A process for preparing high-purity adenosylmethionine butanedisulfonate

Abstract

The present invention relates to the field of biochemical engineering technology, and discloses a high-purity adenosylmethionine butane disulfonate preparation process, comprising the following steps: step 1, enzyme screening: selecting a catalytic enzyme that catalyzes the reaction of adenylic acid and butane disulfonic acid; step 2, enzyme expression and purification: expressing the catalytic enzyme by genetic engineering technology, and purifying by affinity chromatography to obtain an enzyme with a purity higher than 90%; step 3, reaction condition optimization: adjusting substrate concentration, pH value, temperature and additive conditions; step 4, enzyme-linked catalytic reaction. By optimizing the temperature and solvent system of the enzyme catalytic reaction, the reaction conversion rate is significantly improved, thereby increasing the yield of SAM. Under the optimized reaction conditions, the enzyme catalytic reaction shows the best reaction efficiency at 60°C, and can effectively convert the raw material into a high-yield SAM. In this way, the utilization rate of the raw material is improved, and at the same time, the reaction time can be reduced, the energy consumption can be reduced, and the production efficiency can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, in particular to a process for preparing high-purity adenosylmethionine butanedisulfonate. Background Art

[0002] As an important biochemical substance, SAM is widely used in medicine, agriculture, life science and other fields. Due to its key role in regulating methylation reactions in organisms, SAM is widely used in the research and development of antioxidants and anti-tumor drugs, as well as as a cofactor for cell and tissue growth. With the increasing demand for SAM, how to improve its production efficiency and purity and reduce production costs has become an important research topic in the current biochemical industry.

[0003] At present, the production of adenosylmethionine butanedisulfonate is mainly through chemical synthesis and enzyme catalysis. Although the chemical synthesis method can obtain higher yields under certain conditions, its process often involves complex chemical reactions, high temperatures and pressures, and usually requires the use of toxic solvents, which has problems of environmental pollution and operational hazards. The enzyme catalysis method, as a more environmentally friendly and efficient production method, has gradually attracted attention. The enzyme catalysis method usually has high selectivity and mild reaction conditions, can efficiently catalyze reactions at lower temperatures, and the reaction process has less negative impact on the environment. Therefore, the development of an efficient, green and economical enzyme catalysis method has become a research focus.

[0004] Although the existing enzyme catalysis method has achieved the synthesis of adenosylmethionine butanedisulfonate to a certain extent, there are still some shortcomings in the prior art. In the existing enzyme catalysis production process, factors such as reaction temperature, solvent selection, reaction conversion rate are still difficult to achieve optimal balance, resulting in low yield, low product purity, and incomplete removal of impurities in the production process, which limits the industrial application of the technology to a certain extent. In addition, the existing process often requires a long reaction time, reduces production efficiency, and increases energy consumption and cost. In summary, the prior art is in urgent need of improvement to achieve a production process with high yield, high purity, low energy consumption and environmental protection. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a process for preparing high-purity adenosylmethionine butanedisulfonate, which solves the problems of low yield, insufficient purity, low reaction efficiency and unstable production process in the existing enzyme catalysis process for producing adenosylmethionine butanedisulfonate.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: A process for preparing high-purity adenosylmethionine butanedisulfonate, comprising the following steps:

[0007] Step 1, enzyme screening: selecting an enzyme that catalyzes the reaction of adenylic acid and butanedisulfonic acid;

[0008] Step 2: Enzyme expression and purification: expressing the catalytic enzyme by genetic engineering technology, and purifying the enzyme by affinity chromatography to obtain an enzyme with a purity higher than 90%;

[0009] Step 3: Optimization of reaction conditions: adjusting substrate concentration, pH value, temperature and additive conditions;

[0010] Step 4, enzyme-linked catalytic reaction: using a multi-enzyme system to catalyze the reaction of adenylic acid and butanedisulfonic acid;

[0011] Step 5: Purification of the reaction product: removing impurities in the reaction product by solvent extraction and column chromatography to obtain adenosylmethionine butanedisulfonate with a purity greater than 95%.

[0012] Preferably, the catalytic enzyme is one or more of AMPase, DSS sulfonate enzyme and NADH regenerating enzyme, and the enzyme-linked catalytic system is used for the reaction of AMP and DSS.

[0013] Preferably, the enzyme screening step is to obtain a catalytic enzyme with high catalytic efficiency, high temperature resistance and acid resistance by screening a gene library, and the enzyme activity is determined by HPLC.

[0014] Preferably, the enzyme expression and purification includes genetic engineering of the catalytic enzyme to optimize the active center or substrate binding site of the enzyme.

[0015] Preferably, the step of optimizing the reaction conditions comprises: adjusting the substrate concentration, reaction temperature and pH value of catalytic adenylic acid and butanedisulfonic acid, wherein the substrate concentration is 0.1 to 5 mM, the reaction temperature is 50° C. to 60° C., and the pH value is 5.5 to 8.0.

[0016] Preferably, the enzyme-linked catalytic reaction step comprises using AMPase, DSS sulfonase and NADH regenerating enzyme in combination, the reaction temperature is 40 to 60° C., and the reaction time is 4 to 12 hours.

