阅读说明：本技术 一种循环制备核苷酸盐的方法 (Method for circularly preparing nucleotide ) 是由 任洪发 黄励坚 严盟 陈华强 时英爽 王帅 于 2021-09-30 设计创作，主要内容包括：本发明属于生物催化技术领域,具体涉及一种循环制备核苷酸盐的方法,步骤为：A、将核苷、缩合磷酸盐与水混合进行酶催化反应,得反应液；B、将步骤A反应液调pH值至碱性,降温析出结晶体,得核苷与核苷酸的混合液；C、将步骤B核苷与核苷酸的混合液经过活性炭填充柱,吸附核苷,流出核苷酸溶液,降温结晶,得核苷酸盐；D、用洗脱液将步骤C的活性炭填充柱洗脱得到核苷、核苷酸收集液；E、将步骤D的核苷、核苷酸收集液泵入蒸馏塔,塔顶收集蒸馏出的洗脱液,塔底排出核苷、核苷酸釜底液；F、将步骤E的洗脱液循环到步骤D中,将步骤E的核苷、核苷酸釜底液循环到步骤A中。本发明循环利用核苷和洗脱液,核苷利用率高,核苷酸盐产品收率高、纯度高。(The invention belongs to the technical field of biocatalysis, and particularly relates to a method for circularly preparing nucleotide salt, which comprises the following steps: A. mixing nucleoside, condensed phosphate and water to perform enzyme catalytic reaction to obtain reaction liquid; B. regulating the pH value of the reaction liquid in the step A to be alkaline, and cooling to precipitate crystals to obtain a mixed liquid of the nucleoside and the nucleotide; C. b, the mixed solution of the nucleoside and the nucleotide in the step B passes through an activated carbon packed column to adsorb the nucleoside, the solution of the nucleotide flows out, and the solution is cooled and crystallized to obtain the nucleotide salt; D. c, eluting the activated carbon packed column in the step C by using an eluent to obtain a nucleoside and nucleotide collecting solution; E. pumping the nucleoside and nucleotide collecting solution in the step D into a distillation tower, collecting the distilled eluent at the tower top, and discharging nucleoside and nucleotide kettle bottom solution at the tower bottom; F. and E, recycling the eluent in the step E to the step D, and recycling the nucleoside and nucleotide kettle bottom liquid in the step E to the step A. The invention recycles the nucleoside and the eluent, has high utilization rate of the nucleoside, high yield of the nucleotide salt product and high purity.)

1. A cyclic nucleotide preparation method, comprising the steps of:

A. mixing nucleoside, condensed phosphate and water, stirring uniformly, adding acid phosphatase to perform enzyme catalytic reaction to obtain reaction liquid;

B. adjusting the pH value of the reaction solution in the step A to be alkaline, cooling to precipitate crystals, and separating phosphate to obtain a mixed solution of nucleoside and nucleotide;

C. b, enabling the mixed solution of the nucleoside and the nucleotide in the step B to pass through an activated carbon packed column to adsorb the nucleoside, enabling an effluent liquid to be a nucleotide solution, neutralizing with hydrochloric acid, adding activated carbon, carrying out heat preservation and decoloration, and carrying out cooling crystallization to obtain a nucleotide salt;

D. c, eluting the activated carbon packed column in the step C by using an eluent, and collecting a nucleoside solution containing the nucleotide to obtain a nucleoside and nucleotide collecting solution;

E. pumping the nucleoside and nucleotide collecting solution in the step D into a distillation tower, collecting the distilled eluent at the tower top, and discharging nucleoside and nucleotide kettle bottom solution at the tower bottom;

F. recycling the eluent in the step E to the step D, and recycling the nucleoside and nucleotide kettle bottom liquid in the step E to the step A;

the molar ratio of the condensed phosphate to the nucleoside in the step A is (1.0-6.0): 1.0; the pH initial value of the enzyme catalysis reaction in the step A is 4.0-5.5, the reaction temperature is 15-30 ℃ in the reaction time period from the initial enzyme catalysis stage to the front (1/15-2/3) of the reaction, and the reaction temperature is 30-50 ℃ in the subsequent reaction time period.

2. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: the molar ratio of the condensed phosphate to the nucleoside in the step A is (1.2-5.0): 1.0.

3. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: the nucleoside in the step A is one of inosine and guanosine or a mixture of the inosine and the guanosine.

4. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: the condensed phosphate in the step A is at least one of sodium acid pyrophosphate, sodium pyrophosphate and sodium tripolyphosphate.

5. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: the pH value range in the step B is 11-11.5.

6. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: the activated carbon in the step C is one of coconut shell particle activated carbon, coal particle activated carbon and shell particle activated carbon.

7. The cyclic production method of a nucleotide salt according to claim 1, characterized in that: and D, eluting the eluent in the step D by one of a methanol solution and an ethanol solution.

8. The cyclic production method for a nucleotide salt according to claim 7, characterized in that: the weight percentage concentration of the methanol solution and the ethanol solution is 5 to 35 percent.

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for circularly preparing nucleotide salt.

Background

The nucleotide product mainly takes disodium 5' -Inosinate (IMP), disodium 5' -Guanylate (GMP) and disodium 5' -flavor nucleotide (I + G), and is mainly used as a food freshener and applied to seasonings such as monosodium glutamate, chicken essence, soy sauce and the like to improve freshness. Along with the continuous rising of the market demand of the seasonings, the usage amount of the nucleotide is also continuously increased, and the market demand is gradually increased year by year.

The existing production method mainly comprises a synthesis method, a fermentation method, a nucleic acid hydrolysis method and a biological catalysis method, and the biological catalysis method has the characteristic of mild reaction conditions and has a great application and popularization prospect.

However, the existing methods for preparing nucleotides by biocatalysis mainly have the following problems: firstly, due to the characteristics of a biocatalysis method, nucleoside phosphorylation reaction has a reaction equilibrium state, the reaction of substrate nucleoside is incomplete, and the nucleoside utilization rate of a reaction solution is lower than 90 percent according to the report disclosed by the invention patent CN 101265490B; secondly, because nucleoside and nucleotide exist in the reaction liquid at the same time, the removal of the unreacted nucleoside by crystallization is difficult, and the purity of the nucleotide salt is 94.5-98.1% according to the invention patent CN 111286521A.

Disclosure of Invention

Aiming at the technical defects, the invention solves the problem of low utilization rate of the nucleoside in the reaction solution in the existing preparation process, and simultaneously ensures the quality of the extracted nucleotide. The invention provides a method for circularly preparing nucleotide salt.

In order to solve the technical problems, the technical scheme of the invention is as follows: a cyclic nucleotide preparation method comprising the steps of:

A. mixing nucleoside, condensed phosphate and water, stirring uniformly, adding acid phosphatase to perform enzyme catalytic reaction to obtain reaction liquid;

B. adjusting the pH value of the reaction solution in the step A to be alkaline, cooling to precipitate crystals, and separating phosphate to obtain a mixed solution of nucleoside and nucleotide;

C. b, enabling the mixed solution of the nucleoside and the nucleotide in the step B to pass through an activated carbon packed column to adsorb the nucleoside, enabling an effluent liquid to be a nucleotide solution, neutralizing with hydrochloric acid, adding activated carbon, carrying out heat preservation and decoloration, and carrying out cooling crystallization to obtain a nucleotide salt;

D. c, eluting the activated carbon packed column in the step C by using an eluent, and collecting a nucleoside solution containing a small amount of nucleotides to obtain a nucleoside and nucleotide collecting solution;

E. pumping the nucleoside and nucleotide collecting solution in the step D into a distillation tower, collecting the distilled eluent at the tower top, and discharging nucleoside and nucleotide kettle bottom solution at the tower bottom;

F. recycling the eluent in the step E to the step D, and recycling the nucleoside and nucleotide kettle bottom liquid in the step E to the step A.

The molar ratio of the condensed phosphate to the nucleoside in the step A is (1.0-6.0): 1.0; the pH initial value of the enzyme catalysis reaction in the step A is 4.0-5.5, the reaction temperature is 15-30 ℃ in the reaction time period from the initial enzyme catalysis stage to the front (1/15-2/3) of the reaction, and the reaction temperature is 30-50 ℃ in the subsequent reaction time period. In biochemistry, most enzymatic reactions are homogeneous reactions that occur in solution phase, and these reactions are reversible in nature from a physicochemical standpoint. When the reaction system reaches chemical equilibrium, the concentration of one reaction substrate is increased, and the conversion rate of the other reaction substrate is increased. In the reaction of condensed phosphate with nucleoside, it is effective to increase the concentration of condensed phosphate in order to increase the conversion rate of nucleoside. When the nucleoside concentration is constant, the concentration of the condensed phosphate can be increased by increasing the molar ratio of the condensed phosphate to the nucleoside. The condensed phosphate is less, and the nucleoside conversion rate is low; condensed phosphates are more, and the conversion rate of nucleosides is high; however, excessive condensed phosphates lead to increased raw material input and subsequent separation costs. The conversion rate of the reaction is increased along with the increase of the molar ratio of the condensed phosphate to the nucleoside, but the conversion rate is not increased after the reaction reaches a certain ratio, and the molar ratio of the condensed phosphate to the nucleoside is (1.0-6.0): 1.0 is proper after repeated experiments.

