CN106929553B

CN106929553B - Method for synthesizing vidarabine by enzyme method

Info

Publication number: CN106929553B
Application number: CN201511028553.9A
Authority: CN
Inventors: 沈波; 魏民志
Original assignee: Shanghai Xinxin Biological Technology Co ltd
Current assignee: Shanghai Xinxin Biological Technology Co ltd
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2021-02-19
Anticipated expiration: 2035-12-31
Also published as: CN106929553A

Abstract

The invention relates to a method for synthesizing vidarabine by an enzyme method. The invention provides a method for preparing vidarabine by a two-step method, which fully utilizes different actions of uridine phosphorylase and purine nucleoside phosphorylase to enable the reaction to be carried out in one direction, eliminates the occurrence of reverse reaction and obviously improves the conversion rate of vidarabine.

Description

Method for synthesizing vidarabine by enzyme method

Technical Field

The invention belongs to the field of biochemical engineering, and particularly relates to a method for synthesizing vidarabine by an enzymatic method.

Background

Adenosine Arabinoside (araA for short) is a natural antiviral compound, has broad-spectrum antiviral activity, and has been widely used for treating diseases caused by cytomegalovirus, hepatitis b virus and herpes virus in clinic.

In the prior art, the synthesis method of vidarabine comprises chemical synthesis and biotransformation synthesis. The chemical method has complex process, harsh reaction conditions, high raw material cost and heavy metal reagent usage, thus polluting the environment. The basic principle of the biotransformation process is that arabinoside uridine and phosphate ions are reacted by uridine phosphorylase (EC 2.4.2.3) to form arabinoside 1-phosphate and the corresponding bases; the arabinose 1-phosphate and adenine are subjected to purine nucleoside phosphorylase (EC 2.4.2.1) to generate arabinose adenosine and phosphate ions. The biotransformation method has the advantages of mild reaction conditions, simple reaction steps, no use of toxic and harmful reagents, no environmental pollution and the like.

Attempts to synthesize vidarabine using biotransformation have been made in China since the 90 s of the 20 th century. The yield of vidarabine synthesized by biotransformation of Escherichia coli B23 at Von Wanxiang, Zhouxing university of eastern science and technology corresponds to adenine conversion rate of 34% and uridine conversion rate of 12% [ Industrial microorganism, 1994, 24(4):11-14 ]; guo Youli et al hope to increase the conversion rate by optimizing the formulation of fermentation medium of Enterobacter aerogenes and inducing the expression of uridine phosphorylase of the strain with cytidine or cytidylic acid [ Biotechnology, 2006, 16(1):32-35] [ journal of China pharmaceutical industry, 2010, 41(6): 255-. The activities of purine nucleoside phosphorylase and uridine phosphorylase of Weixiao jade, Dingqingbao and the like are improved by mutagenesis of Enterobacter aerogenes ATCC13048 [ Chinese patent application No. 200710043257.5], the reaction yield corresponds to 90 percent of the conversion rate of adenine and 30 percent of the conversion rate of arabinouridine.

In the above works, a method of mixing two enzymes for use and synthesizing a target substance in one step is adopted, although the pollution problem caused by chemical synthesis is solved, the method has a reverse reaction, namely, when a substance at one end of the reaction reaches a certain concentration, the reaction is carried out in a reverse direction, so that the dosage of substrates with different molarity is caused, namely, in the reaction, the molar concentration of adenine is 3 times that of uridine; the consumption of the substrate is large; low target conversion rate, and can not replace chemical synthesis fundamentally.

Therefore, there is a need in the art to further optimize the synthesis method of vidarabine, so as to further reasonably match the substrate dosage, improve the vidarabine conversion rate and improve the yield.

Disclosure of Invention

The invention aims to solve the problems and provides a method for synthesizing vidarabine by an enzyme method.