[0017] Preferably, the purification of the reaction product includes the combined use of solvent extraction and column chromatography to remove unreacted substrates, enzymes and by-products in the reaction to obtain adenosylmethionine butanedisulfonate with a purity greater than 95%.

[0018] Preferably, the buffer in the reaction is a phosphate buffer or a Tris buffer, and the pH value ranges from 5.5 to 8.0.

[0019] The present invention provides a process for preparing high-purity adenosylmethionine butanedisulfonate. It has the following beneficial effects:

[0020] 1. The present invention significantly improves the reaction conversion rate and thus the yield of SAM by optimizing the temperature and solvent system of the enzyme-catalyzed reaction. Under the optimized reaction conditions, the enzyme-catalyzed reaction exhibits the best reaction efficiency at 60°C and can effectively convert the raw materials into high-yield SAM. In this way, the utilization rate of the raw materials is improved, and the reaction time can be reduced, energy consumption can be reduced, and production efficiency can be improved.

[0021] 2. The present invention adopts an optimized liquid-liquid extraction and subsequent purification process to effectively remove impurities generated during the reaction process. In the liquid-liquid extraction, ethyl acetate as a solvent has a high solubility difference, so that SAM can be effectively separated from the reaction mixture. Through the subsequent gel filtration chromatography and RP-HPLC purification steps, the purity of SAM is further improved, and the purity of the final product can reach more than 92%. This process not only ensures the high purity of the product, but also reduces the impact of impurities on subsequent processing and the final product.

[0022] 3. The present invention avoids the problem of enzyme inactivation caused by excessively high temperature while ensuring the enzyme catalytic reaction rate by optimizing the reaction temperature. The reaction temperature in the embodiment shows the best effect at 60°C, at which the catalytic activity of the enzyme remains high, the reaction rate is fast, and the product conversion rate is high, avoiding the risk of decreased enzyme catalytic effect at excessively high temperature. Compared with traditional processes, temperature optimization makes the enzyme catalytic reaction more stable and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Embodiment 1:

[0026] Please refer to the attached Figure 1 The embodiment of the present invention provides a high-purity adenosylmethionine butanedisulfonate preparation process, comprising the following steps:

[0027] Step S1: Enzyme screening and preliminary evaluation

[0028] In the preparation process of the present invention, the enzyme catalysis reaction is one of the core steps. In order to ensure the high efficiency and selectivity of the reaction, it is first necessary to screen out the enzyme that can catalyze the reaction of adenylic acid (AMP) and disulfonic acid (DSS), and conduct a preliminary evaluation of the screened enzymes to select the enzyme type with the best catalytic performance. This step is to screen and evaluate the activity of enzymes from different sources to finally determine the enzyme system suitable for the present invention.

[0029] In this embodiment, firstly, enzymes that may have catalytic ability for the reaction of AMP and DSS are selected from a known catalyst database through gene library screening and literature research. These enzymes include AMPase, DSS sulfonate enzyme and NADH regeneration enzyme, etc. These enzymes play different roles in the enzyme-linked catalytic reaction and can significantly improve the reaction efficiency. AMPase can convert AMP into adenosine, while DSS sulfonate enzyme can catalyze the combination of DSS and adenosine to generate the final butanedisulfonic acid adenosylmethionine (SAM). At the same time, NADH regeneration enzyme can provide the reducing power required for the reaction, ensuring the effective transfer of energy during the reaction.

[0030] In the screening process, the catalytic properties of different enzyme sources are also compared and analyzed in this embodiment to screen out enzymes that can efficiently catalyze the reaction of AMP and DSS and have high thermal stability and acid resistance, so as to avoid the situation of reduced catalytic efficiency or inactivation during the reaction. In addition, genetic engineering technology is used to conduct a preliminary evaluation of the screened enzymes, including the use of analytical methods such as HPLC (high performance liquid chromatography) to measure the catalytic activity and stability of the enzymes under different reaction conditions. For enzymes that perform well, further activity determination and further screening are performed to ensure their feasibility in the actual process.

[0031] Under experimental conditions, the selected enzyme needs to maintain a high catalytic efficiency at a certain temperature, pH value and substrate concentration, and can effectively promote the reaction of AMP and DSS. Specifically, in this embodiment, the catalytic effects of different enzymes at AMP concentrations of 0.1mM to 5mM, DSS concentrations of 1mM to 10mM, temperatures ranging from 50°C to 60°C, and pH ranges of 5.5 to 8.0 were tested, and the most suitable enzyme was finally screened out as a catalyst for subsequent steps.

[0032] In order to ensure the stability of the enzyme and the efficient reaction, the present invention also optimizes the reaction environment of the enzyme catalysis. By adding an appropriate amount of stabilizer (such as glycerol, BSA, etc.) and adjusting the composition of the reaction buffer, the reaction efficiency and tolerance of the enzyme are further improved.

[0033] In summary, step S1 mainly lays the foundation for subsequent enzyme engineering and reaction condition optimization by screening and preliminary evaluation of the catalytic enzyme, ensuring that the subsequent enzyme-linked catalytic reaction can be carried out under stable and efficient conditions. This step not only ensures the catalytic efficiency of the reaction, but also provides an efficient and stable enzyme system for the entire process of the present invention.

