CN113584104B

CN113584104B - Method for synthesizing fludarabine phosphate by biocatalysis

Info

Publication number: CN113584104B
Application number: CN202110889369.2A
Authority: CN
Inventors: 张坤晓; 毛联岗; 侯学雯; 王媛; 王睿君; 杨玉梅
Original assignee: Jari Pharmaceutical Co ltd; Jiangsu Ocean University
Current assignee: Jari Pharmaceutical Co ltd; Jiangsu Ocean University
Priority date: 2021-08-04
Filing date: 2021-08-04
Publication date: 2023-11-17
Anticipated expiration: 2041-08-04
Also published as: CN113584104A

Abstract

The method for preparing fludarabine phosphate by using the enzyme method comprises the steps of preparing fludarabine phosphate by using fludarabine as a raw material through an enzyme catalytic reaction in the presence of inorganic salt and coenzyme, wherein the enzyme used in the enzyme catalytic reaction is at least one selected from phosphotransferase, aldehyde ketone reductase, alcohol oxidase or alcohol dehydrogenase, the pH of the enzyme catalytic reaction is 7.3-7.6, the reaction temperature is 37 ℃, and the reaction is 1-4 h; ATP is selected as coenzyme. The invention solves the technical problems existing in some existing chemical processes by adopting a new enzymatic process for preparing fludarabine phosphate, and improves the existing enzymatic process; the production cost is reduced; the process is environment-friendly, and the reaction condition is mild.

Description

Method for synthesizing fludarabine phosphate by biocatalysis

Technical Field

The invention relates to the technical field of compound synthesis, in particular to a preparation method of fludarabine phosphate.

Technical Field

Fludarabine phosphate (9-beta-D-arabinofuranosyl-2-fluoroadenine-5' -phosphate), english name; fludarabine phosphate, molecular formula: C ₁₀ H ₁₃ FN ₅ O ₇ P, molecular weight: 365.2117, its structural formula is

Is an antimetabolite fluorinated purine nucleoside analogue, which was first marketed in the united states in 1991, and is a chemotherapeutic agent for the treatment of leukemias and lymphomas (including chronic lymphocytic leukemia, non-hodgkin's lymphoma, acute myelogenous leukemia and acute lymphocytic leukemia) administered by intravenous injection or oral injection. After the product enters a body, the metabolite 9-beta-fluoroarabinoadenosine (F-Ara-A) is generated by dephosphorylation, and then the metabolite is phosphorylated into F-Ara-ATP with anti-tumor activity. The anti-tumor mechanism of the product involves joining a tumor cell DNA or RNA chain to stop the chain from extending so as to inhibit the synthesis of the DNA or RNA, thereby achieving the purpose of promoting the apoptosis of the tumor cell to improve the remission rate of the disease. It can be used in combination with cytarabine, mitoxantrone, etc. to enhance the killing effect on tumor cells.

The preparation of fludarabine phosphate can be found in a number of patent documents, all of which are prepared using fludarabine as starting material. Most of the existing methods for preparing fludarabine phosphate in patent and industrial production are chemical preparation methods, and the preparation methods of the chemical methods are generally more in steps, relatively complex in operation, harsh in reaction conditions, difficult to separate generated byproducts and high in cost.

For example, US5110919 uses a process in which trimethyl phosphate and phosphorus oxychloride are reacted at 0 ℃ by adding water and methylene chloride, standing with stirring until the two phases separate, removing the methylene chloride by decantation to give a pale yellow gummy residue which is dissolved in hot water at 50 ℃ to leave it to precipitate. The crude product obtained is characterized by a decomposition point (200 ℃ C. -250 ℃ C.), purified by thin layer chromatography, the secondary product is recovered by means of a resin, and the solid obtained is recrystallized from water.

The disadvantages of this process are the complex operation, low yields and the difficulty of implementation of the process instrumentation used in an industrially large scale.

For example, CN200480030273.5 is prepared by placing triethyl phosphate and fludarabine in a reactor at-15/-20deg.C, dropping phosphorus oxychloride in the reactor for about one hour, reacting at-10/-15deg.C for 48 hours, adding cold toluene to precipitate the product to obtain crude product, purifying with resin, and recrystallizing.

The disadvantage of this process is that the reaction conditions are severe; the operation is complex; the use of phosphorus oxychloride and toluene has great danger and toxicity, and is unfavorable for industrial production.