In a first aspect of the present invention, there is provided a method of preparing vidarabine, the method comprising:

(1) reacting arabinouridine with a uridine phosphorylase enzyme (preferably EC 2.4.2.3) in an aqueous solution containing phosphate ions to obtain an arabino 1-phosphate and uracil containing solution 1;

(2) treating the solution containing the arabinose 1-phosphate and uracil obtained in the step (1) to remove uridine phosphorylase and phosphate ions therein to obtain a solution 2 containing the arabinose 1-phosphate and uracil;

(3) adding adenine and purine nucleoside phosphorylase (preferably EC 2.4.2.1) to the solution 2 containing arabinose 1-phosphate and uracil obtained in (2), and reacting to obtain arabinose adenosine product.

In a preferred embodiment, step (2) is performed after the uridine is completely consumed in step (1).

In another preferred embodiment, step (3) is preceded by the step of diluting the solution 2 containing arabinose 1-phosphate and uracil obtained in step (2).

In another preferred embodiment, in step (2), the removal of phosphate ions by adding metal ions to bind phosphate to form a low-solubility phosphate precipitate comprises; preferably, the precipitate is removed by filtration, ultrafiltration or centrifugation.

In another preferred embodiment, the metal ions include, but are not limited to: calcium ion, magnesium ion, barium ion, silver ion; preferably, the metal ions include, but are not limited to, the following salts formed in solution: calcium chloride, magnesium bromide, barium chloride; more preferably calcium chloride, magnesium chloride.

In another preferred embodiment, the final concentration of metal ions is 5-1000 mM; preferably 100-1000mM, and most preferably 300-800 mM.

In another preferred example, in step (2), the uridine phosphorylase is removed by ultrafiltration. Preferably, the ultrafiltration is low temperature ultrafiltration.

In another preferred embodiment, the ultrafiltration is carried out at a temperature of 2 to 20 deg.C (preferably 4 to 15 deg.C, such as 6, 8, 10, 12, 14 deg.C).

In another preferred embodiment, the phosphate ions include, but are not limited to, phosphate ions produced from an aqueous solution prepared from the following salts: sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate.

In another preferred embodiment, the pH of the aqueous solution containing phosphate ions is 6.0-9.0; preferably pH 6.0-8.0.

In another preferred embodiment, in the step (1), the concentration of phosphate in the aqueous solution of phosphate ions is 5-1000 mM; preferably 100-1000mM, and most preferably 300-600 mM.

In another preferred embodiment, the concentration of uridine is 10-1000 mM; preferably 10-500mM, most preferably 30-200 mM.

In another preferred example, in step (1), the uridine phosphorylase is a pure enzyme or a uridine phosphorylase expressed by a microorganism; preferably, the concentration of the uridine phosphorylase in the reaction system is 20-1000U/ml; more preferably 20-500U/ml; such as 50, 100, 200, 300U/ml.

In another preferred example, in the step (3), the purine nucleoside phosphorylase is a pure enzyme or a purine nucleoside phosphorylase expressed by a microorganism; preferably, the concentration of the purine nucleoside phosphorylase in the system is 20-1000U/ml; more preferably 100-700U/ml; such as 150, 200, 300, 500U/ml.

In another preferred embodiment, in the step (3), the concentration of adenine is 2-400 mM; preferably 5-200mM, most preferably 10-70 mM; such as 10, 20, 50, 70 mM.

In another preferred example, in the step (1) or the step (3), the reaction temperature is 30-80 ℃; preferably 40-65 ℃; more preferably 45-60 deg.c.

In another preferred example, in the step (1), the mixed solution is shaken at the temperature of 55 +/-1 ℃ for reaction for 8 +/-1 hours; in the step (3), the mixed solution is shaken at 50 +/-1 ℃ to react for 4 +/-1 hours.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Detailed Description

The inventor aims to optimize the synthesis method of the vidarabine, and through intensive research, the inventor firstly and unexpectedly discovers that the vidarabine-1-phosphate can be obtained by adding thallus containing uridine phosphorylase into an aqueous solution containing vidarabine and phosphate ions independently, removing the uridine phosphorylase and the phosphate ions after the vidarabine is completely consumed, and then adding adenine and purine nucleoside phosphorylase for reaction.

