A method for synthesizing medical-grade L-threonate magnesium

Abstract

This invention relates to the field of biocatalytic synthesis technology and discloses a method for synthesizing medical-grade L-threonate magnesium. The method employs a three-layer magnetic support to immobilize L-threonase, and utilizes a two-stage series reactor to achieve pH gradient control. The first-stage reactor, at pH 7.0±0.2, performs enzyme-catalyzed oxidation, while the second-stage reactor, at pH 8.0±0.2, performs a magnesium complexation reaction. Finally, the support is recovered via magnetic separation. This method increases the conversion rate from 70-80% to 88±2%, and the product purity from 90-95% to 99.0±0.3%. The support can be recycled 6-10 times, and the enzyme activity retention rate is ≥75% after the sixth use, achieving efficient and green synthesis of medical-grade L-threonate magnesium.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic synthesis technology, and in particular to a method for synthesizing medical-grade L-threonate magnesium using a magnetically immobilized enzyme catalytic system. Background Technology

[0002] Magnesium L-threonate, as a novel antioxidant and cell protectant, has significant application value in the pharmaceutical field. With in-depth research into its bioactivity, the market demand for medical-grade magnesium L-threonate is growing rapidly, placing higher demands on product purity and quality.

[0003] Through searching and analysis, several relevant technical solutions were found in the field of magnetic carrier enzyme immobilization technology. Brazilian patent BR102020016736A2 discloses a magnetic carrier enzyme immobilization method, which uses porous adsorbent materials and magnetic oxides to prepare a magnetic carrier, and immobilizes lipase through mechanical mixing, without surface pretreatment or heating conditions. This technology mainly targets lipase immobilization, uses a single enzyme system, and does not involve multi-enzyme synergistic catalysis. Chinese patent CN110438116B discloses a magnetic immobilization method for laccase, which prepares laccase by co-precipitation. Microparticles, after silanization modification, immobilize laccase, improving the enzyme's catalytic activity and reusability. However, this technology is also limited to single laccase systems and lacks design for complex multi-step reactions. Chinese patent CN115678881A discloses a method for preparing magnetically cross-linked enzyme aggregates, using non-functionally modified... The particles form magnetic cross-linked enzyme aggregates through PEI mediation, exhibiting good structural stability and magnetic properties.

[0004] However, existing technologies mainly focus on the immobilization of single enzymes, with limited attention paid to the design and optimization of multi-step catalytic reaction systems. In the synthesis of magnesium L-threonate, traditional chemical synthesis methods suffer from harsh reaction conditions, numerous side reactions, and poor stereoselectivity, with conversion rates typically around 70-80% and product purity only 90-95%, failing to meet the requirements for medical-grade products. While microbial fermentation methods offer better stereoselectivity, the fermentation cycle is long (7-10 days), requiring significant equipment investment and resulting in low production efficiency. Single-enzyme catalysis methods, although operating under mild conditions, suffer from poor enzyme stability, high costs, and the inability to achieve one-step synthesis from raw materials to the final product.

[0005] The main technical challenges faced by existing technologies in enzyme catalysis systems include: activity decay due to the mismatch between the optimal reaction conditions of the enzyme and subsequent chemical reaction conditions; difficulties in carrier separation and recovery and low enzyme recycling rates; technical control challenges during process scale-up; and difficulties in controlling product purity and quality stability. These problems limit the application of enzyme catalysis technology in the synthesis of medical-grade products. Summary of the Invention

[0006] Technical issues Existing L-threonate magnesium synthesis technologies suffer from the following technical drawbacks: low conversion rate, typically only 70-80%, failing to meet the economic requirements of industrial production; insufficient product purity, generally 90-95%, not meeting the medical-grade product purity standard of ≥99%; numerous side reactions, poor stereoselectivity, and difficulty in separating D / L isomers; poor single-enzyme catalytic stability, high enzyme cost, and limited recycling times; differences in optimal pH conditions between the enzyme-catalyzed reaction and subsequent chemical reactions leading to overall low catalytic efficiency; lack of effective enzyme carrier separation and recovery technologies, resulting in poor process continuity; long production cycle, large equipment investment, and severe environmental pollution; and a lack of assurance regarding the stability and safety of the carrier during long-term use.

[0007] Technical solution To solve the above-mentioned technical problems, the present invention provides a method for synthesizing medical-grade L-threonate magnesium, comprising the following steps: Step 1: Preparation of magnetic support using a co-precipitation method. Magnetic core, through and Prepared by co-precipitation in an alkaline environment at 80℃ under a molar ratio of 2:1, yielding particles with a size of 30–40 nm and a crystallinity of 85–95%. Magnetic core.