In CN201680078610.0, fludarabine phosphate is prepared by adopting an enzyme method, in a four-mouth flask, 100ml of tap water is added with ammonium acetate, mgCl2.6H2O, acetyl lithium phosphate, ATP disodium salt and fludarabine, the mixture is stirred uniformly, the pH is regulated to 8.0 by NaOH, the temperature is raised to 40 ℃, then an enzyme-containing wet thallus solution is added into a reaction system, the temperature is kept at 37-40 ℃, and the pH is controlled to 7.5-8.5 by NaOH in the reaction process, so that the reaction is completed.

Compared with the chemical synthesis method, the method has the advantages of safety, operation and yield improvement; but still has the defects that materials added into a catalytic reaction system are excessive and are required to be operated step by step, and the reaction time is long; and the preparation of the wet thalli containing the enzyme is complex and the cost is high.

Disclosure of Invention

The invention aims to solve the technical problems and series of defects of the chemical synthesis process, and provides an enzymatic catalytic synthesis process of fludarabine, which is simple to operate, mild in reaction condition, environment-friendly and efficient, and meets the synthesis requirement of fludarabine phosphate.

The invention aims to solve the technical problems existing in the synthesis of fludarabine phosphate, and adopts the technical scheme that the method specifically comprises the following steps:

firstly, selecting raw materials and enzymes; selecting fludarabine as a raw material, and selecting at least one of enzymes shown in a sequence table as catalytic enzymes.

Secondly, extracting, separating and purifying the expression of the enzyme; after the selected enzyme is successfully constructed, the enzyme is expressed in escherichia coli, bacteria are cracked to obtain enzyme-containing lysate, and 35000g/min is centrifuged for 30min to obtain supernatant containing the enzyme, and the supernatant can be used as crude enzyme solution for reaction; the enzyme contained in the supernatant can be purified, the supernatant flows through a Ni column and then is eluted by imidazole solutions with different gradients, then the obtained Ni column eluent with the highest enzyme content flows through a Q column, then the elution is carried out by salt solutions (the main component is KCL) with different gradients, so as to obtain a primarily purified enzyme-containing solution, and the primarily purified enzyme-containing solution is dialyzed, so that the purified enzyme solution is obtained.

Thirdly, catalyzing and synthesizing fludarabine phosphate; using fludarabine as raw material, adding MgSO into phosphate buffer solution ₄ And (3) carrying out reaction on the coenzyme ATP and the enzyme solution obtained in the last step for 1-4 hours at the pH of 7.3-7.6 and the temperature of 37 ℃ to obtain the fludarabine phosphate.

The reaction formula of the enzyme catalysis reaction is as follows:

the preparation method of the invention is to directly phosphorylate fludarabine in the presence of coenzyme ATP by enzyme catalytic reaction to prepare fludarabine phosphate. In the preparation method of the invention, the fludarabine phosphate product can be obtained with high conversion rate through enzyme catalysis reaction. Compared with a plurality of existing chemical methods, the preparation method provided by the invention is simple to operate, mild in condition, high in yield, good in selectivity and low in cost.

Drawings

FIG. 1 is a high performance liquid chromatogram of fludarabine.

FIG. 2 is a high performance liquid chromatogram of fludarabine phosphate.

FIG. 3 is a high performance liquid chromatogram of a purified enzyme solution

FIG. 4 is a high performance liquid chromatogram of fludarabine phosphate prepared by the enzyme-catalyzed method of the invention.

Detailed Description

The following description of the present invention provides further details of the embodiments described herein, which are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.

Example 1

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, then coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, growing bacterial colonies, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on an ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line regions on the plate as corresponding marks, and then stirring in a 20 mu l PCR Mix system added with primers for PCR amplification, wherein the PCR reaction conditions are as follows: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 1); after the enzyme is expressed in the escherichia coli, the bacteria are crushed by a high-pressure cell crusher to obtain enzyme-containing lysate, and the supernatant is extracted after centrifugation for 30min at 35000g/min to obtain crude enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) and ddH were added ₂ O the reaction system was made up to 10ml, and finally 1ml (14 mg) of crude enzyme solution (SEQ ID NO: 1) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant, carrying out high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), dissolving 3.4g of potassium dihydrogen phosphate in water, diluting to 1000ml, regulating pH to 3.0 with 10% phosphoric acid, carrying out suction filtration to prepare a mobile phase of potassium dihydrogen phosphate: methanol=94:6, wherein the flow rate: 0.5ml/min, the column temperature: 25 ℃, the sample injection amount: 10 mu l, and the wavelength of 210nm for detection, wherein the detection conversion rate is more than 60%.