The invention aims to break the balance between adenine and a target object generated in a method for producing vidarabine by an enzymatic reaction, fully utilize different actions of uridine phosphorylase and purine nucleoside phosphorylase to enable the reaction to be carried out in one direction, and eliminate the occurrence of reverse reaction. So that the substrate is fully consumed and the conversion rate of the product reaches a higher level.

In view of the above, the present invention provides a novel enzymatic synthesis method of arabinosyladenosine, which is shown in the reaction formula (I) and the reaction formula (II), comprising the steps of:

(1) adding uridine and uridine phosphorylase to an aqueous solution of phosphate ions to perform a reaction as in reaction formula (I);

in formula (I): Ara-U represents uridine arabinoside;

EC 2.4.2.3 represents uridine phosphorylase;

p represents phosphate ion;

uracil stands for Uracil;

Ara-1-P represents arabinose-1-phosphate;

(2) after the uridine is completely consumed, uridine phosphorylase and phosphate ions are removed, and arabinose-1-phosphate is stabilized;

(3) adding adenine and purine nucleoside phosphorylase into the solution 2 containing the arabinose 1-phosphate and uracil obtained in the step (2), and carrying out the reaction shown in the formula (II) to obtain an arabinose adenosine product;

in the formula (II): adenine represents Adenine;

EC2.4.2.1 denotes a purine nucleoside phosphorylase;

Ara-A represents vidarabine;

p represents a phosphate ion.

In the step (2), phosphate ions can be removed by various methods, and various methods for removing phosphate ions in a solution in the art can be applied to the present invention. In a preferred embodiment of the present invention, a metal ion is added so that the metal ion binds to phosphate and forms a phosphate salt having low solubility, thereby causing precipitation. The metal ions and the phosphate ions are combined to form phosphate precipitate, and the precipitate can be removed by centrifugation or ultrafiltration. The metal ions include but are not limited to: calcium ion, magnesium ion, barium ion, silver ion; preferably, the metal ions include, but are not limited to, the following salts formed in solution: calcium chloride, magnesium bromide, barium chloride; more preferably calcium chloride, magnesium chloride.

In step (2), the uridine phosphorylase can be removed by various methods. In a preferred embodiment of the present invention, a low-temperature ultrafiltration method is used. The inventor finds that low-temperature ultrafiltration is beneficial to stabilizing the arabinose-1-phosphoric acid, and is better applied to subsequent reactions to obtain higher conversion rate. Furthermore, cells and enzymes expressed by them can be inactivated by various biological methods (e.g., adjusting the temperature to a temperature at which the cell or enzyme activity is affected, adjusting the pH to a pH at which the cell or enzyme activity is affected, some heavy metals also can inactivate enzymes), which methods can also be included in the methods of the present invention, as long as they do not affect the subsequent steps of the methods of the present invention.

As a preferred mode of the invention, the temperature of the solution in the ultrafiltration process is controlled to be 2-20 ℃; preferably 2 to 15 ℃, and most preferably 6 to 12 ℃.

In the present invention, the uridine phosphorylase may be a pure enzyme or an enzyme expressed by cells. It may be present in the reaction system in a free state or may be an immobilized enzyme. Various forms of uridine phosphorylase can be used in the present invention as long as they have a good enzymatic activity. In a preferred embodiment of the present invention, the enzyme is an enzyme expressed by bacterial cells.

In the invention, the phosphate ions can be generated by preparing an aqueous solution from the following salts: the phosphate is prepared into an aqueous solution with a stable pH value, and the pH value of the aqueous solution is 6.0-9.0 as a preferred mode of the invention; preferably pH 6.0-8.0.

