阅读说明：本技术 一种胞磷胆碱钠的合成方法 (Synthesis method of citicoline sodium ) 是由 何魁芳 胡东晓 马克·博科拉 蔡宝琴 张城孝 胡虎 罗霄 彭沁利 崔琴燕 刘思彤 于 2020-08-31 设计创作，主要内容包括：本发明公开了一种胞磷胆碱钠的合成方法,包括以下步骤：向反应容器中加入5’-胞苷酸,氯化磷酸胆碱钙盐,ATP,氯化镁水溶液,Tris-HCl缓冲液,混合均匀；接着,调节pH值至6.0～8.0,并加入CMP磷酸激酶、核苷二磷酸激酶和磷酸胆碱-胞苷酰转移酶,在25～50℃下搅拌反应4～10小时,高速离心后,取上清液,即得所述胞磷胆碱钠；与传统的胞磷胆碱钠合成工艺相比,本发明提供的合成路线对环境友好,产率稳定,有利于节省能耗和保护环境,因此适用于大规模工业化生产。(The invention discloses a method for synthesizing citicoline sodium, which comprises the following steps: adding 5' -cytidylic acid, choline calcium chlorophosphate, ATP, magnesium chloride aqueous solution and Tris-HCl buffer solution into a reaction container, and uniformly mixing; then, adjusting the pH value to 6.0-8.0, adding CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase, stirring and reacting for 4-10 hours at the temperature of 25-50 ℃, centrifuging at a high speed, and taking supernatant to obtain the citicoline sodium; compared with the traditional citicoline sodium synthesis process, the synthesis route provided by the invention is environment-friendly, has stable yield, is beneficial to saving energy consumption and protecting the environment, and is suitable for large-scale industrial production.)

1. A method for synthesizing citicoline sodium is characterized by comprising the following steps:

adding 5' -cytidylic acid, choline calcium chlorophosphate, ATP, magnesium chloride aqueous solution and Tris-HCl buffer solution into a reaction container, and uniformly mixing; then, adjusting the pH value to 6.0-8.0, adding CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase, stirring and reacting for 4-10 hours at the temperature of 25-50 ℃, centrifuging at a high speed, and taking supernatant to obtain the citicoline sodium;

wherein the amino acid sequence of the CMP phosphokinase is shown as SEQ ID NO.1, the amino acid sequence of the nucleoside diphosphate kinase is shown as SEQ ID NO.2, and the amino acid sequence of the phosphorylcholine-cytidine acyl transferase is shown as SEQ ID NO. 3.

2. The method for synthesizing citicoline sodium according to claim 1, wherein the concentrations of said CMP phosphokinase, said nucleoside diphosphate kinase and said phosphorylcholine-cytidine acyltransferase in the reaction system are 1 to 50g/L, respectively.

3. The method for synthesizing citicoline sodium according to claim 1, wherein the concentration of 5' -cytidylic acid in the reaction system is 5-150 mmol/L.

4. The method for synthesizing citicoline sodium according to claim 1, wherein the concentration of the choline chloride calcium salt is 15-200 mmol/L in the reaction system.

5. The method for synthesizing citicoline sodium according to claim 1, wherein the ATP concentration in the reaction system is 6-200 mmol/L.

6. The method for synthesizing citicoline sodium according to claim 1, wherein said CMP phosphate kinase, said nucleoside diphosphate kinase, and said phosphorylcholine-cytidine acyltransferase are prepared by methods comprising:

s1: cloning a gene corresponding to the CMP phosphokinase or the nucleoside diphosphate kinase or the phosphorylcholine-cytidine acyltransferase onto a pACYC-DUET1 vector, and transforming into a host cell of BL21(DE3) Escherichia coli;

s2: inoculating the cloned engineering bacteria seeds of the enzyme into a culture system, culturing for 18-24 hours, and centrifugally collecting thalli after the culture is finished;

s3: and (3) suspending the thalli in 50mM Tris-HCl buffer solution, crushing to obtain a crushing solution, and performing high-speed centrifugation on the crushing solution to obtain an enzyme solution supernatant.

7. The method for synthesizing citicoline sodium according to claim 6, wherein in step S3, the pH value of the Tris-HCl buffer solution is 7.5.

