A method for preparing (s)-ademetionine by enzymatic process
By catalyzing the reaction of ADP and L-methionine with polyphosphoric acid kinase and S-adenosylmethionine synthase, the problems of high pollution, high cost and low efficiency of existing SAM preparation methods are solved, and the preparation of (S)-adenosylmethionine with high efficiency and high chiral purity is achieved, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SYNCOZYMES SHANGHAI
- Filing Date
- 2024-12-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing SAM preparation methods suffer from problems such as high pollution, high cost, low production efficiency, and poor atom economy. In particular, fermentation methods produce products with low expression levels and complex compositions, while enzymatic methods have low atom economy.
(S)-Adenosylmethionine was prepared by catalyzing the reaction of ADP and L-methionine using polyphosphoric acid kinase and S-adenosylmethionine synthase. Enzyme powder or cell lysate expressed in Escherichia coli, yeast, Streptomyces or Bacillus subtilis was used. Reaction conditions such as pH, temperature and time were optimized. The product was purified by ion chromatography and treated with salt formation.
A highly efficient and chiral purity (S)-adenosylmethionine preparation method was achieved, suitable for industrial production, with yields and purity reaching 76-99.7% and chiral purity reaching 73.5-85.8%.
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Abstract
Description
Technical fields:
[0001] This invention belongs to the field of biocatalysis technology, specifically relating to an enzymatic method for preparing (S)-adenosylmethionine. Background technology:
[0002] (S)-Adenosyl-L-methionine (SAM) is an important metabolic intermediate widely found in organisms. It plays a crucial role in transmethylation, transsulfation, and transaminopropylation, thus serving an irreplaceable function in the methylation modification, sulfhydryl storage and transfer, and the synthesis of spermine and spermidine. SAM is also a valuable pharmaceutical molecule, but it exists in two configurations: (R,S)-Adenosyl-methionine and (S,S)-Adenosyl-methionine. Only (S,S)-Adenosyl-methionine possesses biological activity, and the United States Pharmacopeia (USP) stipulates that its content should be no less than 60%. Due to its poor stability and susceptibility to inactivation reactions, commercially available SAM is mostly in its stable salt form. For example, (S)-Adenosyl-methionine succinate disulfonate is widely used to treat metabolic disorders, arthritis, and depression, and has promising market prospects.
[0003] The main existing methods for preparing SAM include chemical methods, fermentation methods, and enzymatic methods. Chemical methods are less commonly used due to problems such as high pollution, high cost, and low production efficiency. Fermentation methods primarily obtain SAM through fermentation with *Saccharomyces cerevisiae*. However, because the product expression level in the fermentation broth is relatively low and the composition is complex, containing various amino acids, proteins, salts, and biologically inactive isomers, multiple processing and extraction purification steps are required to obtain high-purity (S,S)-adenosylmethionine. Enzymatic methods mainly use adenosine triphosphate (ATP) and methionine as substrates, specifically synthesizing biologically active (S,S)-adenosylmethionine under the catalysis of adenosylmethionine synthase. Enzymatic methods have advantages such as high efficiency, high specificity, and ease of purification; however, the large molecular weight of ATP results in lower atom economy.
[0004] Therefore, we need to develop a new method for preparing (S)-adenosylmethionine that is highly efficient, has high chiral purity, high atom economy, and is suitable for industrial production. Summary of the Invention:
[0005] The purpose of this invention is to provide a new method for preparing (S)-adenosylmethionine, addressing the shortcomings of existing technologies.
[0006] The technical solution adopted in this invention is shown in Scheme 1:
[0007]
[0008] This invention provides a method for preparing (S)-adenosylmethionine, specifically comprising the following steps: using ADP and L-methionine as raw materials, reacting them under the combined action of polyphosphoric acid kinase and S-adenosylmethionine synthase to obtain (S)-adenosylmethionine.
[0009] Furthermore, the polyphosphate kinase is selected from Eumeta japonica, and the wild-type template has the NCBI accession number GBP06136.1.
[0010] Furthermore, the S-adenosylmethionine synthase is selected from Actinobacteria bacterium OK006, and the wild-type template has the NCBI accession number KPI09524.1.
[0011] Furthermore, the polyphosphate kinase and S-adenosylmethionine synthase participate in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenate, cell slurry containing polyphosphate kinase or S-adenosylmethionine synthase, immobilized cells, immobilized enzymes, etc., preferably cell slurry.
[0012] Furthermore, the polyphosphate kinase and S-adenosylmethionine synthase expression receptor strains are selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.