The inventors also optimized the concentration of each raw material in the reaction system to improve the efficiency of the reaction.

As a preferred mode of the present invention, the concentration of uridine ranges from 10 to 1000 mM; preferably 10-500mM, most preferably 30-200 mM.

In a preferred embodiment of the present invention, the concentration of phosphate in the aqueous solution of phosphate ions is 5 to 1000 mM; preferably 100-1000mM, and most preferably 300-600 mM.

As a preferred mode of the present invention, the concentration of uridine phosphorylase in the system is 20-1000U/ml; preferably 20-500U/ml, such as 50, 100, 200, 300U/ml. Preferably 50-200U/ml.

As a preferred mode of the present invention, the final concentration of the metal ions added for removing phosphate ions is 5 to 1000 mM; preferably 100-1000mM, and most preferably 300-800 mM.

As a preferred mode of the present invention, the concentration of adenine is 2 to 400 mM; preferably 5-200mM, most preferably 10-70 mM.

As a preferred mode of the present invention, the concentration of purine nucleoside phosphorylase in the system is 20 to 1000U/ml; preferably 100-700U/ml, and most preferably 200-400U/ml.

In a preferred embodiment of the present invention, the temperature in the uridine phosphorylase reaction is 30 to 80 ℃; preferably 40 to 70 ℃, and most preferably 50 to 60 ℃.

In a preferred embodiment of the present invention, the temperature in the purine nucleoside phosphorylase reaction is 30 to 80 ℃; preferably 40 to 70 ℃, and most preferably 50 to 60 ℃.

The method for measuring the content of the arabinose-1-phosphoric acid generated in the formula I uses high performance liquid chromatography (ELSD) to calculate the specific content through a quantitative standard curve and an actually measured peak area. The method for measuring the content of the vidarabine generated in the formula II uses high performance liquid chromatography (UV) to calculate the specific content through a quantitative standard curve and an actually measured peak area. Unless otherwise stated, the conversion rate of vidarabine referred to in the present invention means that the molar amount of vidarabine in the reaction solution is measured by a high performance liquid (UV) standard quantitative method and then divided by the molar amount of vidarabine to be fed. It is to be understood that other assay devices and assay methods applicable to determining the content of a substance may also be applied to the present invention.

The features mentioned above, or mentioned by way of example, in the present invention may be combined in any combination. All the features disclosed in this specification may be combined in any combination and any combination may be substituted for any combination which provides equivalent, equivalent or similar purpose for each feature disclosed in this specification. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The main advantages of the invention are:

(a) the two-step enzyme synthesis method provided by the invention has low cost, and the required equipment is common equipment in biological product production.

(b) The two-step enzyme synthesis method provided by the invention enables the conversion rate of the vidarabine to reach more than 90%.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.

The unit of enzyme activity and the unit of concentration of each raw material involved in the present invention are well known to those skilled in the art.

The bacterial cells used in the following examples were recombinant E.coli expressing uridine phosphorylase (cloned according to Biosci.Biotech.Biochem.,59(10), 1987. sup. 1990, 1995) and recombinant E.coli expressing purine phosphorylase (cloned according to Biosci.Biotech.Biochem.,59(10), 1987. sup. 1990, 1995).

High performance liquid phase (ELSD) conditions in the following examples of the invention:

the instrument comprises the following steps: waters 2695+ Waters 2420 ELSD detector (evaporative light scattering),

and (3) analyzing the column: YMC-AQ 250 x 4.6mm,

flow rate: 0.8ml/min of the mixture is added,

column temperature: 5 ℃ is adopted.