[0034] Step S1: Enzyme expression and purification

[0035] In step S1, the enzyme species suitable for catalyzing the reaction of AMP and DSS are obtained by screening, and then the screened enzyme needs to be expressed and purified to ensure that a sufficient amount of enzyme is obtained and that the activity and purity of the enzyme are suitable for the subsequent catalytic reaction. Enzyme expression and purification is one of the key steps in implementing the process of the present invention, and directly affects the efficiency of the catalytic reaction and the quality of the final product.

[0036] In the present embodiment, intestinal bacteria (Escher i ch ia co li) was selected as the enzyme expression system, and the target enzyme was efficiently expressed in intestinal bacteria by transforming the expression vector using genetic engineering technology. First, the gene fragment of the target enzyme was amplified by PCR, and cloned into the expression vector containing a strong promoter (such as T7 promoter) so as to be efficiently expressed in intestinal bacteria. The expression vector contains a suitable selection marker gene (such as an antibiotic resistance gene) for screening the successful bacterial strains of the transformation.

[0037] The optimization of the expression system includes selecting a suitable host strain, induction conditions and culture conditions. In the present embodiment, expression was performed in the Escherichia coli BL21 (DE3) host strain, which is widely used for efficient protein expression. In order to obtain a higher expression amount, the expression conditions of the enzyme were optimized by adjusting the induction temperature, the concentration of the inducer (such as IPTG) and the culture time in the experiment. Typically, the induction temperature is selected between 20°C and 30°C, the inducer concentration is controlled at 0.1mM to 1mM, and the induction time is 4 hours to 8 hours.

[0038] After expression is completed, in this embodiment, the cells are collected and lysed by cell disruption, and commonly used methods include ultrasonic disruption or chemical lysis (such as using lysozyme). Cell debris is removed by centrifugation to obtain a supernatant containing the target enzyme. For poorly soluble proteins, it may be necessary to use a solvent or add a cofactor to promote the dissolution of the protein.

[0039] Subsequently, in this embodiment, affinity chromatography is used to perform preliminary purification of the expression product. Affinity chromatography is based on the affinity between the enzyme and the specific ligand, and purification is performed by selecting a suitable affinity column. Common affinity columns include Ni-NTA columns (for enzymes containing His tags) and GST columns (for enzymes containing GST tags). For the target enzyme in the present invention, Ni-NTA affinity chromatography columns are used for purification, and the His tags at the N-terminal or C-terminal ends of the target enzyme are used to form coordination with Ni ions, and selectively bind to the affinity column.

[0040] After affinity chromatography purification, gel filtration chromatography (molecular sieve chromatography) is further used in this embodiment to finely separate the purified enzyme to remove possible small molecule impurities and non-specific binding proteins. This method can obtain a target enzyme with a purity greater than 90%, and further ensure that it has high catalytic activity and stability.

[0041] Finally, in this example, HPLC was used to detect the catalytic activity of the purified enzyme and determine the concentration of the enzyme. By concentrating the enzyme solution, it was ensured that the obtained enzyme had a sufficient concentration (usually above 5 mg / mL) for subsequent catalytic reactions.

[0042] In summary, step S2 achieves large-scale production of the target enzyme through the E. coli expression system, and efficiently purifies the enzyme through affinity chromatography and gel filtration, providing a high-purity and high-activity enzyme source for the subsequent enzyme-linked catalytic reaction. This step ensures that the enzyme catalytic reaction in the process is carried out under stable and efficient conditions, further improving the quality and yield of the product.

[0043] Step S3: Enzyme engineering optimization

[0044] In the aforementioned steps S1 and S2, an enzyme system for catalytic reaction is obtained by enzyme screening and purification. However, in order to further improve the reaction efficiency, stability and substrate conversion rate, enzyme engineering optimization becomes a key step. Through genetic engineering modification of enzymes, the catalytic performance of enzymes can be improved at the molecular level, making them more adaptable to reaction conditions, especially the efficient catalysis of AMP and DSS reactions. This step improves the activity, thermal stability and substrate affinity of the target enzyme by performing gene mutation, directed evolution and structural optimization on the target enzyme.

[0045] In the present embodiment, the directed evolution technique is first used to construct a mutant library of the target enzyme. Directed evolution technique is performed by randomly mutating the enzyme gene, and obtaining enzyme variants with higher catalytic efficiency or better stability through screening. When constructing the enzyme mutant library, the mismatched PCR technique is adopted to randomly introduce point mutations to generate a large number of different enzyme variants. Then, the enzyme variants with better catalytic performance under specific reaction conditions are screened out by high-throughput screening.

[0046] In the screening process, multiple screening criteria are set in this embodiment, including the catalytic rate, substrate affinity, heat resistance and stability of the enzyme under different pH conditions. Through the comprehensive consideration of these criteria, the enzyme variants most suitable for the reaction of AMP and DSS are screened out. In order to ensure the high efficiency of the mutant enzyme, high performance liquid chromatography (HPLC) and mass spectrometry are used to monitor the catalytic efficiency and substrate conversion rate of the mutant enzyme in real time. By optimizing the experimental conditions and screening strategies, several enzyme variants that can significantly improve the reaction efficiency are successfully selected.

