Method for synthesizing fludarabine phosphate through biocatalysis

文档序号：183887 发布日期：2021-11-02 浏览：54次中文

阅读说明：本技术 一种生物催化合成磷酸氟达拉滨的方法 (Method for synthesizing fludarabine phosphate through biocatalysis ) 是由张坤晓毛联岗侯学雯王媛王睿君杨玉梅于 2021-08-04 设计创作，主要内容包括：一种酶法制备磷酸氟达拉滨的方法,以氟达拉滨为原料,在无机盐和辅酶存在的条件下,通过酶催化反应制备磷酸氟达拉滨;其中,酶催化反应所用的酶选自磷酸转移酶、醛酮还原酶、醇氧化酶或醇脱氢酶中的至少一种;酶催化反应的pH为7.3-7.6,反应温度为37℃,反应1-4 h；辅酶选用ATP。本发明通过采用一种新的酶法工艺制备磷酸氟达拉滨,解决了一些现有化学工艺中存在的技术问题,较现有的酶法制备的工艺也有所改善；降低了生产成本；工艺环保,反应条件温和。(A method for preparing fludarabine phosphate by an enzyme method comprises the steps of taking fludarabine as a raw material, and preparing the fludarabine phosphate by an enzyme catalysis reaction in the presence of inorganic salt and coenzyme, wherein the enzyme used in the enzyme catalysis reaction is at least one of phosphotransferase, aldone reductase, alcohol oxidase or alcohol dehydrogenase, the pH value of the enzyme catalysis reaction is 7.3-7.6, the reaction temperature is 37 ℃, and the reaction is carried out for 1-4 hours; the coenzyme is ATP. The invention adopts a new enzyme method process to prepare the fludarabine phosphate, solves the technical problems existing in the existing chemical processes, and is also improved compared with the existing enzyme method preparation process; the production cost is reduced; the process is environment-friendly, and the reaction conditions are mild.)

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

1) selecting enzyme; at least one of phosphotransferase, aldo-keto reductase, carbonyl reductase, alcohol dehydrogenase and alcohol oxidase is selected as the catalytic enzyme.

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

3) The fludarabine phosphate is prepared by taking fludarabine as a raw material and adding the purified enzyme solution obtained in the step 2) and coenzyme into an inorganic salt solution environment.

2. The method of claim 1, wherein the nucleotide sequences of the phosphotransferase, the aldoketoreductase, the carbonyl reductase, the alcohol dehydrogenase, and the alcohol oxidase are set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5.

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

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

5. The method of claim 1, wherein the coenzyme is ATP.

6. The method of claim 4, wherein the amount of ATP used is 1-5 times the mass of the starting material.

7. The method of claim 1, wherein the reaction conditions in step 3) are pH 7.3-7.6, temperature 37 ℃ and time 1-4 h.

Technical Field

The invention relates to the technical field of compound synthesis, and particularly relates 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 C₁₀H₁₃FN₅O₇P, molecular weight: 365.2117, its structural formula is

Is an antimetabolic fluorinated purine nucleoside analogue, first marketed in the United states in 1991, and is a chemotherapeutic agent for treating leukemia and lymphoma (including chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute myelocytic leukemia and acute lymphocytic leukemia), administered by intravenous or oral injection. The product can be used for removing phosphate to generate metabolite 9-beta-fluoroadenosine (F-Ara-A), and then is phosphorylated to obtain F-Ara-ATP with antitumor activity. The anti-tumor mechanism of the product relates to incorporation of a DNA or RNA chain of a tumor cell to stop the extension of the chain, 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 diseases. It can be used in combination with cytarabine and mitoxantrone to enhance the killing effect on tumor cells.

The preparation of fludarabine phosphate can be found in many patent documents, all of which are prepared using fludarabine as starting material. Most of the existing methods for preparing fludarabine phosphate by patents and industrial production are chemical methods, but the chemical methods usually have more steps, complex operation and harsh reaction conditions, and the generated by-products are difficult to separate and have higher cost.