Example 2

Connecting the nucleotide sequence shown in the sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in ice bath into 20 mu l of the connecting product, then carrying out ice bath for 30min and heat shock for 60s at 42 ℃, then carrying out ice bath for 5min, adding 300 mu l of a 37 ℃ nonresistant culture solution into the tube, repairing for 1h by a 200r shaking table at 37 ℃, coating the tube on an ampicillin-resistant solid LB plate, culturing at 37 ℃, growing bacterial colonies, picking single bacterial colonies by using autoclaved toothpicks after bacterial colonies, firstly drawing lines on an ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line regions on the plate as corresponding marks, and then stirring in a 20 mu l PCR Mix system added with primers for PCR amplification, wherein the PCR reaction conditions are as follows: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 2); after the enzyme is expressed in the escherichia coli, the bacteria are crushed by a high-pressure cell crusher to obtain enzyme-containing lysate, and the supernatant is extracted after centrifugation for 30min at 35000g/min to obtain crude enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/L), 400. Mu.L of ATP sodium salt (0.1 mol/L), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/L), 3mL of fludarabine (0.01 mol/L) and ddH were added ₂ O the reaction system was made up to 10ml, and finally 1ml (14 mg) of crude enzyme solution (SEQ ID NO: 2) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant, carrying out high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), dissolving 3.4g of potassium dihydrogen phosphate in water, diluting to 1000ml, regulating pH to 3.0 with 10% phosphoric acid, carrying out suction filtration to prepare a mobile phase of potassium dihydrogen phosphate: methanol=94:6, wherein the flow rate: 0.5ml/min, the column temperature: 25 ℃, the sample injection amount: 10 mu l, and the wavelength of 210nm for detection, wherein the conversion rate is more than 80%.

Example 3

Connecting the nucleotide sequence shown in the sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in ice bath into 20 mu l of the connecting product, then carrying out ice bath for 30min and heat shock for 60s at 42 ℃, then carrying out ice bath for 5min, adding 300 mu l of a 37 ℃ nonresistant culture solution into the tube, repairing for 1h by a 200r shaking table at 37 ℃, coating the tube on an ampicillin-resistant solid LB plate, culturing at 37 ℃, growing bacterial colonies, picking single bacterial colonies by using autoclaved toothpicks after bacterial colonies, firstly drawing lines on an ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line regions on the plate as corresponding marks, and then stirring in a 20 mu l PCR Mix system added with primers for PCR amplification, wherein the PCR reaction conditions are as follows: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 3); after the enzyme is expressed in the escherichia coli, the bacteria are crushed by a high-pressure cell crusher to obtain enzyme-containing lysate, and the supernatant is extracted after centrifugation for 30min at 35000g/min to obtain crude enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/L), 400. Mu.L of ATP sodium salt (0.1 mol/L), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/L), 3mL of fludarabine (0.01 mol/L) and ddH were added ₂ O the reaction system was made up to 10ml, and finally 1ml (14 mg) of crude enzyme solution (SEQ ID NO: 3) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant, carrying out high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), dissolving 3.4g of potassium dihydrogen phosphate in water, diluting to 1000ml, regulating pH to 3.0 with 10% phosphoric acid, carrying out suction filtration to prepare a mobile phase of potassium dihydrogen phosphate: methanol=94:6, wherein the flow rate: 0.5ml/min, the column temperature: 25 ℃, the sample injection amount: 10 mu l, and the wavelength of 210nm for detection, wherein the detection conversion rate is more than 90%.