Further: in the above cyclic production method of a nucleotide salt, the molar ratio of the condensed phosphate to the nucleoside in the step A is preferably (1.2 to 5.0): 1.0.

The nucleoside in the step A is one of inosine and guanosine or a mixture of the inosine and the guanosine.

The condensed phosphate in the step A is at least one of sodium acid pyrophosphate, sodium pyrophosphate and sodium tripolyphosphate.

The pH value range in the step B is 11-11.5.

The activated carbon in the step C is one of coconut shell particle activated carbon, coal particle activated carbon and shell particle activated carbon.

And D, eluting the eluent in the step D by one of a methanol solution and an ethanol solution.

The weight percentage concentration of the methanol solution and the ethanol solution is 5 to 35 percent.

Compared with the prior art, the method has the advantages that the mixed solution of the nucleoside and the nucleotide is treated by the activated carbon packed column, the nucleoside which is not completely reacted is adsorbed, the purity of the collected nucleotide is high, the pure crystal liquid is beneficial to the precipitation of crystals, the content of effective components in the crystal mother liquor is reduced, and the quality and the yield of the nucleotide are improved.

The invention elutes the nucleoside adsorbed on the activated carbon packed column, distills the eluent, and discharges the nucleoside and nucleotide kettle bottom liquid at the bottom of the tower for enzyme catalysis reaction, so that the utilization rate of the nucleoside is improved. The nucleoside and nucleotide kettle bottom liquid contains a small amount of nucleotides, the conversion rate of the nucleoside cannot be influenced, if the nucleoside and nucleotide kettle bottom liquid is not recovered, the utilization rate of the nucleoside is about 90 percent, the nucleoside and nucleotide kettle bottom liquid is recovered, after the enzymatic reaction is put into use, the unreacted nucleoside is utilized, and the utilization rate of the nucleoside is greatly improved to over 97 percent.

The pH initial value of the enzyme catalysis reaction is set to be 4.0-5.5, phosphoric acid is generated along with the subsequent reaction, the pH value is naturally reduced, the nucleoside conversion rate is not obviously influenced, the pH value is prevented from being frequently adjusted in the reaction process in order to simplify the operation, and acid and alkali are not required to be added in order to maintain the pH value of the reaction liquid in the reaction process.

The initial temperature of the enzyme catalysis reaction (in the reaction time period from the initial enzyme catalysis stage to the previous reaction stage (1/15-2/3)) is set to be 15-30 ℃, so that the problems of high viscosity, agglomeration, difficulty in stirring and dispersion and inconvenience for uniform mass transfer of a reaction liquid caused by excessively high conversion speed in the initial reaction stage can be solved, the reaction speed is controlled in the initial stage, the mass transfer and heat transfer of the reaction are facilitated, the smooth proceeding of the reaction is facilitated, the temperature is increased to 30-50 ℃ in the remaining reaction time period of the enzyme catalysis, the reaction speed is facilitated to be accelerated, and the reaction is complete. The reaction time is not fixed due to the amount of the substrate, the enzyme activity, etc., so the total reaction time is usually 5 to 50 hours.

According to the invention, the pH value of the reaction solution is adjusted to 11-11.5, and the reaction solution is cooled and crystallized, wherein the generated sodium phosphate is mainly sodium phosphate, has lower solubility than disodium hydrogen phosphate, and is beneficial to separation and removal of phosphate.

The invention adopts the purification by the activated carbon packed column, and the nucleoside and nucleotide kettle bottom liquid is circularly used for the purification process of the enzyme catalysis reaction, thereby being beneficial to the improvement of quality and yield and the reduction of cost. According to the characteristics of the nucleotide, the concentration, the temperature and the pH value parameters in the reaction and purification stages are effectively regulated, so that the unreacted impurities can be effectively removed, the nucleoside can be recycled, the nucleoside utilization rate is improved, and the quality of the nucleotide salt is further improved by means of control of feeding formulas, adsorption purification and the like in each stage. The invention has important significance for industrial large-scale production of nucleotide.