Gradient conditions:

time (min)	0.01M phosphate buffer (pH6.0)	Methanol
			0	100％	0％
2	100％	0％
			10	80％	20％
15	80％	20％
			42	100％	0％
50	100％	0％

Standard curve: Y3100168.94X +33400.24(R2 1.00),

y-area of the peak; x-mole;

detection effective range: 0.0049 mol to 0.0295 mol;

high performance liquid phase (UV) conditions in the following examples of the invention:

the instrument comprises the following steps: waters 2695+ Waters 996UV detector,

and (3) analyzing the column: YMC-AQ 150 x 4.6mm,

flow rate: 1.0ml/min of the mixture is added,

detection wavelength: 260 nm;

gradient conditions:

standard curve: Y3188562.59X-27005.28 (R)²＝1.00)，

Y-area of the peak; x-mole;

detection effective range: 0.8876 mmol-6.1357 mmol;

example 1 two step Process reaction 1

The reaction is carried out in two steps, which are as follows:

reaction I:

to 1000ml of 300mM sodium phosphate solution pH6.8, 0.04 mol of uridine arabinoside was added to a concentration of 40mM, and then 1.56g of cells (32200U per gram of uridine phosphorylase contained) were added to a concentration of 50U/ml uridine phosphorylase; the mixture was placed in a shaker and shaken at 55 ℃ for 8 hours.

Taking out the reaction liquid, cooling to 10 ℃, adding 34g of anhydrous calcium chloride solid, stirring for 30 minutes, carrying out ultrafiltration to remove precipitated uracil, calcium phosphate and other insoluble substances, wherein the insoluble substances comprise thallus containing uridine phosphorylase, keeping the ultrafiltration solution at 5-10 ℃, collecting the ultrafiltration solution, washing the membrane by 500ml of double-distilled water after the ultrafiltration is finished, washing out residual reaction liquid remaining in the membrane, combining the ultrafiltration liquid and the membrane washing liquid (both parts of the liquids contain products, and the combination can achieve higher rate), and obtaining 1420ml of liquid.

After the liquid obtained by ultrafiltration is diluted by 3 times, the peak area of the arabinose-1-phosphoric acid is 73185.74 by quantitative analysis of high performance liquid phase (ELSD), and the number is substituted into a standard curve formula to calculate: the conversion of this solution containing 0.0385 mole of arabinose-1-phosphate divided by 0.04 mole of arabinouridine was 96.3%.

And (2) reaction II:

1580ml of double distilled water is added into the solution obtained in the first reaction for dilution, and then 0.04 mol of adenine is added to enable the concentration of the adenine to reach 13.3 mM; adding 21.6g of thallus (containing 27800U of purine nucleoside phosphorylase per gram) to make the concentration of purine nucleoside phosphorylase reach 200U/ml; the mixture was placed in a shaker and shaken at 50 ℃ for 4 hours.

After the reaction solution is diluted by 10 times, the high performance liquid (UV) quantitative analysis shows that the peak area of vidarabine is 11961990.05, and the number is substituted into a standard curve formula to calculate: the conversion of the solution containing 0.0376 moles of vidarabine divided by 0.04 moles of uridine was 94%.

Example 2 two-step reaction 2

The reaction is carried out in two steps, which are as follows:

reaction I:

to 1000ml of 600mM sodium phosphate solution pH6.8, 0.2 mol of uridine arabinoside was added to a concentration of 200mM, and then 6.21g of cells (32200U/g uridine phosphorylase contained) were added to a concentration of 200U/ml uridine phosphorylase; the mixture was placed in a shaker and shaken at 55 ℃ for 8 hours.

Taking out the reaction solution, cooling to 10 ℃, and adding 1000ml of double distilled water to dilute the reaction solution; adding anhydrous calcium chloride solid 68g, stirring for 30 min, ultrafiltering to remove uracil, calcium phosphate and other insoluble substances including thallus containing uridine phosphorylase; keeping the ultrafiltration solution at 5-10 deg.C, washing the membrane with 500ml double distilled water, washing out the residual reaction solution in the membrane, and mixing the ultrafiltration solution and the membrane washing liquid to obtain 2390 ml.