Example 4

Connecting the nucleotide sequence shown in the sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in ice bath into 20 mu l of the connecting product, then carrying out ice bath for 30min and heat shock for 60s at 42 ℃, then carrying out ice bath for 5min, adding 300 mu l of a 37 ℃ nonresistant culture solution into the tube, repairing for 1h by a 200r shaking table at 37 ℃, coating the tube on an ampicillin-resistant solid LB plate, culturing at 37 ℃, growing bacterial colonies, picking single bacterial colonies by using autoclaved toothpicks after bacterial colonies, firstly drawing lines on an ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line regions on the plate as corresponding marks, and then stirring in a 20 mu l PCR Mix system added with primers for PCR amplification, wherein the PCR reaction conditions are as follows: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 4); after the enzyme is expressed in the escherichia coli, the bacteria are crushed by a high-pressure cell crusher to obtain enzyme-containing lysate, and the supernatant is extracted after centrifugation for 30min at 35000g/min to obtain crude enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/L), 400. Mu.L of ATP sodium salt (0.1 mol/L), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/L), 3mL of fludarabine (0.01 mol/L) and ddH were added ₂ O the reaction system was made up to 10ml, and finally 1ml (14 mg) of crude enzyme solution (SEQ ID NO: 4) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

Example 5

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 5); after the enzyme is expressed in the escherichia coli, the bacteria are crushed by a high-pressure cell crusher to obtain enzyme-containing lysate, and the supernatant is extracted after centrifugation for 30min at 35000g/min to obtain crude enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/L), 400. Mu.L of ATP sodium salt (0.1 mol/L), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/L), 3mL of fludarabine (0.01 mol/L) and ddH were added ₂ O the reaction system was made up to 10ml, and finally 1ml (14 mg) of crude enzyme solution (SEQ ID NO: 5) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

Example 6

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 1); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) were sequentially added, the reaction system was made up to 10mL by adding ddH2O, and finally 5mL (7 mg) of purified enzyme solution (SEQ ID NO: 1) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g of monopotassium phosphate, adding water for dissolving and diluting to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, carrying out suction filtration to prepare mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detection conversion rate reaching 70%.

Example 7

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 2); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) were sequentially added, the reaction system was made up to 10mL by adding ddH2O, and finally 5mL (7 mg) of purified enzyme solution (SEQ ID NO: 2) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g monopotassium phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, suction filtering, preparing into mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, detection conversion rate up to 98.7%.

Example 8

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 3); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) were sequentially added, the reaction system was made up to 10mL by adding ddH2O, and finally 5mL (7 mg) of purified enzyme solution (SEQ ID NO: 3) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g monopotassium phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, suction filtering, preparing into mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, detection conversion rate up to 98.8%.

Example 9

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 4); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) were sequentially added, the reaction system was made up to 10mL by adding ddH2O, and finally 5mL (7 mg) of purified enzyme solution (SEQ ID NO: 4) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g monopotassium phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, suction filtering, preparing into mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, detection conversion rate up to 99.9%.

Example 10

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 5); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; into a 100mL glass reaction flask, 100. Mu.L of phosphate buffer (pH=7.3, 2 mol/l), 400. Mu.L of ATP sodium salt (0.1 mol/l), 40. Mu.L of anhydrous magnesium sulfate solution (1 mol/l), 3mL of fludarabine (0.01 mol/l) were sequentially added, the reaction system was made up to 10mL by adding ddH2O, and finally 5mL (7 mg) of purified enzyme solution (SEQ ID NO: 5) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

Example 11

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 1); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; in a 100mL glass reaction flask, 33. Mu.l of phosphate buffer (pH=7.3, 2 mol/l), 67. Mu.l of ATP sodium salt (0.1 mol/l), 40. Mu.l of anhydrous magnesium sulfate solution (1 mol/l), 1mL of fludarabine (0.01 mol/l), ddH2O was added to make up the reaction system to 3.3mL, and finally 1.6mL (3.5 mg) of purified enzyme solution (SEQ ID NO: 1) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g of monopotassium phosphate, adding water for dissolving and diluting to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, carrying out suction filtration to prepare mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detection conversion rate reaching 60%.

Example 12

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 2); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; in a 100mL glass reaction flask, 33. Mu.l of phosphate buffer (pH=7.3, 2 mol/l), 67. Mu.l of ATP sodium salt (0.1 mol/l), 40. Mu.l of anhydrous magnesium sulfate solution (1 mol/l), 1mL of fludarabine (0.01 mol/l), ddH2O was added to make up the reaction system to 3.3mL, and finally 1.6mL (3.5 mg) of purified enzyme solution (SEQ ID NO: 2) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g of monopotassium phosphate, adding water for dissolving and diluting to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, carrying out suction filtration to prepare mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detection conversion rate up to 95%.