[0047] In addition to random mutations, the three-dimensional structure of the enzyme is further optimized in this embodiment by combining molecular docking and molecular dynamics simulation. By conducting an in-depth analysis of the active site of the target enzyme, the key amino acid residues that may affect the catalytic performance are identified. By mutating these amino acid residues, the substrate binding site of the enzyme is further optimized to improve its affinity for AMP and DSS. For example, introducing appropriate hydrophilic or hydrophobic amino acids in the substrate binding pocket of the enzyme can improve the binding stability and reaction conversion rate of the substrate.

[0048] In this example, the optimized enzyme was also tested for thermal stability, and its stability was evaluated by the half-life of the enzyme (the time required for the enzyme activity to be reduced by half at a specific temperature). By reacting at high temperature or extreme pH conditions, the heat resistance and acid resistance of the enzyme were further optimized to ensure its stability in the actual production process. The test showed that the enzyme after engineering optimization can maintain good catalytic performance at 60°C and pH 5.5 to 8.0.

[0049] Through enzyme engineering optimization, a series of enzyme variants with significantly improved catalytic efficiency and better stability were finally screened in this example. These enzymes showed higher conversion rate and better reaction selectivity in the reaction of AMP and DSS, providing a more efficient enzyme source for subsequent enzyme-linked catalytic reactions.

[0050] In summary, step S3 uses directed evolution and molecular optimization technology to engineer the selected enzymes, effectively improving the catalytic efficiency, substrate affinity and thermal stability of the enzymes. This optimization process not only enhances the efficiency of the reaction, but also ensures the stability of the reaction process, laying a solid foundation for the efficient implementation of subsequent enzyme-linked catalytic reactions.

[0051] Step S4: Optimization of reaction conditions

[0052] In step S3, an efficient and stable enzyme variant is obtained through enzyme engineering optimization. Next, the various conditions of the enzyme-catalyzed reaction need to be optimized to ensure efficient conversion under the optimal environment. The optimization of reaction conditions involves multiple factors such as temperature, pH value, substrate concentration, enzyme concentration, etc., with the aim of improving the conversion rate, selectivity and purity of the product of the reaction. This step optimizes each reaction condition through systematic experimental design to achieve the maximum efficiency of the reaction process.

[0053] In this embodiment, the optimization of reaction conditions starts with temperature and pH. Since the catalytic activity of the enzyme is usually strongly affected by temperature and pH, it is necessary to select a suitable temperature and pH range to ensure maximum enzyme activity. Through preliminary screening in the laboratory, the catalytic activity of the enzyme at different temperatures is determined. Experiments show that the optimized enzyme variants show higher catalytic activity in the range of 55°C to 65°C, so 60°C is selected as the optimal temperature for the reaction in this embodiment.

[0054] Meanwhile, in this example, by measuring the effect of different pH values ​​on enzyme activity, it was found that the enzyme variant exhibited the best catalytic effect between pH 6.5 and 7.5. The pH value was maintained by adding a suitable buffer to ensure the best catalytic environment for the enzyme during the reaction.

[0055] Secondly, in this example, the substrate concentration and enzyme concentration are optimized. In the reaction of AMP and DSS, too low a substrate concentration will result in a slow reaction rate, while too high a substrate concentration may lead to enzyme saturation, thereby affecting the reaction efficiency. Through multiple experimental measurements, the optimal concentration of AMP was selected to be 1.5 mM and the optimal concentration of DSS was selected to be 2.0 mM to ensure that the reaction has a high conversion rate and the enzyme is not inhibited by the substrate.

[0056] For enzyme concentration, it is found in this embodiment that the concentration of the enzyme has a significant effect on the reaction rate. Within a certain range, the increase of enzyme concentration can improve the reaction rate, but too high enzyme concentration will cause waste of resources and the reaction rate improvement is limited. Through experimental testing, when the enzyme concentration is selected to be 2.0 mg / mL, it is possible to achieve better economic benefits while ensuring a high conversion rate.

[0057] In addition to temperature, pH value and substrate concentration, the solvent effect in the reaction system is also considered in this example. Since the solubility of disulfonic acid and adenylic acid in aqueous solution is quite different, the selection of a suitable solvent system is crucial to improving the efficiency of the reaction. This example uses an aqueous solution containing 10% (v / v) ethanol as the reaction solvent. The addition of ethanol can effectively increase the solubility of DSS without significantly affecting the catalytic activity of the enzyme.

[0058] In this embodiment, in order to further improve the reaction selectivity, an appropriate amount of metal ions or cofactors are added to the reaction system to promote the catalytic effect of the enzyme. For example, adding an appropriate amount of Mg2+ or Ca2+ can enhance the substrate binding ability and catalytic efficiency of the enzyme. In addition, by adding some surfactants, such as Tween 80, the solubility of the substrate can also be effectively increased, and the smooth progress of the reaction can be promoted.

[0059] Finally, in this embodiment, the reaction time is optimized, and the test shows that at 60°C, the highest product yield can be achieved when the reaction time is 4 hours. Under this condition, the conversion rate of the reaction is stable, and the purity of the product is high. Too long a reaction time may lead to a decrease in enzyme activity or the occurrence of side reactions, so 4 hours is selected as the optimal reaction time.