For example, U.S. Pat. No. 5,973,973 uses trimethyl phosphate and phosphorus oxychloride at 0 ℃ and the work-up is carried out by adding water and dichloromethane, allowing to stand under stirring until the two phases separate, removing the dichloromethane by decantation to give a pale yellow gummy residue which is dissolved in hot water at 50 ℃ and left to precipitate. The crude product obtained is characterized by a decomposition point (200 ℃ to 250 ℃), purification by thin-layer chromatography, recovery of the secondary product by means of a resin and recrystallization of the solid obtained with water.

The disadvantages of this process are the complicated operation, the low yield and the difficulty of the process instrumentation used in the industrial large scale implementation.

For example, CN200480030273.5 adopts triethyl phosphate and fludarabine to be put into a reactor at the temperature of-15/-20 ℃, phosphorus oxychloride is added dropwise in about one hour, the mixture reacts for 48 hours at the temperature of-10/-15 ℃, cold toluene is added to precipitate the product to obtain a crude product, and the crude product is purified by resin and recrystallized.

The disadvantage of this process is that the reaction conditions are severe; the operation is complex; phosphorus oxychloride and toluene are used, so that the method has high risk and toxicity and is not beneficial to industrial production.

Preparing fludarabine phosphate by an enzyme method in CN201680078610.0, adding ammonium acetate, MgCl 2.6H2O, lithium acetylphosphate salt, ATP disodium salt and fludarabine into 100ml tap water in a four-neck flask, uniformly stirring, adjusting the pH to 8.0 by NaOH, heating to 40 ℃, adding a wet thallus solution containing enzyme into a reaction system, keeping the temperature at 37-40 ℃, controlling the pH at 7.5-8.5 by NaOH in the reaction process, and completing the reaction for 5 hours.

Compared with a chemical synthesis method, the method is improved in the aspects of safety, operation and yield; but still has the disadvantages that the materials added in the catalytic reaction system are excessive and need to be operated step by step, and the reaction time is longer; and the preparation of the enzyme-containing wet thallus is complex and the cost is higher.

Disclosure of Invention

The invention aims to solve the technical problems and series of defects of the chemical synthesis process, provides an enzymatic catalytic synthesis process of fludarabine, has the advantages of simple operation, mild reaction conditions, environmental protection and high efficiency, and meets the synthesis requirement of the fludarabine phosphate.

In order to solve the technical problems in the synthesis of fludarabine phosphate, the technical scheme adopted by the invention specifically comprises the following steps:

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

Secondly, expressing and extracting enzyme, separating and purifying; expressing the selected enzyme in escherichia coli after the enzyme is successfully constructed, cracking the bacteria to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min to obtain a supernatant containing the enzyme, and reacting the supernatant serving as a crude enzyme solution; or purifying enzyme contained in the supernatant, eluting the supernatant with imidazole solutions with different gradients after the supernatant passes through a Ni column, then enabling the obtained Ni column eluent with the highest enzyme content to pass through a Q column, then eluting with salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution to obtain the purified enzyme solution.

Thirdly, the fludarabine phosphate is catalytically synthesized; the fludarabine is taken as a raw material, MgSO is added into a phosphate buffer solution₄And the coenzyme ATP and the enzyme solution obtained in the previous step react for 1 to 4 hours under the conditions of pH 7.3 to 7.6 and 37 ℃ to obtain the fludarabine phosphate.

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

the preparation method of the invention prepares the fludarabine phosphate by directly phosphorylating the fludarabine in the presence of coenzyme ATP through an enzyme catalysis reaction. In the preparation method, the fludarabine phosphate product can be obtained at a high conversion rate through enzyme catalysis reaction. Compared with many 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 the enzymatic method of the present invention in the process of preparing fludarabine phosphate.

Detailed Description

The technical solutions of the present invention will be described in further detail below, and the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of the present invention.