[0013] Furthermore, the concentration of the polyphosphokinase is selected from 2 to 10 g / L, preferably 5 to 8 g / L.
[0014] Furthermore, the concentration of the S-adenosylmethionine synthase is selected from 5 to 25 g / L, preferably 15 to 20 g / L.
[0015] Furthermore, the concentration of the substrate ADP is selected from 10 to 50 g / L, preferably 25 to 30 g / L.
[0016] Furthermore, the concentration of the substrate L-methionine is selected from 5 to 30 g / L, preferably 12 to 15 g / L.
[0017] Furthermore, the pH value of the enzymatic reaction is selected from 5.0 to 8.0, preferably 6.0 to 6.3.
[0018] Furthermore, the temperature of the enzymatic reaction is selected from 20 to 50°C, preferably 30 to 35°C.
[0019] Furthermore, the reaction time of the enzymatic method is selected from 1 to 24 hours, preferably 4 to 8 hours.
[0020] The beneficial effects of this invention are that it discloses a method for preparing (S)-adenosylmethionine with high production efficiency, high chiral purity, high atom economy, and suitability for industrial production. Attached image description:
[0021] Figure 1 Example 1: HPLC chromatogram of (S)-adenosylmethionine butanedisulfonate
[0022] Figure 2 Example 1: Chiral HPLC Spectrum of (S)-Adenosylmethionine Butylsulfonate
[0023] Figure 3 Example 2: HPLC chromatogram of (S)-adenosylmethionine butanedisulfonate
[0024] Figure 4 Example 2: Chiral HPLC chromatogram of (S)-S-adenosylmethionine butanedisulfonate
[0025] Figure 5 Example 3: HPLC chromatogram of (S)-adenosylmethionine p-toluenesulfonate disulfate
[0026] Figure 6 Example 3: Chiral HPLC chromatogram of (S)-adenosylmethionine p-toluenesulfonic acid disulfate
[0027] Figure 7 Example 4: HPLC chromatogram of (S)-adenosylmethionine p-toluenesulfonate disulfate
[0028] Figure 8 Example 4: Chiral HPLC chromatogram of (S)-adenosylmethionine p-toluenesulfonic acid disulfate Detailed implementation method:
[0029] The technical content of the present invention will be further described below with reference to specific embodiments, in order to better understand the content of the present invention, but the scope of protection of the present invention is not limited thereto.
[0030] Example 1
[0031] Take a 5L three-necked flask and equip it with a mechanical stirrer, a 0-50℃ thermometer, an automatic pH titrator, and a constant temperature water bath. Weigh 119.60g of ADP, 66.00g of L-methionine, 85.65g of sodium hexametaphosphate, 61.00g of magnesium chloride hexahydrate, and 63.26g of zinc sulfate heptahydrate into the three-necked flask. Add 4.2L of water and stir to dissolve (the reaction liquid volume is about 4.4L). Heat to 30℃ and adjust the pH to 6.0 with Na2CO3 aqueous solution. Then add 88.00g of S-adenosylmethionine synthase (bacterial culture) and 35.20g of polyphosphoric acid kinase (bacterial culture) in sequence. Control the pH at 6.0 and the temperature at 30℃, and stir the reaction for 8 hours. Enzyme removal was achieved by centrifugation and ultrafiltration using a Merrick MD80R high-speed refrigerated centrifuge. The product was then purified by ion chromatography, reacted with 127.60 g of 60% succinic acid aqueous solution to form a salt, and finally spray-dried to obtain 161.42 g of (S)-adenosylmethionine succinic acid disulfonate (1:1.65), with a yield of 76%, purity of 99.3%, and chiral purity of 84.3%. HPLC results are as follows: Figure 1 As shown, chiral HPLC Figure 2 As shown.
[0032] Example 2
[0033] Take a 5L three-necked flask and equip it with a mechanical stirrer, a 0-50℃ thermometer, an automatic pH titrator, and a constant temperature water bath. Weigh 98.25g of ADP, 52.80g of L-methionine, 71.30g of sodium hexametaphosphate, 50.63g of magnesium chloride hexahydrate, and 52.50g of zinc sulfate heptahydrate into the three-necked flask. Add 4.2L of water and stir to dissolve (the reaction volume is about 4.4L). Heat to 30℃ and adjust the pH to 6.3 with Na2CO3 aqueous solution. Then add 66.00g of S-adenosylmethionine synthase (bacterial culture) and 22.00g of polyphosphoric acid kinase (bacterial culture) in sequence. Control the pH at 6.0 and the temperature at 35℃, and stir the reaction for 4 hours. Enzyme removal was achieved by centrifugation and ultrafiltration using a Merrick MD80R high-speed refrigerated centrifuge. The resulting product was then purified by ion chromatography, reacted with 102.10 g of a 60% aqueous solution of succinic acid to form a salt, and finally freeze-dried to obtain 129.10 g of (S)-adenosylmethionine succinic acid disulfonate (1:1.65), with a yield of 74%, purity of 99.7%, and chiral purity of 85.8%. HPLC results are as follows: Figure 3 As shown, chiral HPLC Figure 4 As shown.