[0060] In summary, step S4 determines the optimal conditions of the reaction of the present invention by optimizing the reaction temperature, pH value, substrate concentration, enzyme concentration and solvent system, thereby ensuring an efficient and stable enzyme-catalyzed reaction. Under these optimized conditions, the enzyme can catalyze the reaction of AMP and DSS with optimal performance, thereby obtaining high-purity butanedisulfonic acid adenosylmethionine (SAM).

[0061] Step S5: Enzyme-catalyzed reaction

[0062] In the aforementioned step S4, the conditions of the enzyme catalytic reaction have been optimized so that the reaction can be carried out efficiently and the target product can be obtained. Next, the goal of step S5 is to extract high-purity adenosylmethionine (SAM) from the reaction mixture by appropriate separation and purification means. Product separation and purification is one of the key steps of the entire preparation process and is directly related to the quality and output of the final product. To this end, a strategy combining multiple separation techniques is adopted to ensure the purity, yield and operability of the product.

[0063] In this embodiment, a preliminary separation is first performed by liquid-liquid extraction. Since the reaction system contains both the target product SAM and the reaction substrates AMP, DSS and some by-products, in order to effectively remove these impurities, this embodiment adopts a water-organic solvent extraction method. After the enzyme-catalyzed reaction is completed, the reaction solution is added with an appropriate amount of organic solvent (such as ethyl acetate or an alcohol solvent) and fully oscillated. Since the polarity of SAM is relatively low, and the substrate and by-products have a higher polarity, SAM will tend to the organic phase, thereby separating from the polar substances. After phase separation through a separatory funnel, the organic phase is collected to remove impurities in the aqueous phase.

[0064] After liquid-liquid extraction, the organic phase is further concentrated in this embodiment. The solvent is removed using a rotary evaporator to obtain a relatively concentrated SAM solution. Next, the concentrated solution is analyzed using thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) to detect whether the target product has been purified. If it is found that there are still impurities in the solution, the subsequent separation and purification steps are continued.

[0065] After confirming that the main component in the solution is SAM, gel filtration chromatography was used in this embodiment to further purify the target product. Gel filtration chromatography uses molecular weight differences to separate the mixture, and can effectively remove smaller or larger impurity molecules based on the molecular weight differences between SAM and other substances. In this process, the reaction solution is subjected to column chromatography using common gel fillers such as Sephadex G-25 or G-50, SAM is separated from other small molecule impurities (such as residual substrates or by-products), and finally the fraction containing high-purity SAM is collected.

[0066] In this embodiment, during the gel filtration process, a suitable mobile phase is selected to ensure the separation effect. The mobile phase is often a low-salt buffer solution (such as 0.05M PBS buffer, pH 7.4) to maintain the stability of SAM and prevent the degradation of SAM molecules. The purity of the separated SAM is confirmed by ultraviolet spectroscopy, and the purity can usually reach more than 90%.

[0067] In order to further improve the purity of SAM, reverse phase high performance liquid chromatography (RP-HPLC) is used in this embodiment to finely purify the separated product. RP-HPLC uses reverse phase filler and gradient elution method to further remove impurity molecules by controlling the polarity change of the solvent. By adjusting the solvent gradient, SAM is effectively separated in the column, and the purity is finally confirmed by the detector. RP-HPLC can not only separate SAM from impurities, but also accurately measure the concentration of SAM, thereby ensuring the quality of the product.

[0068] Finally, in this embodiment, the purified SAM is freeze-dried to obtain a solid high-purity SAM powder. The freeze-drying process can effectively remove moisture, prevent hydrolysis or degradation of SAM during storage, and facilitate subsequent storage and transportation.

[0069] In summary, step S5 successfully separates and purifies high-purity adenosylmethionine butanedisulfonate from the reaction mixture by multiple purification steps such as liquid-liquid extraction, gel filtration chromatography, and RP-HPLC. The entire process can effectively remove reaction impurities, ensure that the purity and quality of the final product meet the application requirements, and ensure the stability of the product during storage by freeze-drying.

[0070] Step S6: Product purification and separation

[0071] In the aforementioned step S4, the conditions of the enzyme catalytic reaction have been optimized so that the reaction can be carried out efficiently and the target product can be obtained. Next, the goal of step S5 is to extract high-purity adenosylmethionine (SAM) from the reaction mixture by appropriate separation and purification means. Product separation and purification is one of the key steps of the entire preparation process and is directly related to the quality and output of the final product. To this end, a strategy combining multiple separation techniques is adopted to ensure the purity, yield and operability of the product.

[0072] In this embodiment, a preliminary separation is first performed by liquid-liquid extraction. Since the reaction system contains both the target product SAM and the reaction substrates AMP, DSS and some by-products, in order to effectively remove these impurities, this embodiment adopts a water-organic solvent extraction method. After the enzyme-catalyzed reaction is completed, the reaction solution is added with an appropriate amount of organic solvent (such as ethyl acetate or an alcohol solvent) and fully oscillated. Since the polarity of SAM is relatively low, and the substrate and by-products have a higher polarity, SAM will tend to the organic phase, thereby separating from the polar substances. After phase separation through a separatory funnel, the organic phase is collected to remove impurities in the aqueous phase.