[0008] Using the sol-gel method Surface coating The intermediate layer is formed by the hydrolysis and condensation of tetraethyl orthosilicate under ammonia catalysis, resulting in a thickness of 12–18 nm and a porosity of 15–25%. Intermediate layer.

[0009] The surface is then modified with the silane coupling agent 3-aminopropyltriethoxysilane. Functional groups were grafted at a density of 2.5–3.5 mmol / g to obtain Magnetic carrier.

[0010] The magnetic carrier has a three-layer structure, a saturation magnetization of 8–15 emu / g, and a specific surface area of ​​120–150 m². 2 / g, with a particle size of 90-110nm, a dispersion stability in aqueous solution with a Zeta potential of +25 to +35mV, a magnetic response time of less than 30 seconds, and a magnetic separation efficiency of 90-95%.

[0011] Step 2: Enzyme immobilization. L-Threooxidase was immobilized on a magnetic support using glutaraldehyde cross-linking. The immobilization density was 8–15 mg / g of support, and the support concentration was 18–22 mg / mL. A composite immobilization method was used, employing glutaraldehyde cross-linking for covalent bonding followed by chitosan encapsulation: glutaraldehyde concentration was 2.5%, and covalent cross-linking was performed at 4°C for 4 hours; chitosan concentration was 1%, molecular weight 100,000, degree of deacetylation ≥90%, and encapsulation was performed for an additional 2 hours, forming a double protective structure to improve enzyme stability and activity retention.

[0012] Step 3: Enzymatic catalytic reaction. The immobilized enzyme carrier is introduced into the first-stage reactor for catalytic reaction. L-Threose oxidase catalyzes the oxidation of L-threose to L-threonic acid. The pH of the first-stage reactor is controlled at 7.0±0.2, and the reaction temperature is controlled at 37±2℃. This method is suitable for L-threose concentrations of 10~100mM and single-batch reaction volumes of 0.1~10L.

[0013] Step 4: Chemical complexation reaction. In the second-stage reactor, magnesium chloride or magnesium sulfate is added, and the reaction is carried out for 30 to 60 minutes at pH 8.0 ± 0.2 to achieve the complexation of L-threonic acid with magnesium ions to form magnesium L-threonate.

[0014] Step 5: Magnetic separation and recovery. The carrier is separated and recovered from the reaction solution using an external magnetic field with a strength of 0.30±0.05T. After magnetic separation and recovery and washing with buffer, the carrier can be recycled 6–10 times. The enzyme activity retention rate is no less than 75% when used for the 6th time and no less than 65% when used for the 10th time.

[0015] The first-stage and second-stage reactors are connected in series to form a two-stage series reactor system, employing a continuous flow design. The optimal residence time ratio for each reactor is determined based on reaction kinetics. The reactors are connected via peristaltic pumps, with the flow rate controlled between 0.5 and 1.5 mL / min. Magnetic stirring devices are installed within the reactors, rotating at 200–300 rpm. Magnetic separation devices, employing a permanent magnet array structure, are located at the outlet of each reactor stage, with a magnetic field gradient of 50 T / m.

[0016] Gradient pH microenvironment regulation is achieved by setting pH control systems in the first and second stage reactors respectively. A multi-parameter collaborative optimization control system is adopted, using a PID control system to adjust the pH value of each stage reactor with a pH control accuracy of ±0.2 and a system response time of less than 3 minutes; the coordinated control of parameters such as temperature, pH, magnetic field strength, and flow rate is achieved through a data bus.

[0017] Medical-grade L-threonate magnesium was obtained through recrystallization, activated carbon decolorization, and vacuum drying. The recrystallization solvent was an ethanol-water mixture with a volume ratio of 3:1; activated carbon was used at 2-5% of the product weight; and vacuum drying was performed at 60℃, -0.08 MPa, for 12 hours. Glutaraldehyde residue was controlled to be below 1 ppm.

[0018] The method achieves a magnesium L-threonate conversion rate of 88±2% and a product purity of 99.0±0.3%; the product stereoselectivity ee value is not less than 95%; the production cycle of the method is 48 to 72 hours; the heavy metal content does not exceed 5 ppm; and the microbial limits meet the relevant standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0019] The iron ion leakage of the carrier during use does not exceed 2 ppm, and the structural integrity retention rate of the carrier is ≥95% after 10 consecutive uses. The biocompatibility test meets the ISO10993 standard for biological evaluation of medical materials.