Example 13

Connecting the nucleotide sequence shown in the sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in ice bath into 20 mu l of the connecting product, then carrying out ice bath for 30min and heat shock for 60s at 42 ℃, then carrying out ice bath for 5min, adding 300 mu l of a 37 ℃ nonresistant culture solution into the tube, repairing for 1h by a 200r shaking table at 37 ℃, coating the tube on an ampicillin-resistant solid LB plate, culturing at 37 ℃, growing bacterial colonies, picking single bacterial colonies by using autoclaved toothpicks after bacterial colonies, firstly drawing lines on an ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line regions on the plate as corresponding marks, and then stirring in a 20 mu l PCR Mix system added with primers for PCR amplification, wherein the PCR reaction conditions are as follows: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 3); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; in a 100mL glass reaction flask, 33. Mu.l of phosphate buffer (pH=7.3, 2 mol/l), 67. Mu.l of ATP sodium salt (0.1 mol/l), 40. Mu.l of anhydrous magnesium sulfate solution (1 mol/l), 1mL of fludarabine (0.01 mol/l), ddH2O was added to make up the reaction system to 3.3mL, and finally 1.6mL (3.5 mg) of purified enzyme solution (SEQ ID NO: 3) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

Example 14

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 4); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; in a 100mL glass reaction flask, 33. Mu.l of phosphate buffer (pH=7.3, 2 mol/l), 67. Mu.l of ATP sodium salt (0.1 mol/l), 40. Mu.l of anhydrous magnesium sulfate solution (1 mol/l), 1mL of fludarabine (0.01 mol/l), ddH2O was added to make up the reaction system to 3.3mL, and finally 1.6mL (3.5 mg) of purified enzyme solution (SEQ ID NO: 4) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

After completion of the above reaction, the reacted solution was centrifuged and filtered through a 0.22 μm filter. Taking 10 mu l of supernatant liquid, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X250 mm,5 mu m), taking 3.4g of monopotassium phosphate, adding water for dissolving and diluting to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, carrying out suction filtration to prepare mobile phase of monopotassium phosphate: methanol=94:6, flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detection conversion rate up to 98%.

Example 15

Connecting a nucleotide sequence shown in a sequence table with a vector pBAD-D, adding 200 mu l of escherichia coli TOP10 competent cells in an ice bath into 20 mu l of a connecting product, then carrying out ice bath 30min, carrying out heat shock at 42 ℃ for 60s, carrying out ice bath 5min, adding 300 mu l of a non-antibiotic culture solution at 37 ℃ into a tube, repairing for 1h by a 200r shaking table at 37 ℃, coating on an ampicillin-resistant solid LB plate, culturing at 37 ℃, after bacterial colonies grow out, picking single bacterial colonies by using autoclaved toothpicks, firstly drawing lines on the ampicillin-resistant plate for seed preservation, using the corresponding bacteria and the drawn line areas on the plate as corresponding marks, and then placing the toothpick into a 20 mu l PCR Mix system which is added into a primer for PCR amplification under the following conditions: denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 1min and extension at 72 ℃ for 30 cycles, heat preservation at 72 ℃ for 5min, and electrophoresis observation after PCR amplification to obtain positive clones, and obtaining the escherichia coli strain containing the target enzyme sequence (SEQ ID NO: 5); after expressing enzymes in escherichia coli, crushing the bacteria by a high-pressure cell disruption instrument to obtain enzyme-containing lysate, centrifuging for 30min at 35000g/min, extracting supernatant, enabling the supernatant to flow through a Ni column, eluting by imidazole solutions with different gradients, enabling the obtained Ni column eluent with the highest enzyme content to flow through a Q column, eluting by salt solutions (with KCL as a main component) with different gradients, obtaining a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; in a 100mL glass reaction flask, 33. Mu.l of phosphate buffer (pH=7.3, 2 mol/l), 67. Mu.l of ATP sodium salt (0.1 mol/l), 40. Mu.l of anhydrous magnesium sulfate solution (1 mol/l), 1mL of fludarabine (0.01 mol/l), ddH2O was added to make up the reaction system to 3.3mL, and finally 1.6mL (3.5 mg) of purified enzyme solution (SEQ ID NO: 5) was added. The temperature of the reaction solution is controlled to be 37 ℃, the pH value of the reaction solution is controlled to be 7.3-7.6, and the reaction is carried out for 3 hours after the uniform stirring.