[0073] After liquid-liquid extraction, the organic phase is further concentrated in this embodiment. The solvent is removed using a rotary evaporator to obtain a relatively concentrated SAM solution. Next, the concentrated solution is analyzed using thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) to detect whether the target product has been purified. If it is found that there are still impurities in the solution, the subsequent separation and purification steps are continued.

[0074] After confirming that the main component in the solution is SAM, gel filtration chromatography was used in this embodiment to further purify the target product. Gel filtration chromatography uses molecular weight differences to separate the mixture, and can effectively remove smaller or larger impurity molecules based on the molecular weight differences between SAM and other substances. In this process, the reaction solution is subjected to column chromatography using common gel fillers such as Sephadex G-25 or G-50, SAM is separated from other small molecule impurities (such as residual substrates or by-products), and finally the fraction containing high-purity SAM is collected.

[0075] In this embodiment, during the gel filtration process, a suitable mobile phase is selected to ensure the separation effect. The mobile phase is often a low-salt buffer solution (such as 0.05M PBS buffer, pH 7.4) to maintain the stability of SAM and prevent the degradation of SAM molecules. The purity of the separated SAM is confirmed by ultraviolet spectroscopy, and the purity can usually reach more than 90%.

[0076] In order to further improve the purity of SAM, reverse phase high performance liquid chromatography (RP-HPLC) is used in this embodiment to finely purify the separated product. RP-HPLC uses reverse phase filler and gradient elution method to further remove impurity molecules by controlling the polarity change of the solvent. By adjusting the solvent gradient, SAM is effectively separated in the column, and the purity is finally confirmed by the detector. RP-HPLC can not only separate SAM from impurities, but also accurately measure the concentration of SAM, thereby ensuring the quality of the product.

[0077] Finally, in this embodiment, the purified SAM is freeze-dried to obtain a solid high-purity SAM powder. The freeze-drying process can effectively remove moisture, prevent hydrolysis or degradation of SAM during storage, and facilitate subsequent storage and transportation.

[0078] In summary, step S5 successfully separates and purifies high-purity adenosylmethionine butanedisulfonate from the reaction mixture by multiple purification steps such as liquid-liquid extraction, gel filtration chromatography, and RP-HPLC. The entire process can effectively remove reaction impurities, ensure that the purity and quality of the final product meet the application requirements, and ensure the stability of the product during storage by freeze-drying.

[0079] Step S7: process scale-up and production verification.

[0080] After completing the quality inspection of the product, the goal of step S7 is to package and store the high-purity adenosylmethionine (SAM) for its subsequent distribution and use. Product packaging and storage are important links to ensure that SAM maintains long-term stability and is easy to transport. In this step, in addition to the choice of packaging form, it is also necessary to consider the impact of environmental factors on SAM and adopt appropriate storage conditions to maximize its shelf life.

[0081] In this embodiment, the product is first packaged. Considering the sensitivity of SAM to moisture and light, a packaging material with good sealing properties, such as a high-density polyethylene (HDPE) bottle or an aluminum foil bag, is used to prevent the external environment from affecting the SAM. The packaging material must have good moisture resistance to prevent moisture from entering and affecting the stability of the SAM. An appropriate amount of desiccant can be added to the packaging bottle to further reduce the impact of moisture on the SAM and ensure the quality of the product during storage and transportation.

[0082] In this embodiment, an appropriate packaging capacity is selected, and SAM is packaged into specifications of 10g, 50g or 100g according to market demand and actual usage. The packaging container should be designed to be moisture-proof and sealed to ensure that the product is not affected by the external environment during the entire circulation process. Each packaging bottle should also be marked with information such as the batch number, production date, expiration date, storage conditions, etc. of the product to ensure that the product is traceable and meets relevant regulatory requirements.

[0083] After the product is packaged, the storage phase is entered in this embodiment. Considering the sensitivity of SAM to temperature and humidity, the product is required to be stored in a dry, cool environment, avoiding direct sunlight. The ideal storage temperature is 2°C to 8°C to reduce the hydrolysis and degradation process of SAM. In order to further ensure the stability of the product, the product can be stored in a constant temperature and humidity environment to ensure that it is in stable storage conditions for a long time.

[0084] In this embodiment, the product storage is also regularly checked to ensure that the storage environment meets the standard requirements, and the inventory products are regularly tested to confirm whether their quality remains within the standard range. If the storage environment is found to be abnormal (such as excessive temperature or humidity), the storage conditions should be adjusted in time to prevent the quality of the SAM from deteriorating.

[0085] In summary, step S7 ensures the quality stability of SAM during transportation and storage through appropriate packaging materials and standardized storage conditions. The key to packaging and storage is moisture protection, light protection and temperature control to extend the shelf life of the product and ensure that the end user obtains high-quality SAM products.

[0086] Embodiment 2:

[0087] In Example 1, high-purity adenosylmethionine (SAM) was successfully obtained by multi-step purification methods such as liquid-liquid extraction, gel filtration chromatography and RP-HPLC. In Example 2, only the solvent used in the liquid-liquid extraction process was changed to explore the effects of different solvents on product purity and reaction efficiency. The purpose of this example is to verify the superiority of ethyl acetate used in Example 1 as a solvent and prove that it is the best choice.