After the liquid obtained by ultrafiltration is diluted by 10 times, the peak area of the arabinose-1-phosphoric acid is 69992.87 by quantitative analysis of high performance liquid phase (ELSD), and the number is substituted into a standard curve formula to calculate: the conversion of the solution containing 0.1855 moles of arabinose-1-phosphate divided by 0.2 moles of uridine was 92.75%.

And (2) reaction II:

adding 610ml of double distilled water into the solution obtained in the first reaction for dilution, and then adding 0.2 mol of adenine to enable the concentration of the adenine to reach 66.6 mM; adding 43g of thallus (containing 27800U of purine nucleoside phosphorylase per gram) to make the concentration of purine nucleoside phosphorylase reach 398U/ml; the mixture was placed in a shaker and shaken at 50 ℃ for 4 hours.

After the reaction solution is diluted by 50 times, the high performance liquid (UV) quantitative analysis shows that the peak area of vidarabine is 10902381.78, and the number is substituted into a standard curve formula to calculate: the conversion of 0.1836 moles of vidarabine divided by 0.2 moles of uridine in this solution was 91.8%.

Example 3 and comparative example 1

The same procedure as in example 1 was followed, except that adenine was added directly after shaking at 55 ℃ for 8 hours in the first reaction, and that the second reaction was carried out without the steps of low temperature ultrafiltration in the first reaction and dilution with double distilled water in the second reaction.

The reaction is carried out in two steps, which are as follows:

reaction I:

After the obtained mixed solution is diluted by 3 times, the high performance liquid chromatography (ELSD) quantitative analysis shows that the peak area of the arabinose-1-phosphoric acid is 72974.04, and the number is substituted into a standard curve formula to calculate: the conversion of this solution containing 0.0381 moles of arabino-1-phosphate divided by 0.04 moles of arabinouridine was 95.3%.

And (2) reaction II:

adding 0.04 mol of adenine into the solution obtained in the first reaction to make the concentration of the adenine reach 40 mM; adding 21.6g of thallus (containing 27800U of purine nucleoside phosphorylase per gram) to make the concentration of purine nucleoside phosphorylase reach 600U/ml; the mixture was placed in a shaker and shaken at 50 ℃ for 4 hours.

After the reaction solution is diluted by 10 times, the high performance liquid (UV) quantitative analysis shows that the peak area of vidarabine is 8774560.76, and the number is substituted into a standard curve formula to calculate: the conversion of this solution, which contained 0.00884 moles of vidarabine divided by 0.04 moles of uridine, was 22.1%.

Although the yield of arabino-1-phosphate in reaction one is as high as 95.3%, the generation of arabino-adenosine is greatly inhibited in reaction two without removing phosphate ions, and the existence of uridine phosphorylase enables the reverse reaction pathway for producing arabinouridine to still exist.

Example 4 and comparative example 2

To 1000ml of 300mM sodium phosphate solution pH6.8, 0.04 mol of uridine was added to a concentration of 40mM, 0.04 mol of adenine was added to a concentration of 40mM, 1.56g of cells (32200U per gram of uridine phosphorylase) were added to a concentration of 50U/ml of uridine phosphorylase, and 21.6g of cells (27800U per gram of purine nucleoside phosphorylase) were added to a concentration of 600U/ml of purine nucleoside phosphorylase; the mixture was placed in a shaker and shaken at 50 ℃ for 12 hours.

After the reaction solution is diluted by 10 times, the high performance liquid (UV) quantitative analysis shows that the peak area of vidarabine is 8624233.32, and the number is substituted into a standard curve formula to calculate: the conversion in this solution was 21.3% with 0.0084 moles of vidarabine divided by 0.04 moles of uridine.

The one-step reaction system has the disadvantage that the reaction is balanced before the substrate is not used up.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any entity or method that is calculated by any person skilled in the art is intended to be encompassed by the claims, if it is the same as or equivalent to that which is claimed. All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference.