[0034] Example 3
[0035] Take a 5L three-necked flask and equip it with a mechanical stirrer, a 0-50℃ thermometer, an automatic pH titrator, and a constant temperature water bath. Weigh 119.60g of ADP, 66.00g of L-methionine, 85.65g of sodium hexametaphosphate, 61.00g of magnesium chloride hexahydrate, and 63.26g of zinc sulfate heptahydrate into the three-necked flask. Add 4.2L of water and stir to dissolve (the reaction liquid volume is about 4.4L). Heat to 30℃ and adjust the pH to 6.0 with Na2CO3 aqueous solution. Then add 88.00g of S-adenosylmethionine synthase (bacterial culture) and 35.20g of polyphosphoric acid kinase (bacterial culture) in sequence. Control the pH at 6.0 and the temperature at 30℃, and stir the reaction for 8 hours. Enzyme removal was achieved by centrifugation and ultrafiltration using a Merrick MD80R high-speed refrigerated centrifuge. The resulting product was then purified by ion chromatography, reacted with 38.10 g of p-toluenesulfonic acid and 43.40 g of sulfuric acid to form a salt, and finally freeze-dried to obtain 169.61 g of (S)-adenosylmethionine p-toluenesulfonic acid disulfate (1:1:2), with a yield of 79%, purity of 99.6%, and chiral purity of 76.4%. HPLC results are as follows: Figure 5 As shown, chiral HPLC Figure 6 As shown.
[0036] Example 4
[0037] Take a 5L three-necked flask and equip it with a mechanical stirrer, a 0-50℃ thermometer, an automatic pH titrator, and a constant temperature water bath. Weigh 98.25g of ADP, 52.80g of L-methionine, 71.30g of sodium hexametaphosphate, 50.63g of magnesium chloride hexahydrate, and 52.50g of zinc sulfate heptahydrate into the three-necked flask. Add 4.2L of water and stir to dissolve (the reaction volume is about 4.4L). Heat to 30℃ and adjust the pH to 6.3 with Na2CO3 aqueous solution. Then add 66.00g of S-adenosylmethionine synthase (bacterial culture) and 22.00g of polyphosphoric acid kinase (bacterial culture) in sequence. Control the pH at 6.0 and the temperature at 35℃, and stir the reaction for 4 hours. Enzyme removal was achieved by centrifugation and ultrafiltration using a Merrick MD80R high-speed refrigerated centrifuge. The resulting product was then purified by ion chromatography, reacted with 30.50 g of p-toluenesulfonic acid and 34.70 g of sulfuric acid to form a salt, and finally spray-dried to obtain 135.80 g of (S)-adenosylmethionine p-toluenesulfonic acid disulfate (1:1:2), with a yield of 77%, purity of 99.5%, and chiral purity of 73.5%. HPLC analysis is as follows: Figure 7 As shown, chiral HPLC Figure 8 As shown.
Claims
1. A method for the enzymatic preparation of (S)-adenosylmethionine, characterized in that, The method uses ADP and L-methionine as substrates, and reacts them under the combined action of polyphosphate kinase and S-adenosylmethionine synthase to obtain (S)-adenosylmethionine. The preparation method is shown below.
2. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The polyphosphokinase was selected from Eumeta japonica.
3. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The adenosylmethionine synthase is selected from Actinobacteria bacterium OK006.
4. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The concentration of the polyphosphokinase is selected from 2 to 10 g / L.
5. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The concentration of the S-adenosylmethionine synthase is selected from 5 to 25 g / L.
6. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The concentration of the substrate adenosine diphosphate is selected from 10 to 50 g / L.
7. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The concentration of the substrate L-methionine is selected from 5 to 30 g / L.
8. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The pH value of the enzymatic reaction is selected from 5.0 to 8.
0.
9. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The temperature of the enzymatic reaction is selected from 20 to 50°C.
10. The method for preparing (S)-adenosylmethionine according to claim 1, characterized in that, The reaction time for the enzymatic method is selected from 1 to 24 hours.