Detailed Description

The method circularly utilizes the nucleoside and the eluent, has the characteristics of low process cost, high nucleoside utilization rate and high nucleotide product purity, overcomes the defects of low nucleoside utilization rate, high cost and low product purity in the prior art, and obviously improves the nucleoside utilization rate and the nucleotide quality.

The nucleoside utilization rate of the invention means: the molar ratio of nucleosides used for nucleotidyl acidification in the starting nucleosides used in nucleotide production. The nucleotide yield of the present invention is the weight ratio of disodium 5' -Inosinate (IMP), disodium 5' -Guanylate (GMP) and disodium 5' -ribonucleotide (I + G) obtained by purifying a nucleotide reaction solution to inosine and guanosine which are added as raw materials for reaction. The nucleoside recovery rate of the invention is as follows: after the reaction solution passes through an activated carbon packed column and is eluted, the nucleoside recovered from the tower bottom accounts for the proportion of the nucleoside substrate put into the reaction.

The acid phosphatase used in the present invention is commercially available or can be produced by a known fermentation method, and for example, the method described in patent application CN103642823B can be used.

The following will explain the present invention in more detail by way of examples. However, the following examples are only illustrative for explaining the present invention in detail, and the scope of the present invention is not limited to these examples.

Example 1

(1) Mixing 124.5g of inosine and 200g of acid sodium pyrophosphate with 350mL of water, uniformly stirring, adding 380mL of inosine eluted from an activated carbon packed column and distilled, and inosinic acid kettle bottom liquid containing 9.5g of inosine, adjusting the pH to 4.5 by using hydrochloric acid, adding 95mL of acid phosphatase, reacting at 26 ℃ for 3 hours, raising the temperature to 32 ℃ and reacting until the inosine conversion rate is 89.5 percent, and reacting for about 25 hours to obtain reaction liquid containing inosinic acid.

(2) And (3) adjusting the pH of the reaction solution to 11.2 by using a 50% sodium hydroxide solution, cooling to 5 ℃, cooling for crystallization, and separating phosphate to obtain a mixed solution of inosine and inosinic acid.

(3) Enabling a mixed solution of inosine and inosinic acid to flow through a 200mL activated carbon packed column, collecting an inosinic acid outflow part, adjusting the pH to 7.0 by hydrochloric acid, heating to 65-75 ℃, adding 0.5% powdered activated carbon, preserving heat for 30 minutes, filtering, cooling filtrate for crystallization, separating and crystallizing at 10 ℃, and drying to obtain 166.8g of finished 5' -disodium inosinate with the detection chromatographic purity of 99.1% and the yield of 124.5%.

(4) Eluting the activated carbon column with 12% ethanol solution, collecting eluent, distilling the eluent with a packed tower, recovering ethanol solution from the top of the tower for preparing the eluent in the step (4), discharging inosine and inosinic acid kettle bottom liquid from the bottom of the tower, recovering 10.5g of inosine, recovering 8% of inosine, and putting the inosine into the reaction system in the subsequent batch step (1) to serve as a reaction substrate. The inosine utilization rate in step (1) was 89.5%, and the inosine utilization rate in steps (1) to (4) was increased to 97.5%. In addition, the conversion rate of inosine in step (1) was not reduced.

Example 2

(1) Guanosine 131.4g, sodium pyrophosphate 265g and 950mL of water were mixed, and the mixture was stirred uniformly, 750mL of a bottom solution of guanosine and guanylic acid eluted from an activated carbon packed column and distilled, the bottom solution containing 10.1g of guanosine, the pH of the mixture was adjusted to 4.3 with hydrochloric acid, 135mL of acid phosphatase was added, the mixture was reacted at 27 ℃ for 3 hours, the temperature was raised to 44 ℃ and the reaction was carried out until the conversion of guanosine was 89.8%, and the reaction solution containing guanylic acid was obtained after about 27 hours of reaction.

(2) And (3) adjusting the pH value of the reaction solution to 11.7 by using a 50% sodium hydroxide solution, cooling to 3 ℃, cooling for crystallization, and separating phosphate to obtain a mixed solution of guanosine and guanylic acid.