[0088] Step S1: Enzyme-catalyzed reaction

[0089] Similar to Example 1, an enzyme catalytic reaction was first carried out at a temperature of 60° C. and in a buffer solution with a pH value of 6.5, using the optimized enzyme variant to catalyze the reaction of AMP and DSS to obtain butanedisulfonate adenosylmethionine (SAM).

[0090] Step S2: Liquid-liquid extraction (changing solvent)

[0091] In Example 2, the solvent of the liquid-liquid extraction was changed. Compared with the use of ethyl acetate in Example 1, this example uses an alcohol solvent (such as isopropanol) as the extraction solvent. After the liquid-liquid extraction, a separatory funnel is used to separate the aqueous phase and the alcohol solvent phase in the reaction mixture. SAM still preferentially enters the alcohol solvent phase due to its relatively low polarity.

[0092] In this embodiment, under this solvent system, the solubility difference of the solvent is smaller than that of ethyl acetate, so the efficiency of SAM transfer from the aqueous phase to the alcohol phase is slightly low, resulting in a relatively low concentration of the product after extraction. After the solvent is removed by rotary evaporation and concentrated, the impurities in the obtained SAM solution are not effectively separated, and the purity is lower than that of Example 1.

[0093] Step S3: Subsequent purification

[0094] Same as the gel filtration chromatography and RP-HPLC in Example 1, this example also carried out the gel filtration chromatography and RP-HPLC purification steps. Although the same separation technology was adopted, due to the change of the solvent system in the liquid-liquid extraction process, the remaining impurities still could not be completely removed during the purification process. Therefore, finally detected by HPLC, the purity of SAM was still lower than that of Example 1, and contained obvious miscellaneous peaks, indicating that the impurities could not be completely removed.

[0095] Step S4: Product quality inspection

[0096] The concentration of SAM was 0.89 g / L as measured by UV spectrophotometer, which was lower than 1.12 g / L in Example 1. In addition, HPLC analysis showed that the product purity was only 85%, which was lower than 92% in Example 1, and the impurity peak area was larger, indicating that the solvent selection during liquid-liquid extraction affected the purity of the final product.

[0097] Step S5: Product packaging and storage

[0098] The packaging and storage process is the same as that of Example 1, using packaging materials with good sealing properties and appropriate storage conditions, but due to the low purity, the stability and shelf life of the product may also be affected to a certain extent. Therefore, in this example, an accelerated stability test is performed on the product, and the test results show that under high temperature and humidity conditions, the stability of SAM decreases more significantly than that of Example 1.

[0099] Table 1 Data comparison table of embodiment 1 and 2

[0100]

[0101]

[0102] Final concentration and purity of SAM: The final concentration of SAM in Example 1 was 1.12 g / L, and the purity reached 92%, indicating that the solvent selection, separation and purification processes were optimized. However, due to the low efficiency of solvent extraction in Example 2, the final concentration of SAM was 0.89 g / L, and the purity was only 85%, and obvious impurity peaks appeared in the HPLC analysis, indicating that impurities were not completely removed during the extraction process.

[0103] Impurity removal: In the purification process of Example 1, most of the impurities were successfully removed by gel filtration chromatography and RP-HPLC, and the impurity peak area was small. In Example 2, the impurities were not completely removed, and the impurity peak area was large, which affected the purity of the final product.

[0104] Product stability and storage: In terms of product stability, the SAM in Example 1 did not undergo significant degradation during long-term storage, meeting the requirements for long-term storage. However, the SAM in Example 2 showed significant degradation under high temperature and high humidity conditions, indicating that the product with lower purity had poor stability.

[0105] Embodiment three:

[0106] Example 1 High-purity adenosylmethionine (SAM) was successfully prepared by enzyme-catalyzed reaction at 60° C. In Example 3, the reaction temperature was changed to study the reaction rate, product purity, yield and other parameters under different temperature conditions, aiming to find a more suitable reaction temperature, thereby optimizing the production process of SAM.

[0107] Step S1: Enzyme-catalyzed reaction (temperature variation)

[0108] In this example, the same enzyme catalytic reaction system was used, but the reaction temperature was changed to four different temperature conditions of 50°C, 55°C, 60°C and 65°C, and experiments were conducted to test their effects on reaction efficiency, conversion rate and product purity. Other reaction conditions (such as pH value, buffer type, etc.) remained the same as in Example 1.

[0109] In this embodiment, the reaction time is set to 4 hours at different temperatures. The experimental results show that at 50°C and 55°C, the reaction rate is slow, the conversion rate is low, and the purity of the SAM product is low. At 60°C, the enzyme-catalyzed reaction is most efficient, the reaction conversion rate is high, and the purity and yield are also ideal, so 60°C is selected as the optimal reaction temperature. Although the reaction temperature of 65°C increases the reaction rate, the high temperature causes the enzyme to be inactivated, the reaction system is not fully converted, and the purity and stability of the final product are reduced.

[0110] Step S2: Liquid-liquid extraction

[0111] After the reaction temperature was determined to be 60°C, Example 3 was the same as Example 1, and ethyl acetate was used as the solvent for liquid-liquid extraction. The extraction process was repeated three times, and finally the aqueous phase and the solvent phase were separated by a separatory funnel. The solvent phase was collected and the solvent was removed by rotary evaporation, and the concentrated liquid was the SAM solution to be purified.

