CN113307832A

CN113307832A - Method for preparing uridylic acid

Info

Publication number: CN113307832A
Application number: CN202110628172.3A
Authority: CN
Inventors: 吕祖平; 吴志卿; 刘存虎; 徐永鑫; 赵小龙; 许勇
Original assignee: Taizhou Houpu Biotechnology Co ltd
Current assignee: Taizhou Houpu Biotechnology Co ltd
Priority date: 2021-06-06
Filing date: 2021-06-06
Publication date: 2021-08-27

Abstract

The present invention provides a method for preparing uridylic acid, the method comprising: taking cytidine (or uridine) and adenosine as main raw materials, transforming by biological cell homogeneous liquid, and then carrying out microfiltration, precipitation, chromatography and crystallization to obtain the uridine nucleotide. The method has the advantages of simple process operation, simple equipment, low cost, environmental friendliness and convenience for industrial production.

Description

Method for preparing uridylic acid

Technical Field

The invention relates to a preparation method of uridine monophosphate, which takes cytidine (or uridine) and adenosine as main raw materials, and the uridine monophosphate is obtained by biological cell homogeneous liquid conversion, microfiltration, precipitation, chromatography and crystallization, and belongs to the technical field of biological engineering.

Background

Uridylic acid, which is called uridine monophosphate, is an important food additive and a medical intermediate.

The currently known patented methods for uridylic acid are mainly: in chinese patent CN110885812A, an enzyme composition is proposed to convert cytidine into uridylic acid, the concentration of cytidine treated by a single reaction is only 16-24 g/L, ATP with a higher unit price needs to be added for the conversion reaction, sodium hexametaphosphate is used as a substrate for phosphoric acid, the price is also higher, and more importantly, the enzyme composition used for the reaction is not expensive, so that in summary, the process has great limitations if the process is successful in industrialization.

In order to develop a process more beneficial to industrial amplification, a biological cell homogenized solution is used as an enzymatic catalyst according to the metabolic relationship among cytidine, uridine and uridylic acid, cytidine is firstly converted into uridine (or uridine is directly used as a substrate), then uridine is converted into uridylic acid, ATP is directly deleted from reaction raw materials in order to reduce cost, low-price adenosine is used as a raw material, adenosine is converted into ATP through enzyme carried in the cell homogenized solution, and phosphoric acid is provided for the uridine.

Disclosure of Invention

The invention provides a method for preparing uridylic acid. The invention has the advantages that: high purity, high yield, simple process, low cost and environmental protection.

The above object of the present invention is achieved by the following technical solutions:

a method for preparing uridylic acid, comprising the steps of:

(1) preparing a certain amount of cytidine or uridine, adenosine, magnesium salt and sodium pyrophosphate into a solution, adding the biological cell homogeneous solution, and reacting for 4-14 hours at a certain pH and temperature, wherein the cytidine or uridine, adenosine and sodium pyrophosphate are added for multiple times to obtain a conversion solution of uridylic acid;

(2) heating the conversion solution, adding calcium salt, precipitating, and filtering to obtain conversion clear solution;

(3) adsorbing the converted clear liquid by anion exchange resin, washing with water and desorbing to obtain uridylic acid eluent, and performing nanofiltration desalination and concentration on the eluent;

(4) heating and decolorizing the eluate with active carbon, filtering to obtain decolorized solution, concentrating the decolorized solution under reduced pressure, slowly adding organic solvent, and crystallizing to obtain uridylic acid crystal;

preferably, the concentration of the cytosine or uridine in the step (1) is 24-60 g/L, the concentration of the adenosine is 2-10 g/L, the concentration of the magnesium salt is 1-10 g/L, and the concentration of the sodium pyrophosphate is 1.2-2 times of the concentration of the cytosine or uridine calculated according to the number of moles of the cytosine or uridine. Wherein the cytosine or uridine, the adenosine and the sodium pyrophosphate are added for a plurality of times, and the concentration of the biological cell homogeneous solution is 0.5-10 g/L.

Preferably, the conversion temperature in the step (1) is 25-40 ℃, preferably 36-38 ℃.

Preferably, the pH value of the conversion in the step (1) is 6.0-9.0, and the pH value is controlled by liquid caustic soda, flake caustic soda or ammonia water, preferably 7.0-8.0.

Preferably, the heating temperature in the step (2) is 50-80 ℃, preferably 60 ℃.

Preferably, the amount of the calcium salt added in the step (2) is measured according to the precipitation condition, namely, after a certain amount of calcium salt is added for precipitation, a small amount of calcium salt is added into the clear liquid obtained by filtration, if the calcium salt still precipitates, the calcium salt is continuously added, the steps are repeated until no new precipitate appears, and the pH value is kept at 6-8 in the precipitation process.