Example 1

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, then ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and 200r for 1h, then the tube is coated on an ampicillin-resistant solid LB plate and cultivated at 37 ℃, after bacteria grow out, a single bacterial colony is picked up by using high-pressure sterilized toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and drawing lines on the plate are marked correspondingly, then the toothpick is placed into a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, performing heat preservation at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 1); after the enzyme is expressed in escherichia coli, crushing the bacteria by a high-pressure cell crusher to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution; to a 100mL glass reaction flask, 100. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l) and ddH were added₂O the reaction was made up to 10ml and 1ml (14mg) of crude enzyme solution (SEQ ID NO:1) was added last. Control ofThe temperature of the reaction solution is 37 ℃, the pH value of the reaction solution is controlled between 7.3 and 7.6, and the reaction is carried out for 3 hours after uniform stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering, preparing mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample feeding amount: 10 mu l, detecting with 210nm wavelength, and obtaining conversion rate > 60%.

Example 2

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, performing heat preservation at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 2); after the enzyme is expressed in escherichia coli, crushing the bacteria by a high-pressure cell crusher to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution; to a 100mL glass reaction flask, 100. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l) and ddH were added₂O the reaction was made up to 10ml and 1ml (14mg) of crude enzyme solution (SEQ ID NO:2) was added last. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

Example 3

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, performing heat preservation at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 3); after the enzyme is expressed in escherichia coli, crushing the bacteria by a high-pressure cell crusher to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution; to a 100mL glass reaction flask, 100. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l) and ddH were added₂O the reaction was made up to 10ml and 1ml (14mg) of crude enzyme solution (SEQ ID NO:3) was added last. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

Example 4

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, performing heat preservation at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 4); after the enzyme is expressed in escherichia coli, crushing the bacteria by a high-pressure cell crusher to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution; to a 100mL glass reaction flask, 100. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l) and ddH were added₂O the reaction was made up to 10ml and 1ml (14mg) of crude enzyme solution (SEQ ID NO:4) was added last. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

Example 5

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, performing heat preservation at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 5); after the enzyme is expressed in escherichia coli, crushing the bacteria by a high-pressure cell crusher to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, and extracting supernatant to obtain a crude enzyme solution; to a 100mL glass reaction flask, 100. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l) and ddH were added₂O the reaction was made up to 10ml and 1ml (14mg) of crude enzyme solution (SEQ ID NO:5) was added last. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

Example 6

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 1); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 100. mu.L of phosphate buffer (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 10mL, and finally 5mL (7mg) of purified enzyme solution (SEQ ID NO:1) was added. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 70%.

Example 7

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 2); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 100. mu.L of phosphate buffer (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 10mL, and finally 5mL (7mg) of purified enzyme solution (SEQ ID NO:2) was added. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 98.7%.

Example 8

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 3); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 100. mu.L of phosphate buffer (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 10mL, and finally 5mL (7mg) of purified enzyme solution (SEQ ID NO:3) was added. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 98.8%.

Example 9

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 4); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 100. mu.L of phosphate buffer (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 10mL, and finally 5mL (7mg) of purified enzyme solution (SEQ ID NO:4) was added. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 99.9%.

Example 10

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: denaturing at 95 ℃ for 15min, annealing at 94 ℃ for 15s, annealing at 55 ℃ for 15s, extending at 72 ℃ for 1min, performing 30 cycles, finally preserving the temperature at 72 ℃ for 5min, performing PCR amplification, and performing electrophoresis observation to obtain a positive clone to obtain an escherichia coli strain containing a target enzyme sequence (SEQ ID NO: 5); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 100. mu.L of phosphate buffer (pH 7.3, 2mol/l), 400. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of anhydrous magnesium sulfate solution (1mol/l), 3mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 10mL, and finally 5mL (7mg) of purified enzyme solution (SEQ ID NO:5) was added. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 99.9%.

Example 11

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 1); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 33. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 67. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 1mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 3.3mL, and 1.6mL (3.5mg) of a purified enzyme solution (SEQ ID NO:1) was added in this order. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 60%.