In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> university of Jiangsu ocean

JARI PHARMACEUTICAL Co.,Ltd.

<120> method for biocatalytically synthesizing fludarabine phosphate

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 690

<212> DNA

<213> unknown

<400> 1

atgcatcatc atcatcacca tagcagcggc aacaactatg gcattccgca gaacgcgatt 60

attaccattg cgggcaccgt gggcgtgggc aaaagcaccc tgacccaggc gctggcggat 120

aaactgaact ttaaaaccag ctttgaaaac gtggaacata acccgtatct ggataaattt 180

tatagcgatt ttgaacgctg gagctttcat ctgcagattt attttctggc ggaacgcttt 240

aaagaacaga aacgcatgtt tgaatatggc ggcggctttg tgcaggatcg cagcatttat 300

gaagatgtgg atatttttgc gaaaatgcat gaagaagaag gcaccatgag caaagaagat 360

tttaaaacct atagcgatct gtttaacgcg atggtgatga ccccgtattt tccgaaaccg 420

gatgtgatga tttatctgga atgcaactat gatgaagtga ttgatcgcat tattgaacgc 480

ggccgcgaaa tggaaattaa caccgatccg gaatattgga aaaaactgtt taaacgctat 540

gatgattgga ttaacagctt taacgcgtgc ccggtggtgc gcattaacat taacgaatat 600

gatattcata aagatccgga tagcctgaac ccgatgattg ataaaattgc gcgcattatt 660

cagacctatc gccaggtgga tacccgctaa 690

<210> 2

<211> 780

<212> DNA

<213> unknown

<400> 2

atgcatcatc atcatcacca tagcagcggc accaccccga ttctgaacag cagcgtgccg 60

ggcaacaaca actatggcat tccgcagaac gcgattatta ccattgcggg caccgtgggc 120

gtgggcaaaa gcaccctgac ccaggcgctg gcggataaac tgaactttaa aaccagcttt 180

gaaaacgtgg aacataaccc gtatctggat aaattttata gcgattttga acgctggagc 240

tttcatctgc agatttattt tctggcggaa cgctttaaag aacagaaacg catgtttgaa 300

tatggcggcg gctttgtgca ggatcgcagc atttatgaag atgtggatat ttttgcgaaa 360

atgcatgaag aagaaggcac catgagcaaa gaagatttta aaacctatag cgatctgttt 420

aacgcgatgg tgatgacccc gtattttccg aaaccggatg tgatgattta tctggaatgc 480

aactatgatg aagtgattga tcgcattatt gaacgcggcc gcgaaatgga aattaacacc 540

gatccggaat attggaaaaa actgtttaaa cgctatgatg attggattaa cagctttaac 600

gcgtgcccgg tggtgcgcat taacattaac gaatatgata ttcataaaga tccggatagc 660

ctgaacccga tgattgataa aattgcgcgc attattcaga cctatcgcca ggtggatacc 720

cgcacctttg gcaacggccc gaccaccaac aaaattatta gcaccccgaa agatctgtaa 780

<210> 3

<211> 765

<212> DNA

<213> unknown

<400> 3

atgcatcatc atcatcacca tagcagcggc accaccccga ttctgaacag cagcgtgccg 60

ggcaacaaca actatggcat tccgcagaac gcgattatta ccattgcggg caccgtgggc 120

gtgggcaaaa gcaccctgac ccaggcgctg gcggataaac tgaactttaa aaccagcttt 180

gaaaacgtgg aacataaccc gtatctggat aaattttata gcgattttga acgctggagc 240

tttcatctgc agatttattt tctggcggaa cgctttaaag aacagaaacg catgtttgaa 300

tatggcggcg gctttgtgca ggatcgcagc atttatgaag atgatatttt tgcgaaaatg 360

catgaagaag aaggcaccat gagcaaagaa gattttaaaa cctatagcga tctgtttaac 420

gcgatgaccc cgtattttcc gaaaccggat gtgatgattt atctggaatg caactatgat 480

gaagtgattg atcgcattat tgaacgcggc cgcgaaatgg aaattaacac cgatccggaa 540

tattggaaaa aactgtttaa acgctatgat gattggatta acagctttaa cgcgtattgc 600

ccggtggtgc gcattaacat taacgaatat aaagatccgg atagcctgaa cccgatgatt 660

gataaaattg cgcgcattat tcagacctat cgccaggtgg atacccgcac ctttggcaac 720

ggcccgacca ccaacaaaat tattagcacc ccgaaagatc tgtaa 765

<210> 4

<211> 765

<212> DNA