8. The method for synthesizing citicoline sodium according to claim 6, wherein in step S3, the suspension ratio of the cell mass to the Tris-HCl buffer solution is 1:10 to 1:20 g/mL.

Technical Field

The invention relates to the technical field of enzyme catalysis, in particular to a method for synthesizing citicoline sodium.

Background

Citicoline sodium is also called citicoline sodium, is a brain metabolism activator, is a nucleoside derivative, is a precursor of phosphatidylcholine, and is a coenzyme necessary for lecithin synthesis. Research shows that the citicoline sodium has the functions of repairing brain injury, resisting anoxia, improving memory and enhancing intelligence, and has wide clinical application.

Therefore, the preparation method of citicoline sodium is a research topic which is relatively concerned by researchers.

Disclosure of Invention

Aiming at various technical defects in the prior art, the invention aims to provide a novel preparation method of citicoline sodium, which takes free enzyme expressed by escherichia coli as a biocatalyst and 5' -cytidylic acid, phosphorylcholine and ATP as reaction raw materials to carry out biotransformation so as to successfully prepare a target product; meanwhile, the technical scheme provided by the invention also solves the technical problems of limited enzyme source and overhigh synthesis cost.

Meanwhile, the invention provides a new synthetic route of citicoline sodium, which is shown as follows:

specifically, the method for synthesizing citicoline sodium comprises the following steps:

adding 5' -cytidylic acid, choline calcium chlorophosphate, ATP, magnesium chloride aqueous solution and Tris-HCl buffer solution into a reaction container, and uniformly mixing; then, adjusting the pH value to 6.0-8.0, adding CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase, stirring and reacting for 4-10 hours at the temperature of 25-50 ℃, centrifuging at a high speed, and taking supernatant to obtain the citicoline sodium;

wherein the amino acid sequence of the CMP phosphokinase is shown as SEQ ID NO.1, the amino acid sequence of the nucleoside diphosphate kinase is shown as SEQ ID NO.2, and the amino acid sequence of the phosphorylcholine-cytidine acyl transferase is shown as SEQ ID NO. 3; see table 1 below for details:

table 1 amino acid sequence listing

Preferably, in the method for synthesizing citicoline sodium, the concentrations of the CMP phosphate kinase, the nucleoside diphosphate kinase and the phosphorylcholine-cytidine acyltransferase in a reaction system are 1 to 50g/L, respectively.

Preferably, in the method for synthesizing citicoline sodium, the concentration of the 5' -cytidylic acid in the reaction system is 5-150 mmol/L.

Preferably, in the method for synthesizing citicoline sodium, the concentration of the choline chloride calcium salt is 15-200 mmol/L in the reaction system.

Preferably, in the method for synthesizing citicoline sodium, the concentration of ATP in the reaction system is 6-200 mmol/L.

It should be noted that the concentrations of the respective substances in the above preferred embodiment, for example, the concentration of 5' -cytidylic acid, the concentration of choline phosphochloridate calcium, the concentration of ATP, etc., are relative to the total volume of the catalyst system (for example, the effective volume of the reaction tank), in other words, the above concentrations describe their respective concentrations relative to the total volume of the catalyst system.

Preferably, in the above method for synthesizing citicoline sodium, the CMP phosphate kinase, the nucleoside diphosphate kinase, and the phosphorylcholine-cytidine acyltransferase are prepared by methods comprising:

s1: cloning a gene corresponding to the CMP phosphokinase or the nucleoside diphosphate kinase or the phosphorylcholine-cytidine acyltransferase onto a pACYC-DUET1 vector, and transforming into a host cell of BL21(DE3) Escherichia coli;

s2: inoculating the cloned engineering bacteria seeds of the enzyme into a culture system, culturing for 18-24 hours, and centrifugally collecting thalli after the culture is finished;

wherein, the volume of the seed liquid of 3 different engineering bacteria is 5-10% of the volume of the culture medium;

s3: suspending the thalli in 50mM Tris-HCl buffer solution, crushing to obtain a crushing solution, and centrifuging the crushing solution at a high speed to obtain enzyme solution supernatants of 3 different enzymes; wherein the disruption is carried out using an ultrasonic disruptor or a homogenizer.

Further preferably, in step S3 of the above method for synthesizing citicoline sodium, the Tris-HCl buffer has a pH of 7.5.