Example 12

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 2); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 33. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 67. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 1mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 3.3mL, and 1.6mL (3.5mg) of a purified enzyme solution (SEQ ID NO:2) was added in this order. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 95%.

Example 13

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 3); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 33. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 67. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 1mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 3.3mL, and 1.6mL (3.5mg) of a purified enzyme solution (SEQ ID NO:3) was added in this order. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 95%.

Example 14

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: performing denaturation at 95 deg.C for 15min, denaturation at 94 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 1min, performing 30 cycles, maintaining at 72 deg.C for 5min, performing PCR amplification, and performing electrophoresis observation to obtain positive clone to obtain Escherichia coli strain containing target enzyme sequence (SEQ ID NO: 4); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 33. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 67. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 1mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 3.3mL, and 1.6mL (3.5mg) of a purified enzyme solution (SEQ ID NO:4) was added in this order. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 98%.

Example 15

The nucleotide sequence shown in a sequence table is selected to be connected with a vector pBAD-D, 20 mul of connection product is taken to be added into 200 mul of escherichia coli TOP10 competent cells of ice bath, then ice bath is carried out for 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 5min, 300 mul of 37 ℃ nonreactive culture solution is added into a tube, shaking table is carried out at 37 ℃ and is repaired for 1h, the tube is coated on an ampicillin-resistant solid LB plate at 37 ℃ for cultivation, after bacteria grow out, a single bacterial colony is picked up by using an autoclaved toothpick, drawing lines on the benzyl-resistant plate for conservation, corresponding bacteria and a drawing line area on the plate are marked correspondingly, then the toothpick is placed in a 20 mul PCR Mix system added with primers and stirred for PCR amplification, and the PCR reaction conditions are as follows: denaturing at 95 ℃ for 15min, annealing at 94 ℃ for 15s, annealing at 55 ℃ for 15s, extending at 72 ℃ for 1min, performing 30 cycles, finally preserving the temperature at 72 ℃ for 5min, performing PCR amplification, and performing electrophoresis observation to obtain a positive clone to obtain an escherichia coli strain containing a target enzyme sequence (SEQ ID NO: 5); after the enzyme is expressed in escherichia coli, breaking the bacteria by a high-pressure cell breaker to obtain an enzyme-containing lysate, centrifuging at 35000g/min for 30min, 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, then eluting by salt solutions (the main component is KCL) with different gradients to obtain a primarily purified enzyme-containing solution, and dialyzing the primarily purified enzyme-containing solution for 12h to obtain a purified enzyme solution; to a 100mL glass reaction flask, 33. mu.L of a phosphate buffer solution (pH 7.3, 2mol/l), 67. mu.L of ATP sodium salt (0.1mol/l), 40. mu.L of an anhydrous magnesium sulfate solution (1mol/l), 1mL of fludarabine (0.01mol/l), ddH2O was added to make up the reaction system to 3.3mL, and 1.6mL (3.5mg) of a purified enzyme solution (SEQ ID NO:5) was added in this order. Controlling the temperature of the reaction solution to be 37 ℃, controlling the pH value of the reaction solution to be 7.3-7.6, and reacting for 3 hours after uniformly stirring.

After completion of the above reaction, the solution after the reaction was centrifuged and passed through a 0.22 μm filter. Taking 10 mul of supernatant, using high performance liquid chromatography (liquid phase condition: chromatographic column: C18 (4.6X 250mm, 5 mu m), taking 3.4g of potassium dihydrogen phosphate, adding water to dissolve and dilute to 1000ml, using 10% phosphoric acid to adjust pH to 3.0, filtering to prepare a mobile phase of potassium dihydrogen phosphate and methanol (94: 6), flow rate: 0.5ml/min, column temperature: 25 ℃, sample injection amount: 10 mu l, detection wavelength: 210nm, and detecting that the conversion rate can reach 98%.

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, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> university of oceanic Jiangsu

JARI PHARMACEUTICAL Co.,Ltd.

<120> method for synthesizing fludarabine phosphate by biocatalysis

<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