[0020] Through the design of a continuous flow reactor, large-scale production can be achieved, with a continuous production capacity of 1 to 50 kg / day.

[0021] The core technological innovation of this invention lies in: through Three-layer structure design The intermediate layer protects the magnetic core from chemical corrosion and provides a stable substrate. The functional layer provides enzyme immobilization sites; the two-stage tandem reactor design enables stepwise optimization of enzyme-catalyzed reactions and chemical complexation reactions. Through pH gradient control technology, each reaction is carried out under its optimal conditions, thereby significantly improving the overall conversion efficiency and product purity.

[0022] Beneficial effects The present invention has the following advantages over the prior art: 1. Significantly improved conversion rate: By immobilizing the enzyme catalytic system on a three-layer functionalized carrier, the conversion rate of L-threonate magnesium is increased from 70-80% in the existing technology to 88±2%, which greatly improves the utilization rate of raw materials and reduces production costs.

[0023] 2. Significantly improved product purity: By adopting a two-stage series reactor pH gradient control technology, the product purity is increased from 90-95% in the existing technology to 99.0±0.3%, meeting the medical grade product standard, with a stereoselectivity ee value of not less than 95%.

[0024] 3. Significantly improved production efficiency: Through the integrated process design of synergistic catalytic purification, the production cycle is shortened from the traditional 7-10 days to 48-72 hours, and the production efficiency is increased by more than 85%.

[0025] 4. Excellent enzyme recycling performance: The three-layer functionalized carrier design allows the enzyme to be recycled 6-10 times. The activity retention rate is ≥75% when used for the 6th time and ≥65% when used for the 10th time. The enzyme cost is reduced by 70%, resulting in significant economic benefits.

[0026] 5. Safety and stability assurance: During use, the iron ion leakage of the carrier is controlled below 2 ppm, the structural integrity retention rate is ≥95% after 10 consecutive uses, and the glutaraldehyde residue is controlled below 1 ppm to ensure product safety.

[0027] 6. Product quality meets medical standards: heavy metal content is controlled below 5 ppm, and microbial limits meet the relevant standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China, ensuring the medical safety and effectiveness of the product.

[0028] 7. Strong large-scale production capacity: Through the design of a continuous flow reactor, large-scale production is achieved, with a continuous production capacity of 1-50 kg / day, meeting the needs of industrial production of different scales. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of a two-stage series reactor system according to an embodiment of the present invention; Figure 2 This is the present invention. @ @ A schematic diagram of the three-layer structure of a magnetic carrier; Figure 3 This is a process flow diagram of the multi-parameter collaborative control system of the present invention. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0031] Example 1 The purpose of this embodiment is to verify the adoption of... A complete technical solution for the synthesis of medical-grade L-threonate magnesium by immobilizing L-threonase on a three-layer magnetic carrier and achieving pH gradient regulation in a two-stage series reactor.

[0032] Preparation of three-layer functionalized carriers: like Figure 2 As shown, The magnetic carrier has a three-layer structure, in which As a magnetic core, As an intermediate layer, The functionalized outer layer is a functionalized group.

[0033] Prepared by coprecipitation method Magnetic core: (2.7g, 10mmol) and (1.0 g, 5 mmol) was dissolved in 50 mL of deionized water and heated to 80 °C under nitrogen protection. 25 mL of ammonia (25%) was quickly added, and the mixture was stirred vigorously for 30 minutes. The black precipitate was separated using a magnet and washed with deionized water until neutral, yielding particles with a diameter of 30-40 nm. Nanoparticles.

[0034] Encapsulation via sol-gel method Intermediate layer: 1g Dispersed in 100 mL of anhydrous ethanol, 2 mL of ammonia and 3 mL of tetraethyl orthosilicate were added, and the mixture was stirred at room temperature for 12 hours. The product was collected by magnetic separation, washed three times with ethanol, and yielded... Core-shell structure, The layer thickness is 12-18nm.

[0035] Surface amino functionalization: (1 g) was dispersed in 80 mL of anhydrous toluene, and 2 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed for 6 hours. The product was collected by magnetic separation, washed successively with toluene and ethanol, and dried under vacuum to obtain the final product. Carrier.

[0036] Characterization results of the carrier: saturation magnetization is 12.5 emu / g, and specific surface area is 135 m². 2 / g, particle size 95nm, amino grafting density 3.0mmol / g (determined by ninhydrin colorimetric method, using glycine as standard, absorbance measured at 570nm wavelength, according to formula). Calculation, where The concentration of amino group is mmol / L. Absorbance The molar absorptivity is L / mmol / cm. (where C is the optical path length of the cuvette in cm, and Dilution factor is the dilution ratio of the sample before measurement).