Preferably, after the anion resin in the step (3) is loaded on a column, the resin is washed by water, then the resin is eluted by 0.1-0.5 mol/L NaCl aqueous solution in a fractional manner, the eluent with the uridylic acid purity of more than 95% is collected, and then the eluent is subjected to nanofiltration desalination and concentration by a nanofiltration machine.

Preferably, the adding amount of the activated carbon in the step (4) is 0.3-3% (w/v), preferably 0.5%, and the decolorizing temperature is 50-80 ℃, preferably 65 ℃.

Preferably, the concentration of the concentrated uridylic acid in the step (4) is 200-400 g/L, and the addition amount of the ethanol is 3-5 times of the volume of the concentrated solution.

The invention has the advantages that:

(1) the invention has simple process, easy control and is beneficial to industrialized mass production;

(2) the raw materials and the used raw materials are cheap and easy to obtain, and the production cost is reduced.

Detailed Description

The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but the scope of the invention is within the scope of the invention as long as they do not depart from the basic idea of the invention.

The reagents used in the invention are all industrial-grade qualified products or the purity of the industrial-grade qualified products is higher than that of the industrial-grade qualified products.

Example 1

Adding 40g magnesium sulfate, 600g cytidine, 40g adenosine and 500g sodium pyrophosphate into 20L water, controlling the stirring speed at 200rpm, adjusting the pH to 7.0, controlling the temperature at 37 ℃, adding 200g biological cell homogenized solution, and controlling the pH to 7.0 by using liquid alkali during the reaction. Taking 60g of adenosine and 500g of sodium pyrophosphate for standby application, adding 12g of adenosine and 100g of sodium pyrophosphate into the reaction solution every 1h until the reaction is completed after 6h, stopping the reaction after 9h, raising the temperature of the reaction solution to 60 ℃, adding phosphate and sulfate in the calcium chloride precipitation reaction solution, controlling the pH to be 6.5-7.0, filtering to obtain clear solution after the precipitation is completed, passing the clear solution through 201 x 4 anion resin, firstly washing the resin with water, then washing the resin with 0.3mol/L NaCl aqueous solution, collecting eluent with the uridylic acid purity of more than 95%, carrying out nanofiltration concentration until the uridylic acid concentration is about 100g/L, then washing a nanofiltration membrane with water to remove salt, adding 15g of activated carbon into the nanofiltration concentrated solution, carrying out decolorization for 12min at 50 ℃, filtering to obtain decolorized solution, concentrating the decolorized solution under reduced pressure until the uridylic acid concentration reaches about 250g/L, slowly adding 12L of ethanol into the concentrated solution, stirring while adding, continuing stirring for 30min after adding, standing for 1h, filtering to obtain uridine acid crystals, and drying to obtain 886g of finished product with the purity of 99.5%.

Example 2

Adding 40g of magnesium sulfate, 600g of uridine, 40g of adenosine and 500g of sodium pyrophosphate into 20L of water, controlling the stirring speed at 200rpm, adjusting the pH to 7.0, controlling the temperature at 37 ℃, adding 200g of biological cell homogenized solution, and controlling the pH to 7.0 by using liquid alkali during the reaction. Taking 60g of adenosine and 500g of sodium pyrophosphate for standby application, adding 12g of adenosine and 100g of sodium pyrophosphate into the reaction solution every 1h until the reaction is completed after 6h, stopping the reaction after 9h, raising the temperature of the reaction solution to 60 ℃, adding phosphate and sulfate in the calcium chloride precipitation reaction solution, controlling the pH to be 6.5-7.0, filtering to obtain clear solution after the precipitation is completed, passing the clear solution through 201 x 4 anion resin, firstly washing the resin with water, then washing the resin with 0.3mol/L NaCl aqueous solution, collecting eluent with the uridylic acid purity of more than 95%, carrying out nanofiltration concentration until the uridylic acid concentration is about 100g/L, then washing a nanofiltration membrane with water to remove salt, adding 15g of activated carbon into the nanofiltration concentrated solution, carrying out decolorization for 12min at 50 ℃, filtering to obtain decolorized solution, concentrating the decolorized solution under reduced pressure until the uridylic acid concentration reaches about 250g/L, slowly adding 12L of ethanol into the concentrated solution, stirring while adding, continuously stirring for 30min after adding, standing for 1h, filtering to obtain uridine acid crystals, and drying to obtain 905g of finished products with the crystal purity of 99.7%.

