate buffer (containing 1mol/L sodium chloride) pH7.0 to obtain immobilized GTP-producing enzyme.
Preferably, the isolated product: preparing 10L reaction liquid containing GMP250g, sodium hexametaphosphate 150g, magnesium chloride 120g, GTP 5g and water in a 20L reaction tank; adjusting the pH of the reaction solution to 7.0 by NaOH, adding 0.5kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 5 hours, and detecting the generation amount of GTP by high performance liquid chromatography; the reaction solution after the reaction is filtered to recover immobilized GTP production enzyme, and the permeation solution is chromatographically separated, crystallized and dried to obtain 230g of Guanosine Triphosphate (GTP).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A method for preparing guanosine triphosphate by an immobilized enzyme method is characterized by comprising the following steps:
(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;
(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;
(3) and (3) separating a product: directly separating the immobilized GTP production enzyme in a reaction tank, recovering the immobilized GTP production enzyme from the separated reaction liquid through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
2. The method for preparing guanosine triphosphate according to claim 1, wherein said preparation of GTP producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing GMPK, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the GTP production enzyme.
3. The method for preparing guanosine triphosphate according to claim 1, wherein said immobilized GTP producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with 10L GTP production enzyme, and stirring at 150rpm for 4-6h at room temperature; filtering and collecting the carrier, and washing with 0.1M phosphate buffer solution (containing 1mol/L sodium chloride) with pH7.0 for 2-4 times to obtain the immobilized GTP production enzyme.
4. The method for preparing guanosine triphosphate according to claim 1, wherein said separation product is: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 200-300g of GMP, 200g of sodium hexametaphosphate, 130g of magnesium chloride 110-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.4-0.6kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 4-6h, and detecting the generation amount of GTP by high performance liquid chromatography; recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122593A (en) * | 2019-12-31 | 2021-07-16 | 安徽古特生物科技有限公司 | Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate by utilizing polyphosphate |
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| CN105647996A (en) * | 2016-03-22 | 2016-06-08 | 深圳市古特新生生物科技有限公司 | Method for preparing adenosine triphosphate with immobilized enzyme method |
| CN109196109A (en) * | 2016-04-06 | 2019-01-11 | 绿光生物科技股份有限公司 | Cell-free generation ribonucleic acid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647996A (en) * | 2016-03-22 | 2016-06-08 | 深圳市古特新生生物科技有限公司 | Method for preparing adenosine triphosphate with immobilized enzyme method |
| CN109196109A (en) * | 2016-04-06 | 2019-01-11 | 绿光生物科技股份有限公司 | Cell-free generation ribonucleic acid |
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| Title |
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| 由洋等: "利用固定化啤酒酵母合成5’-核苷三磷酸", 《工业微生物》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122593A (en) * | 2019-12-31 | 2021-07-16 | 安徽古特生物科技有限公司 | Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate by utilizing polyphosphate |
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