[0037] Composite immobilization process: L-Threooxidase (100 mg, activity ≥50 U / mg, purchased from Sigma-Aldrich) was dissolved in 10 mL of phosphate buffer (pH 7.0, 50 mM). Add... The carrier (1 g) and glutaraldehyde solution (2.5%, 1 mL) were covalently cross-linked by gentle stirring at 4 °C for 4 hours. Then, chitosan solution (1%, 5 mL, molecular weight 100,000, degree of deacetylation ≥90%) was added, and stirring continued for 2 hours for immobilization. The immobilized enzyme carrier was collected by magnetic separation, washed with buffer until no free enzyme remained, and the immobilization density was determined to be 12 mg / g carrier, with an enzyme activity recovery rate of 78%.

[0038] Enzyme activity assay: Using L-threose as a substrate, the reaction was carried out at 37℃ and pH 7.0 for 10 minutes. The amount of L-threonic acid produced was detected by HPLC. One enzyme activity unit was defined as the amount of enzyme required to catalyze the production of 1 μmol of L-threonic acid per minute.

[0039] Second-order tandem catalytic reaction: like Figure 1 As shown, a two-stage series reactor system was constructed. In the first-stage reactor (500 mL), L-threose substrate solution (50 mM, pH 7.0 ± 0.2) was added, followed by an immobilized L-threose oxidase carrier (20 mg / mL). The reaction was carried out at 37 °C with stirring for 2 hours, resulting in the conversion of L-threose to L-threonic acid. The pH value at different locations on the carrier surface was measured using a pH microelectrode, verifying that the pH of the carrier surface in the first-stage reactor was 7.0 ± 0.1.

[0040] In the second-stage reactor (300 mL), the pH was adjusted to 8.0 ± 0.2, and magnesium chloride solution (0.1 M) was added. The reaction was allowed to proceed for 45 minutes to achieve the complexation of L-threonic acid with magnesium ions to form magnesium L-threonate. pH microelectrode measurements confirmed that the pH of the carrier surface in the second-stage reactor was 8.0 ± 0.1, forming an effective micro-pH gradient. A PID control system was used to maintain the pH accuracy of each reactor stage at ± 0.2. The pH sensor was a Mettler Toledo InPro4260i, and the controller was a Siemens PCS7 system. Figure 3 As shown, the multi-parameter collaborative control system includes detection devices such as pH sensors, temperature sensors, and flow meters, which are connected to the central control unit via a data bus to realize real-time monitoring and collaborative adjustment of various parameters. Through the integrated process flow, it realizes the collaborative adjustment and optimized control of parameters such as temperature, pH, and magnetic field strength.

[0041] Magnetic response intelligent separation technology: After the reaction is complete, based on the relationship between the carrier magnetization and the separation efficiency, the following formula is used: Calculate the optimal magnetic field strength, where For magnetic force, The permeability of free space, Let m be the volume of the magnetic carrier. 3 , , is the magnetic susceptibility The applied magnetic field strength is A / m. The magnetic field gradient is A / m 2 In the experiment, a magnetic field strength of 0.30T was applied (using neodymium iron boron permanent magnets with a magnetic field gradient of 50T / m), and the carrier separation was completed within 3 minutes, with a separation efficiency of 96%. The carrier can be directly used in the next round of reaction after washing with phosphate buffer.

[0042] Product Analysis: Product purity and conversion were determined by HPLC. Chromatographic conditions: C18 column (Agilent ZORBAX EclipseXDB-C18, 250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile-water (20:80, containing 0.1% trifluoroacetic acid), flow rate: 1.0 mL / min, detection wavelength: 210 nm, column temperature: 30 °C.

[0043] Stereoselectivity ee value determination: A chiral HPLC column (Daicel Chiralpak AD-H, 250 mm × 4.6 mm) was used, with a mobile phase of n-hexane-isopropanol (90:10), a flow rate of 1.0 mL / min, and a detection wavelength of 254 nm, according to the formula... The calculation is performed, where R and S represent the peak areas of the R-type and S-type enantiomers, respectively, and ee% is the enantiomer excess value, used to evaluate the stereoselectivity of the reaction.

[0044] The results showed that the conversion rate of L-threonate magnesium was 88.5%, the product purity was 99.18%, and the stereoselectivity (ee) was 98.2%. Heavy metal content testing (ICP-MS, Agilent 7700x) showed an iron ion content of 1.8 ppm and a copper ion content of 1.2 ppm, both meeting medical-grade standards (≤5 ppm). Glutaraldehyde residue testing (GC-MS) showed a value of 0.8 ppm, meeting safety requirements.