Example 3

Adding 40g of magnesium sulfate, 800g of uridine, 40g of adenosine and 600g of sodium pyrophosphate into 20L of water, controlling the stirring speed at 200rpm, adjusting the pH to 7.0, controlling the temperature at 37 ℃, adding 300g of biological cell homogenized solution, and controlling the pH to 7.0 by using liquid alkali during the reaction. Taking 120g of adenosine and 1000g of sodium pyrophosphate for standby, adding 12g of adenosine and 100g of sodium pyrophosphate into the reaction solution every 1h until the reaction is completely completed after 11h, stopping the reaction after 14h, raising the temperature of the reaction solution to 60 ℃, adding phosphate and sulfate in the calcium chloride precipitation reaction solution, controlling the pH to be 6.5-7.0, filtering to obtain clear solution after the precipitation is completed, passing the clear solution through 201 x 4 anion resin, firstly washing the resin with water, then washing the resin with 0.3mol/L NaCl aqueous solution, collecting eluent with the uridylic acid purity of more than 95%, carrying out nanofiltration concentration until the uridylic acid concentration is about 100g/L, then washing a nanofiltration membrane with water to remove salt, adding 20g of activated carbon into the nanofiltration concentrated solution, carrying out decolorization for 12min at 50 ℃, filtering to obtain decolorized solution, concentrating the decolorized solution under reduced pressure until the uridylic acid concentration reaches about 250g/L, slowly adding 18L of ethanol into the concentrated solution, stirring while adding, stirring for 30min after adding, standing for 1h, filtering to obtain uridine acid crystal, and oven drying to obtain 1125g finished product with crystal purity of 99.5%.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing uracil nucleotides, comprising the steps of:

s1, preparing a certain amount of cytidine or uridine, adenosine, magnesium salt and sodium pyrophosphate into a solution, adding the biological cell homogenized solution, and reacting for 4-14 hours at a certain pH and temperature, wherein the cytidine or uridine, adenosine and sodium pyrophosphate are added for multiple times to obtain a conversion solution of uridylic acid;

s2, heating the conversion solution, adding calcium salt, precipitating and filtering to obtain a conversion clear solution;

s3, adsorbing the converted clear liquid by anion exchange resin, washing with water and desorbing to obtain uridylic acid eluent, and performing nanofiltration desalination and concentration on the eluent;

s4, heating and decoloring the eluent by using activated carbon, filtering to obtain a decolored solution, decompressing and concentrating the decolored solution, and slowly adding an organic solvent for crystallization to obtain the uridine acid crystals.

2. The method according to claim 1, wherein the cytosine nucleoside used in step S1 is a solid cytosine nucleoside product or a concentrate of an intermediate of cytosine nucleoside, uridine is a solid uracil nucleoside product or a concentrate of an intermediate of uracil nucleoside, adenine nucleoside is a solid adenine nucleoside product or a crude adenine nucleoside product, and the magnesium salt is magnesium sulfate, magnesium chloride, acetic acid enzyme, magnesium nitrate, or the like.

3. The production method of uracil nucleotides according to claim 1, wherein the anion exchange resin of step S3 is a strongly basic anion exchange resin or a weakly basic anion exchange resin.

4. The method according to claim 1, wherein the concentration of cytosine or uridine in step S1 is 25 to 60g/L, the concentration of adenosine is 2 to 10g/L, the concentration of magnesium salt is 1 to 10g/L, and the concentration of sodium pyrophosphate is 1.2 to 2 times as high as the number of moles of cytosine or uridine. The concentration of the biological cell homogeneous solution is 0.5-10 g/L.

5. The step S1, wherein the temperature of the step S1 is 25-40 ℃.

6. The method of claim 1, wherein the conversion pH of step S1 is 6.0-9.0, and the pH is controlled by liquid alkali, flake alkali or ammonia.

7. The method according to claim 1, wherein the heating temperature in step S2 is 50-80 ℃, the concentration of the concentrated uridine in step S4 is 200-400 g/L, and the amount of ethanol added is 3-5 times the volume of the concentrated solution.

8. The method according to claim 1, wherein the amount of calcium salt added in step S2 is measured according to precipitation conditions, i.e., a small amount of calcium salt is added to the filtered clear solution after a certain amount of calcium salt is added for precipitation, and if there is still precipitation, the addition of calcium salt is continued, and the process is repeated until no more new precipitation occurs, and the pH of the precipitation process is maintained at 6-8.

9. The method according to claim 1, wherein after the anion resin is applied to the column in step S3, the resin is washed with water, the resin is eluted with 0.1-0.5 mol/L NaCl solution, the eluate with the purity of uridylic acid higher than 95% is collected, and the eluate is subjected to nanofiltration desalination and concentration by a nanofiltration machine.

10. The method of claim 1, wherein the activated carbon is added in an amount of 0.3 to 3% (w/v), preferably 0.5%, and the decolorization temperature is 50 to 80 ℃ in step S4.