<213> unknown

<400> 4

atgcatcatc atcatcacca tagcagcggc accaccccga ttctgaacag cagcgtgccg 60

ggcaacattc agaaaaaaag cctggaaggc acccatatta ccattgcggg caccgtgggc 120

gtgggcaaaa gcaccctgac ccaggcgctg gcggataaac tgaactttaa aaccagcttt 180

gaaaacgtgg aacataaccc gtatctggat aaattttata gcgattttga acgctggagc 240

tttcatctgc agatttattt tctggcggaa cgctttaaag aacagaaacg catgtttgaa 300

tatggcggcg gctttgtgca ggatcgcagc atttatgaag atgatatttt tgcgaaaatg 360

catgaagaag aaggcaccat gagcaaagaa gattttaaaa cctatagcga tctgtttaac 420

gcgatgaccc cgtattttcc gaaaccggat gtgatgattt atctggaatg caactatgat 480

gaagtgattg atcgcattat tgaacgcggc cgcgaaatgg aaattaacac cgatccggaa 540

tattggaaaa aactgtttaa acgctatgat gattggatta acagctttaa cgcgtattgc 600

ccggtggtgc gcattaacat taacgaatat aaagatccgg atagcctgaa cccgatgatt 660

aaagcggaat atgaaagcat gcgctttatg aaccagatta acccgccgac ctttggcaac 720

ggcccgacca ccaacaaaat tattagcacc ccgaaagatc tgtaa 765

<210> 5

<211> 765

<212> DNA

<213> unknown

<400> 5

atgcatcatc atcatcacca tagcagcggc accaccccga ttctgaacag cagcgtgccg 60

ggcaacattc agaaaaaaag cctggaaggc acccatatta ccattgcggg caccgtgggc 120

gtgggcaaaa gcaccctgac ccaggcgctg gcggataaac tgaactttaa aaccagcttt 180

gaaaacgtgg aacataaccc gtatctggat aaattttata gcgattttga acgctggagc 240

tttcatctgc agatttattt tctggcggaa cgctttaaag aacagcagca gattatttgg 300

caggcgcgcg gctttgtgca ggatcgcagc atttatgaag atgatatttt tgcgaaaatg 360

catgaagaag aaggcaccat gagcaaagaa gattttaaaa cctatagcga tctgtttcag 420

aacctgagca actttatgcg ccgcccggat gtgatgattt atctggatgt gagcccggaa 480

aaaagcctgg aacgcattat tgaacgcggc cgcgaaatgg aaattaacac cgatccggaa 540

tattggaaaa aactgtttaa acgctatcat gaatttctgc aggatattag ccgctattgc 600

ccggtggtgc gcattaacat taacgaatat aaagatccgg atagcctgaa cccgatgatt 660

aaagcggaat atgaaagcat gcgctttatg aaccagatta acccgccgac ctttggcaac 720

ggcccgacca ccaacaaaat tattagcacc ccgaaagatc tgtaa 765

Claims

1. The preparation method of fludarabine phosphate is characterized by comprising the following steps:

1) Selecting enzymes; selecting phosphotransferase as catalytic enzyme;

2) Constructing the catalytic enzyme selected in the step 1), expressing in escherichia coli, and then extracting, separating and purifying;

3) Adding the purified enzyme solution and the coenzyme obtained in the step 2) into an inorganic salt solution environment by taking fludarabine as a raw material to prepare fludarabine phosphate;

the nucleotide sequence of the phosphotransferase is shown as SEQ ID NO. 1;

the coenzyme is ATP.

2. The method of claim 1, wherein the inorganic salt solution environment in step 3) is phosphate buffered saline plus anhydrous magnesium sulfate and purified water.

3. The method of claim 2, wherein the inorganic salt solution comprises Na2HPO4, KH2PO4, naCl, KCl, and anhydrous MgSO4 as main components.

4. The method of claim 1, wherein the ATP is used in an amount of 1 to 5 times the mass of the feedstock.

5. The process of claim 1, wherein the reaction conditions in step 3) are pH 7.3 to 7.6, temperature 37 ℃ and time 1 to 4 hours.