[0045] This embodiment verifies the adoption of... The three-layer magnetic carrier and two-stage series reactor pH gradient control technology can efficiently synthesize medical-grade L-threonate magnesium, with both conversion rate and purity meeting the expected targets, proving the effectiveness of the technical solution of this invention.

[0046] Example 2 The purpose of this embodiment is to verify the effect of carrier dosage in the range of 18-22 mg / mL on the reaction effect, and to evaluate the carrier recycling performance.

[0047] The same carrier preparation and composite immobilization process as in Example 1 was used, with a focus on investigating the effect of different carrier concentrations. Three levels of carrier concentration were set: 18 mg / mL, 20 mg / mL, and 22 mg / mL, while keeping other conditions constant.

[0048] The results showed that the conversion rate was 87.2% and the product purity was 99.05% when the carrier concentration was 18 mg / mL; 88.5% and 99.18% when the carrier concentration was 20 mg / mL; and 88.8% and 99.21% when the carrier concentration was 22 mg / mL. When the carrier concentration was within the range of 18-22 mg / mL, both the conversion rate and purity remained at a high level, verifying the rationality of the carrier concentration range in the claims.

[0049] Cyclic performance test: The immobilized enzyme carrier was used 10 times consecutively. After each reaction, it was recovered by magnetic separation, washed 3 times with phosphate buffer at pH 7.0, and then entered the next round of reaction.

[0050] Results of repeated use: The conversion rate was 88.5% after the first use, 86.8% after the fourth use, 84.2% after the sixth use (enzyme activity retention rate 76.3%), 82.1% after the eighth use, and 79.8% after the tenth use (enzyme activity retention rate 68.2%). The enzyme activity retention rate remained above 75% after the sixth use and above 65% after the tenth use, meeting the requirements for industrial applications.

[0051] Carrier structural stability analysis: Transmission electron microscopy (TEM, JEOL JEM-2100F) was used to observe the morphological changes of the carrier before and after repeated use. The results showed that the three-layer structure of the carrier remained intact, and the particle size distribution remained basically unchanged. After 10 consecutive uses, the structural integrity retention rate of the carrier was 96.2%. Iron ion leakage detection showed that the cumulative leakage after 10 uses was 1.9 ppm, which meets the safety requirements.

[0052] This embodiment verifies the rationality of the optimal range of carrier dosage, as well as the carrier's excellent recyclability and structural stability, providing technical support for industrial applications.

[0053] Example 3 The purpose of this embodiment is to verify the effect of magnetic field strength in the range of 0.30±0.05T on separation efficiency, and the effect of achieving temperature control accuracy of ±2℃.

[0054] Using the same technical solution as in Example 1, the separation effect under different magnetic field strengths was investigated. Three magnetic field strengths were set at 0.25T, 0.30T, and 0.35T respectively, and the carrier separation efficiency and separation time were measured.

[0055] The results showed that the separation efficiency was 94.2% with a separation time of 5 minutes at a magnetic field strength of 0.25 T; 96.1% with a separation time of 3 minutes at 0.30 T; and 97.8% with a separation time of 2 minutes at 0.35 T. Effective separation was achieved within a magnetic field strength range of 0.30 ± 0.05 T, verifying the rationality of the magnetic field strength range specified in the claims.

[0056] Temperature control system verification: A PID temperature controller (Omron E5CC type) combined with a platinum resistance temperature sensor (Pt100) was used to achieve precise control of the reaction temperature at 37±2℃. After 24 hours of continuous monitoring, the temperature fluctuation range was 35.2-38.8℃, and the control accuracy met the ±2℃ requirement.

[0057] Effect of temperature on reaction efficiency: Comparative experiments were conducted at three temperature points: 35℃, 37℃, and 39℃. The results showed that the conversion rate was the highest at 37℃ (88.5%), 85.2% at 35℃, and 86.8% at 39℃, verifying the rationality of the temperature range of 37±2℃.

[0058] This embodiment verifies the rationality of the magnetic field strength and temperature control parameters, and proves the controllability and stability of the technical solution of the present invention.

[0059] Example 4 The purpose of this embodiment is to verify that the biocompatibility test meets the requirements of ISO 10993 standard, and the technical effect of continuous flow design.