(3) Enabling the mixed solution of guanosine and guanylic acid to flow through a 200mL activated carbon packed column, collecting the guanylic acid outflow part, adjusting the pH to 7.2 by hydrochloric acid, heating to 65-75 ℃, adding 0.7% powdered activated carbon, preserving the heat for 30 minutes, filtering, adding 500mL of 90% ethanol solution into the filtrate, cooling for crystallization, separating and crystallizing at 10 ℃, and drying to obtain 172.8g of finished product disodium 5' -guanylic acid, wherein the detection chromatographic purity is 99.3%, and the yield is 122.1%.

(4) Eluting the activated carbon column with 25% methanol solution, collecting eluent, distilling the eluent with a packed tower, recovering the methanol solution from the top of the tower for preparing the eluent in the step (4), discharging guanosine and guanylic acid kettle bottom liquid from the bottom of the tower, recovering 10.6g of guanosine, recovering 7.5% of guanosine, and putting the guanosine into a reaction system in the subsequent batch step (1) to serve as a reaction substrate. The guanosine utilization rate in step (1) was 89.8%, and the guanosine utilization rate was increased to 97.3% by the steps (1) to (4). In addition, the guanosine conversion rate in step (1) was not reduced thereby.

Example 3

(1) 61 inosine, 64.4g guanosine, 155g sodium acid pyrophosphate and 55g sodium tripolyphosphate were mixed with 770mL water, and stirred uniformly, 730mL of inosine, inosinic acid, guanosine and guanylic acid bottom solution eluted from an activated carbon packed column and distilled, containing 6 inosine and 5.6g guanosine, was added to the bottom solution, the pH was adjusted to 4.6 with hydrochloric acid, 155mL of acid phosphatase was added thereto, and the mixture was reacted at 28 ℃ for 3 hours, and then the temperature was raised to 35 ℃ to react at an inosine/guanylic acid conversion rate of 90.2% for about 30 hours, thereby obtaining a reaction solution containing inosinic acid and guanylic acid.

(2) And (3) adjusting the pH of the reaction solution to 11.5 by using a 50% sodium hydroxide solution, cooling to 2 ℃, cooling for crystallization, and separating phosphate to obtain a mixed solution of inosine and inosinic acid and guanosine and guanylic acid.

(3) Enabling a mixed solution of inosine and inosinic acid, guanosine and guanylic acid to flow through a 200mL activated carbon packed column, collecting the outflow parts of inosinic acid and guanylic acid, adjusting the pH to 7.1 by hydrochloric acid, heating to 65-75 ℃, adding 0.6% powdered activated carbon, preserving the temperature for 30 minutes, filtering, adding 500mL 90% ethanol solution into filtrate, cooling for crystallization, separating and crystallizing at 10 ℃, and drying to obtain 169.3g of finished 5' -flavor-developing nucleotide disodium, wherein the detection chromatographic purity is 99.5% and the yield is 123.6%.

(4) Eluting the activated carbon column with 20% ethanol solution, collecting eluate, distilling the eluate with a packed column, recovering ethanol solution from the top of the column, preparing eluate for the step (4), discharging inosine, inosinic acid, guanosine, and guanylic acid kettle bottom solution from the bottom of the column, recovering 5.3 g of inosine and 4.8g of guanosine, and putting the obtained product into the reaction system of the subsequent step (1) to serve as a reaction substrate, wherein the recovery rate of inosine and guanosine is 7.4%. The utilization rate of inosine and guanosine in step (1) was 90.2%, and the utilization rates of inosine and guanosine in steps (1) to (4) were increased to 97.6%. In addition, the conversion rates of inosine and guanosine in step (1) were not reduced.

In examples 1 to 3, the chromatographic purity of disodium 5' -inosinate, disodium 5' -guanylate, or disodium 5' -ribonucleotide was measured by high performance liquid chromatography. The specific method comprises the following steps:

mobile phase: 5.0g of monopotassium phosphate is weighed, placed in a 1000mL beaker, added with 1000mL of ultrapure water, stirred on a magnetic stirrer until the monopotassium phosphate is completely dissolved, and filtered for later use.

A chromatographic column: BDS-C18(5um, 4.6X 250mm)

Column temperature: 35 deg.C

Flow rate: 1.2mL/min

A detector: ultraviolet detector (lambda 254mm)

Sample introduction amount: 2uL

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.