Further preferably, in step S3 of the method for synthesizing citicoline sodium, the suspension ratio of the cell mass to the Tris-HCl buffer solution is 1:10 to 1:20 g/mL. It should be noted that the cell mass herein refers to a wet cell mass.

In summary, compared with the prior art, the technical scheme provided by the invention at least has the following beneficial effects:

3 different enzymes are prepared by adopting escherichia coli engineering bacteria of molecular cloning CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase, and the enzyme sources are stable, so that the defects of workshop environmental pollution and treatment of a large amount of waste yeast residues caused by a reaction system of beer yeast generally adopted in China at present are avoided; the adoption of CMP as one of the synthetic raw materials has a significant cost advantage over the CTP used in the prior art as one of the synthetic raw materials.

In a word, compared with the traditional citicoline sodium synthesis process, the synthesis route provided by the invention is environment-friendly, has stable yield, is beneficial to saving energy consumption and protecting the environment, and is suitable for large-scale industrial production.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 protection scope of the present invention.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The method for synthesizing citicoline sodium according to the preferred embodiment mainly comprises the following steps: utilizing escherichia coli engineering bacteria of molecular cloning CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase to prepare the above 3 enzymes and provide enzyme liquid supernatant of the 3 enzymes; the method comprises the steps of using calcium choline chloride phosphate, ATP and CMP as raw materials, and using the three enzymes to catalyze and generate citicoline sodium together, wherein the amino acid sequence of the used CMP phosphokinase is shown as SEQ ID No.1, the amino acid sequence of the used nucleoside diphosphate kinase is shown as SEQ ID No.2, and the amino acid sequence of the used phosphorylcholine-cytidine acyl transferase is shown as SEQ ID No. 3.

In a preferred embodiment, the method for synthesizing citicoline sodium comprises the following steps:

adding 5' -cytidylic acid, choline calcium chlorophosphate, ATP, magnesium chloride aqueous solution and Tris-HCl buffer solution into a reaction container, and uniformly mixing; then, adjusting the pH value to 6.0-8.0, adding CMP phosphokinase, nucleoside diphosphate kinase and phosphorylcholine-cytidine acyl transferase, stirring and reacting for 4-10 hours at the temperature of 25-50 ℃, centrifuging at a high speed, and taking supernatant to obtain the citicoline sodium;

wherein the amino acid sequence of the CMP phosphokinase is shown as SEQ ID NO.1, the amino acid sequence of the nucleoside diphosphate kinase is shown as SEQ ID NO.2, and the amino acid sequence of the phosphorylcholine-cytidine acyl transferase is shown as SEQ ID NO. 3;

wherein the CMP phosphate kinase, the nucleoside diphosphate kinase, and the phosphorylcholine-cytidine acyltransferase are prepared by methods that respectively comprise:

s1: cloning a gene corresponding to the CMP phosphokinase or the nucleoside diphosphate kinase or the phosphorylcholine-cytidine acyltransferase onto a pACYC-DUET1 vector, and transforming into a host cell of BL21(DE3) Escherichia coli;

s2: inoculating the cloned engineering bacteria seeds of the enzyme into a culture system, culturing for 18-24 hours, and centrifugally collecting thalli after the culture is finished;

s3: and (3) suspending the thalli in 50mM Tris-HCl buffer solution, crushing to obtain a crushing solution, and performing high-speed centrifugation on the crushing solution to obtain an enzyme solution supernatant.

In a further preferred embodiment, the concentrations of the CMP phosphate kinase, the nucleoside diphosphate kinase and the phosphorylcholine-cytidine acyltransferase in the reaction system are 1 to 50g/L, respectively.

In a further preferred embodiment, the concentration of the 5' -cytidylic acid in the reaction system is 5-150 mmol/L.

In a further preferred embodiment, the concentration of the choline phosphocholine chloride calcium salt in the reaction system is 15-200 mmol/L.

In a further preferred embodiment, the ATP concentration in the reaction system is 6-200 mmol/L.

In a further preferred embodiment, in step S3, the Tris-HCl buffer has a pH of 7.5.

In a further preferred embodiment, in step S3, the suspension ratio of the cell mass to the Tris-HCl buffer solution is 1: 10-1: 20 g/mL.