[0060] Biocompatibility testing: Cytotoxicity testing was conducted according to ISO 10993-5 standards: L929 mouse fibroblasts were used, and the carrier extract was co-cultured with the cells at different dilutions (100%, 50%, 25%, and 12.5%) for 24 hours. Cell viability was detected by the MTT assay. The results showed that the cell viability was 89.2% with 100% extract, 94.6% with 50% extract, 97.8% with 25% extract, and 98.5% with 12.5% ​​extract, all exceeding the safety threshold of 70% and meeting the requirements of ISO 10993-5 standards.

[0061] Irritation tests were conducted according to ISO 10993-10 standard: A rabbit eye irritation test was performed by instilling the carrier extract into the conjunctival sac of rabbit eyes, and observing the irritation response at 24, 48, and 72 hours. Results showed no obvious redness, swelling, or increased discharge, and the irritation score was 0 (no irritation), meeting the requirements of ISO 10993-10 standard.

[0062] Continuous flow design: A continuous flow reaction system was constructed, including a feed pump (Lange BT100-1F peristaltic pump), a mixer, two-stage reactors in series, a magnetic separator, and a product collector. The flow rate was set at 1.0 mL / min, and the residence time was 120 min. Based on the kinetic constants of the L-threose oxidation and magnesium complexation reactions, the optimal volume ratio of the first-stage reactor to the second-stage reactor was determined to be 5:3 to achieve optimal residence time matching for each reaction step.

[0063] Results after 72 hours of continuous operation: the conversion rate remained stable at 87.8±1.2%, the product purity was 99.05±0.15%, and the RSD of the process stability was 1.4%, demonstrating the feasibility and stability of the continuous flow design. The continuous production capacity reached 25 kg / day, meeting the requirements for industrial production.

[0064] The online monitoring device includes a pH sensor, temperature sensor, flow meter, and turbidity meter. It achieves real-time monitoring and automatic adjustment of parameters through a data acquisition system (Advantech ADAM-4000 series). The data bus uses the RS-485 communication protocol to connect each sensor and actuator to the central control unit. Real-time data exchange and coordinated control of parameters such as temperature, pH, magnetic field strength, and flow rate are achieved through PLC programming, ensuring stable operation of each parameter within the set range.

[0065] This embodiment verifies that the biocompatibility of the carrier meets medical standards, and the continuous flow design enables stable industrial production, providing technical support for the industrial application of this invention.

[0066] Comparative Example 1 The purpose of this comparative example is to verify the lack of... The influence of the bilayer carrier in the middle layer on enzyme immobilization and catalytic performance highlights the necessity of the three-layer structure design.

[0067] Use only A comparative experiment was conducted on a bilayer carrier. The preparation method was as follows: Nanoparticles are directly modified with APTES, skipping... The coating process and other conditions are the same as in Example 1.

[0068] Characterization results of the carrier: The saturation magnetization is 18.2 emu / g (higher than the three-layer structure), but the specific surface area is only 85 m². 2 / g, with an amino grafting density of 1.8 mmol / g, both lower than those of the three-layer structure carrier.

[0069] Enzyme immobilization results: The immobilization density was 8.5 mg / g of the carrier, which was significantly lower than the 12 mg / g of the trilayer structure; the enzyme activity recovery rate was 62%, which was lower than the 78% of the trilayer structure.

[0070] Catalytic reaction results: The conversion rate of L-threonate magnesium was 81.2%, the product purity was 97.8%, and the stereoselectivity (ee) was 95.1%, all lower than those of the three-layered support. After six cycles of support recycling, the enzyme activity retention rate was only 58%, indicating poor stability. The iron ion leakage reached 8.5 ppm, exceeding the safety standard.

[0071] The comparison shows that the lack of The intermediate layer leads to a decrease in the specific surface area of ​​the carrier, a reduction in enzyme immobilization density, a decrease in catalytic efficiency, and insufficient safety, proving that... The necessity and advantages of the three-layer structure design.

[0072] Comparative Example 2 The purpose of this comparative example is to verify the effect of using a single reactor instead of a two-stage series reactor system on the reaction effect, highlighting the advantages of pH gradient control technology.

[0073] The same carrier and enzyme immobilization method as in Example 1 was used, but the reaction was carried out in a single reactor with the pH set at 7.5 (between the pH of the two-stage reactor), while other conditions remained unchanged.

[0074] Reaction results: The conversion rate of L-threonate magnesium was 79.8%, significantly lower than the 88.5% of the series reactor; the product purity was 96.5%, lower than the 99.18% of the series reactor. The by-product content increased to 8.2%, and the stereoselectivity ee value decreased to 94.1%.