[0112] In this embodiment, since the reaction temperature is optimized to 60° C., the reaction conversion rate is high and the yield of liquid-liquid extraction is also improved. Compared with Example 1, more target products are obtained under the same conditions.

[0113] Step S3: Subsequent purification

[0114] Similar to Example 1, further purification was performed using gel filtration chromatography and RP-HPLC in Example 3. After liquid-liquid extraction, the target product was initially separated by gel filtration chromatography and then further purified by RP-HPLC.

[0115] In this example, the SAM concentration obtained at 60° C. is relatively high, and the purity reaches 91%, which is slightly lower than 92% in Example 1. However, considering the reaction efficiency after the reaction temperature is optimized, the purity can still be considered to be relatively high.

[0116] Step S4: Product quality inspection

[0117] The concentration of SAM in Example 3 was 1.10 g / L as detected by UV spectrophotometer, slightly lower than 1.12 g / L in Example 1, but generally in compliance with the standard. The HPLC analysis results showed that the purity of SAM was 91%, and the impurity peak area was small, meeting the quality requirements.

[0118] In this embodiment, although the purity is slightly low, considering the improved reaction efficiency and yield after the reaction temperature is optimized, the overall effect is good.

[0119] Step S5: Product packaging and storage

[0120] After the product quality test is qualified, the SAM in Example 3 is also packaged by sealing, adding desiccant and marking the production date, batch number and other information. The packaging container is a polyethylene bottle that meets the requirements to ensure that it is moisture-proof and light-proof, and finally stored at 2-8°C.

[0121] In this embodiment, due to the optimization of the reaction temperature, the product has high purity and good stability, and no obvious degradation occurs during storage.

[0122] Table 2, experimental data table of Examples 2 and 3

[0123]

[0124]

[0125] Liquid-liquid extraction efficiency: Example 3 achieved a liquid-liquid extraction efficiency of 92% at 60°C, which is slightly higher than 90% of Example 1. However, considering the improvement of the reaction conversion rate after temperature optimization, the overall effect is still relatively superior.

[0126] Purity and concentration of SAM: The purity of SAM in Example 3 is 91%, which is not much different from that in Example 1, and the final concentration is 1.10 g / L, which meets the production requirements. Due to improper solvent selection in Example 2, the purity and concentration are relatively low.

[0127] Stability and storage: Since the reaction temperature of Example 3 was 60° C., the high purity and stability of SAM were ensured, and no obvious degradation was observed during storage.

[0128] Example 3 achieved a higher reaction conversion rate and better product purity by optimizing the reaction temperature, proving that 60° C. is the optimal reaction temperature. However, considering the reaction efficiency, product purity, cost and stability, Example 1 (ethyl acetate as solvent and 60° C. reaction temperature) is superior in all aspects and is still the best solution.

[0129] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for preparing high-purity adenosylmethionine butanedisulfonate, characterized in that: The following steps are involved: Step 1, enzyme screening: selecting an enzyme that catalyzes the reaction of adenylic acid and butanedisulfonic acid; Step 2: Enzyme expression and purification: expressing the catalytic enzyme by genetic engineering technology, and purifying the enzyme by affinity chromatography to obtain an enzyme with a purity higher than 90%; Step 3: Optimization of reaction conditions: adjusting substrate concentration, pH value, temperature and additive conditions; Step 4, enzyme-linked catalytic reaction: using a multi-enzyme system to catalyze the reaction of adenylic acid and butanedisulfonic acid; Step 5: Purification of the reaction product: removing impurities in the reaction product by solvent extraction and column chromatography to obtain adenosylmethionine butanedisulfonate with a purity greater than 95%.

2. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The catalytic enzyme is one or more of AMP enzyme, DSS sulfonate enzyme and NADH regeneration enzyme, and the enzyme-linked catalytic system is used for the reaction of AMP and DSS.

3. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The enzyme screening step involves obtaining a catalytic enzyme with high catalytic efficiency, high temperature resistance and acid resistance through gene library screening, and the enzyme activity is determined by HPLC.

4. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: Enzyme expression and purification include genetic engineering of the catalytic enzyme and optimization of the enzyme's active center or substrate binding site.

5. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The reaction condition optimization step includes: adjusting the substrate concentration, reaction temperature and pH value of catalytic adenylic acid and butanedisulfonic acid, wherein the substrate concentration is 0.1 to 5 mM, the reaction temperature is 50° C. to 60° C., and the pH value is 5.5 to 8.

0.

6. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The enzyme-linked catalytic reaction step includes using AMP enzyme, DSS sulfonate enzyme and NADH regeneration enzyme in combination, the reaction temperature is 40 to 60° C., and the reaction time is 4 to 12 hours.

7. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The purification of the reaction product includes the combined use of solvent extraction and column chromatography to remove unreacted substrates, enzymes and by-products in the reaction to obtain adenosylmethionine butanedisulfonate with a purity greater than 95%.

8. A process for preparing high-purity adenosylmethionine butanedisulfonate according to claim 1, characterized in that: The buffer in the reaction is phosphate buffer or Tris buffer, and the pH value ranges from 5.5 to 8.0.

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