Claims

1. A method of making vidarabine, comprising:

(1) reacting arabinouridine with uridine phosphorylase in an aqueous solution containing phosphate ions to obtain a solution 1 containing arabino-1-phosphate and uracil; the uridine phosphorylase is a uridine phosphorylase expressed by a microorganism;

(2) treating the solution 1 containing the arabinose 1-phosphate and uracil obtained in the step (1) to remove uridine phosphorylase and phosphate ions therein to obtain a solution 2 containing the arabinose 1-phosphate and uracil; phosphate ions are removed by adding metal ions to combine with phosphate radicals to form phosphate precipitates with low solubility; the metal ions are calcium ions;

(3) adding adenine and purine nucleoside phosphorylase into the solution 2 containing the arabinose 1-phosphate and uracil obtained in the step (2), and reacting to obtain an arabinose adenosine product; the purine nucleoside phosphorylase is a microorganism-expressed purine nucleoside phosphorylase.

2. The method of claim 1, wherein in step (2), the precipitate is removed by filtration.

3. The method of claim 1, wherein in step (2), the precipitate is removed by ultrafiltration.

4. The method of claim 1, wherein in step (2), the precipitate is removed by centrifugation.

5. The method of claim 1, wherein the metal ion is a metal ion formed in solution from the following salts: calcium chloride.

6. The method of claim 1, wherein the final concentration of metal ions is 5 to 1000 mM.

7. The method of claim 6, wherein the final concentration of metal ions is 100 and 1000 mM.

8. The method of claim 7, wherein the final concentration of metal ions is 300-800 mM.

9. The method of claim 1, wherein in step (2), the uridine phosphorylase is removed by ultrafiltration.

10. The method of claim 9, wherein ultrafiltration is performed at a temperature of 2 to 20 ℃.

11. The method of claim 1, wherein the phosphate ions comprise phosphate ions produced from an aqueous solution of: sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate.

12. The method of claim 1 or 11, wherein said aqueous solution containing phosphate ions has a PH of 6.0 to 9.0.

13. The method of claim 12, wherein said aqueous solution containing phosphate ions has a PH of 6.0 to 8.0.

14. The method of claim 1 or 11, wherein in step (1), the concentration of phosphate in the aqueous solution of phosphate ions is 5 to 1000 mM.

15. The method as claimed in claim 14, wherein the concentration of phosphate in the aqueous solution of phosphate ions is 100 and 1000 mM.

16. The method of claim 15, wherein the concentration of phosphate in the aqueous solution of phosphate ions is 300-600 mM.

17. The method of claim 1, wherein the concentration of uridine is 10 to 1000 mM.

18. The method of claim 1, wherein the concentration of uridine is 10 to 500 mM.

19. The method of claim 1, wherein the concentration of uridine is 30 to 200 mM.

20. The method according to claim 1, wherein in step (1), the uridine phosphorylase is uridine phosphorylase expressed by recombinant E.coli.

21. The method according to claim 20, wherein the concentration of the uridine phosphorylase in the reaction system is 20 to 1000U/ml.

22. The method according to claim 21, wherein the concentration of the uridine phosphorylase in the reaction system is 20 to 500U/ml.

23. The method according to claim 1, wherein in the step (3), the purine nucleoside phosphorylase is purine nucleoside phosphorylase expressed by Escherichia coli.

24. The method of claim 23, wherein said purine nucleoside phosphorylase is present in the system at a concentration of 20 to 1000U/ml.

25. The method as set forth in claim 24, wherein the concentration of said purine nucleoside phosphorylase in the system is 100-700U/ml.

26. The method of claim 1, wherein in step (3), the concentration of adenine is 2 to 400 mM.

27. The method of claim 26, wherein the concentration of adenine is 5 to 200 mM.

28. The method of claim 27, wherein the concentration of adenine is 10 mM to 70 mM.

29. The process according to claim 1, wherein the reaction temperature in step (1) or step (3) is from 30 ℃ to 80 ℃.