[0075] Analysis of the reasons: A single pH condition cannot simultaneously meet the optimal requirements for L-threon oxidation and magnesium complexation reactions, resulting in reduced reaction efficiency. The activity of L-threon oxidase at pH 7.5 is only 85% of that at the optimal pH 7.0, while the efficiency of the magnesium complexation reaction at pH 7.5 is only 78% of that at the optimal pH 8.0.

[0076] The comparison shows that a single reactor system cannot optimize each step of the reaction, resulting in a significant decrease in overall performance. This demonstrates the superiority and necessity of a two-stage series reactor pH gradient control system.

[0077] Comparative Example 3 The purpose of this comparative example is to verify the difference in effectiveness between traditional chemical synthesis methods and the enzyme catalysis method of this invention, highlighting the advantages of biocatalysis technology.

[0078] L-Threonate magnesium was synthesized using a traditional chemical oxidation method: L-Threose was used as a raw material and oxidized with potassium permanganate under acidic conditions (pH 2.0) at a reaction temperature of 60°C for 4 hours. Then, it was neutralized and complexed with magnesium hydroxide to generate L-Threonate magnesium.

[0079] Chemical synthesis results: conversion rate 76.3%, product purity 93.2%, stereoselectivity (ee) 89.5%. The reaction process generated numerous byproducts, including D-threonic acid and peroxidation products, which were difficult to separate and purify. The reaction conditions were relatively harsh, energy consumption was high, and manganese-containing wastewater was produced.

[0080] Compared with the method of this invention, the enzyme-catalyzed method of this invention improves the conversion rate by 12.2%, purity by 5.8%, and stereoselectivity by 8.7%. The reaction conditions are mild (37℃, near-neutral pH), no organic solvents are used, and it is environmentally friendly. The production cycle is shortened from 3-4 days using chemical methods to 2-3 days.

[0081] The comparison shows that the enzyme catalysis method of the present invention is significantly superior to traditional chemical synthesis methods in terms of conversion rate, product purity, stereoselectivity and environmental friendliness, demonstrating the advantages of biocatalysis technology.

[0082] Comparative Example 4 The purpose of this comparative example is to verify the effect of the amount of carrier on the reaction effect and to determine the optimal range of amounts.

[0083] Using the same method as in Example 1, four levels of carrier dosage were set: 10 mg / mL, 15 mg / mL, 25 mg / mL, and 30 mg / mL, and compared with the 18-22 mg / mL range in Example 2.

[0084] The results showed that the conversion rate was 78.5% and the product purity was 97.2% when the carrier concentration was 10 mg / mL; 85.2% and 98.6% when the carrier concentration was 15 mg / mL; 89.1% and 99.25% when the carrier concentration was 25 mg / mL; and 89.3% and 99.28% when the carrier concentration was 30 mg / mL.

[0085] When the carrier concentration is below 18 mg / mL, the conversion rate and purity decrease significantly; when the carrier concentration exceeds 22 mg / mL, the improvement in conversion rate is not significant, but the cost increases significantly. Considering both economic efficiency and effectiveness, the optimal carrier concentration is determined to be 18-22 mg / mL, verifying the rationality of the carrier concentration range in the claims.

[0086] This comparative example determined the optimal range of carrier dosage, providing data support for process optimization and demonstrating the controllability and practicality of the technical solution of this invention.

[0087] Based on the above embodiments and comparative examples, the present invention, through... The innovative core technologies, including a three-layer functionalized carrier design, pH gradient control in a two-stage tandem reactor, and an integrated synergistic catalytic purification process, have successfully solved key problems in existing L-threonate magnesium synthesis technologies, such as low conversion rate, insufficient purity, complex processes, and inadequate safety. This technological solution achieves efficient, green, safe, and economical synthesis of medical-grade L-threonate magnesium, with excellent carrier recycling performance and a stable and controllable process, providing crucial technical support for the industrial production of related products.

[0088] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A method for synthesizing medical-grade L-threonate magnesium, characterized in that, Includes the following steps: Step 1: Preparation of magnetic support using a co-precipitation method. Magnetic core, through sol-gel method in Surface coating The intermediate layer is then modified on the surface with a silane coupling agent. Functional groups, to obtain @ @ Magnetic carrier; Step 2: Enzyme immobilization. L-Threo oxidase was immobilized on the magnetic support using the glutaraldehyde cross-linking method, with an immobilization density of 8–15 mg / g support. Step 3: Enzymatic catalytic reaction. The immobilized enzyme carrier is put into the first-stage reactor for catalytic reaction. L-Threose oxidase catalyzes the oxidation of L-Threose to L-Threonic acid. The pH of the first-stage reactor is controlled at 7.0±0.

2. Step 4: Chemical complexation reaction. In the second-stage reactor, magnesium chloride or magnesium sulfate is added, and the reaction is carried out for 30 to 60 minutes at pH 8.0 ± 0.2 to achieve the complexation of L-threonic acid with magnesium ions to form magnesium L-threonate. Step 5: Magnetic separation and recovery, using an external magnetic field to separate and recover the carrier from the reaction solution; The magnetic carrier has a three-layer structure and a saturation magnetization of 8–15 emu / g; The first-stage reactor and the second-stage reactor are connected in series to form a two-stage series reactor system. By setting pH control systems in the first-stage and second-stage reactors respectively, the gradient pH microenvironment can be regulated.

2. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The specific surface area of ​​the magnetic carrier is 120-150 m². 2 / g, with a particle size of 90-110nm; the enzyme immobilization is first performed by glutaraldehyde cross-linking and covalent binding, followed by chitosan encapsulation; the carrier dosage is 18-22mg / mL.

3. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The reaction temperature of the enzyme-catalyzed reaction is controlled at 37±2℃; the magnetic separation adopts a magnetic field strength of 0.30±0.05T; the carrier can be recycled 6 to 10 times after magnetic separation recovery and buffer washing, and the enzyme activity retention rate is not less than 75% when used for the 6th time and not less than 65% when used for the 10th time.

4. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, A multi-parameter collaborative optimization control system is adopted. The pH value of each reactor is adjusted by the PID control system, with a pH control accuracy of ±0.2 and a system response time of less than 3 minutes. The temperature, pH, magnetic field strength and flow parameters are coordinated and controlled by the data bus.

5. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The Magnetic core through and The particles were prepared by co-precipitation at 80°C in an alkaline environment under a molar ratio of 2:1, with a particle size of 30–40 nm. The intermediate layer is formed by the hydrolysis and condensation of tetraethyl orthosilicate under ammonia catalysis, and has a thickness of 12–18 nm; The functional groups were obtained by modification with 3-aminopropyltriethoxysilane, with a grafting density of 2.5–3.5 mmol / g.

6. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The method achieves a magnesium L-threonate conversion rate of 88±2% and a product purity of 99.0±0.3%; the product has a stereoselectivity ee value of not less than 95%; the production cycle of the method is 48 to 72 hours; the heavy metal content does not exceed 5 ppm; it is suitable for L-threonate concentrations of 10 to 100 mM and single batch reaction volumes of 0.1 to 10 L.

7. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The two-stage series reactor adopts a continuous flow design. The volume ratio of each reactor is determined according to the reaction kinetics to obtain the optimal residence time ratio. The reactors are connected by peristaltic pumps, and the flow rate is controlled at 0.5-1.5 mL / min. A magnetic stirring device is installed in the reactor with a rotation speed of 200-300 rpm. The magnetic separation device is located at the outlet of each reactor and adopts a permanent magnet array structure with a magnetic field gradient of 50 T / m.

8. The method for synthesizing medical-grade L-threonate magnesium according to claim 1, characterized in that, The method also includes a product purification step, in which medical-grade L-threonate magnesium product is obtained through recrystallization, activated carbon decolorization, and vacuum drying. The recrystallization solvent is an ethanol-water mixture with a volume ratio of 3:

1. The amount of activated carbon used is 2-5% of the product weight. The vacuum drying conditions are 60℃, -0.08MPa, and 12 hours. The residual amount of glutaraldehyde is controlled below 1ppm. Through the design of a continuous flow reactor, large-scale production can be achieved, with a continuous production capacity of 1-50kg / day.

9. A magnetic carrier for use in the synthesis method of medical-grade L-threonate magnesium according to claim 1, characterized in that, have @ @ Three-layer structure, among which As a magnetic core, As an intermediate layer, The functionalized outer layer consists of functionalized groups; the saturation magnetization of the carrier is 8–15 emu / g, and the specific surface area is 120–150 m². 2 / g, with a particle size of 90-110nm; the dispersion stability zeta potential of the carrier in aqueous solution is +25 to +35mV; the iron ion leakage of the carrier during use does not exceed 2ppm, and the structural integrity retention rate of the carrier is ≥95% after 10 consecutive uses; the... The magnetic core has a particle size of 30–40 nm and a crystallinity of 85–95%; The intermediate layer has a thickness of 12–18 nm and a porosity of 15–25%; The grafting density of functional groups is 2.5–3.5 mmol / g; the magnetic response time of the carrier is less than 30 seconds, and the magnetic separation efficiency reaches 90–95%.

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