Synthetic method of medical magnesium L-threonate

Abstract

The invention relates to the technical field of biological catalytic synthesis, and discloses a medical magnesium L-threonate synthesis method, which adopts three layers of magnetic carriers to immobilize L-threose oxidase, and realizes pH gradient regulation and control through a two-stage series reactor. The pH value of the first-stage reactor is 7.0 + / -0.2, enzyme catalytic oxidation is carried out, the pH value of the second-stage reactor is 8.0 + / -0.2, a magnesium complex reaction is carried out, and finally the carrier is recovered through magnetic separation. According to the method, the conversion rate is increased from 70-80% to 88 + / -2%, the product purity is increased from 90-95% to 99.0 + / -0.3%, the carrier can be recycled 6-10 times, the enzyme activity retention rate is larger than or equal to 75% when the carrier is used for the sixth time, and efficient and green synthesis of medical grade magnesium L-threonate is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biocatalytic synthesis, and particularly relates to a method for synthesizing medical L-magnesium threonate by using a magnetic carrier immobilized enzyme catalytic system. BACKGROUND

[0002] As a new type of antioxidant and cell protective agent, L-magnesium threonate has important application value in the medical field. With the in-depth study of its biological activity, the market demand for medical grade L-magnesium threonate is rapidly growing, and higher requirements are put forward for product purity and quality.

[0003] After searching and analyzing, several related technical solutions are found in the field of magnetic carrier enzyme immobilization technology. Brazilian patent BR102020016736A2 discloses a magnetic carrier enzyme immobilization method, which uses a porous adsorbent material and a magnetic oxide to prepare a magnetic carrier, and fixes a lipase by mechanical mixing without surface pretreatment and heating conditions. This technology mainly aims at the immobilization of lipase, and uses a single enzyme system, without involving multi-enzyme cooperative catalysis. Chinese patent CN110438116B discloses a method for magnetic immobilization of laccase, which prepares particles by coprecipitation, and fixes laccase after silanization modification, thereby improving the catalytic activity and reusability of the enzyme. This technology is also limited to a single laccase system, and lacks design for complex multi-step reactions. Chinese patent CN115678881A discloses a preparation method of magnetic cross-linked enzyme aggregates, which uses particles without functional group modification, and forms magnetic cross-linked enzyme aggregates through PEI mediation, thereby having good structural stability and magnetic properties.

[0004] However, the existing technology mainly focuses on the immobilization technology of single enzyme, and less involves the design and optimization of multi-step catalytic reaction system. In the synthesis of L-magnesium threonate, the traditional chemical synthesis method has problems such as harsh reaction conditions, many side reactions, poor stereoselectivity, etc. The conversion rate is usually 70-80%, and the product purity is only 90-95%, which is difficult to meet the requirements of medical grade products. Although the microbial fermentation method has good stereoselectivity, the fermentation period is as long as 7-10 days, the equipment investment is large, and the production efficiency is low. Although the single enzyme catalytic method has mild conditions, the enzyme stability is poor, the cost is high, and it cannot realize one-step synthesis from raw materials to final products.

[0005] The main technical challenges in the application of enzyme catalytic system in the existing technology include: activity decay caused by the mismatch between the optimum reaction conditions of the enzyme and the subsequent chemical reaction conditions; difficulty in carrier separation and recovery and low enzyme recycling rate; technical control difficulties in process scale-up; and difficulty in controlling product purity and quality stability. These problems limit the application of enzyme catalytic technology in the synthesis of medical grade products. SUMMARY​

[0006] Technical problems The existing L-magnesium threonate synthesis technology has the following technical defects: low conversion rate, usually only 70-80%, which cannot meet the economic requirements of industrial production; insufficient product purity, generally 90-95%, which cannot reach the standard of medical grade product purity ≥ 99%; many side reactions, poor stereoselectivity, and difficult separation of D / L isomers; poor single enzyme catalytic stability, high enzyme cost, and few recycling times; the optimal pH conditions of enzyme catalysis reaction and subsequent chemical reaction are different, resulting in low overall catalytic efficiency; lack of effective enzyme carrier separation and recovery technology, poor process continuity; long production cycle, large equipment investment, and serious environmental pollution; and lack of stability and safety guarantee of the carrier during long-term use.

[0007] Technical solutions To solve the above technical problems, the present application provides a synthesis method of medical L-magnesium threonate, comprising the following steps: Step one: preparing a magnetic carrier, using a coprecipitation method to prepare Magnetic core, by And Prepared by coprecipitation in alkaline environment at 80℃ under the condition of molar ratio 2:1, the particle size is 30-40nm, the crystallinity is 85-95%, and the Magnetic core is obtained.

[0008] The intermediate layer is coated on the surface by sol-gel method The intermediate layer is coated on the surface by sol-gel method The intermediate layer is coated on the surface by sol-gel method The intermediate layer is coated on the surface by sol-gel method

[0009] Then the functional groups are modified on the surface with silane coupling agent 3-aminopropyl triethoxysilane, the grafting density is 2.5-3.5mmol / g, and the Magnetic carrier is obtained. Magnetic carrier is obtained.

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

[0011] Step two: enzyme immobilization, L-sugar oxidase is immobilized on the magnetic carrier by glutaraldehyde cross-linking method, the immobilization density is 8-15 mg / g carrier, and the carrier dosage is 18-22 mg / mL. The composite fixation method of glutaraldehyde cross-linking covalent combination and chitosan embedding is adopted: the concentration of glutaraldehyde is 2.5%, and the covalent cross-linking is carried out at 4°C for 4 hours; the concentration of chitosan is 1%, the molecular weight is 100,000, and the deacetylation degree is ≥90%, and the embedding fixation is continued for 2 hours to form a double protection structure, thereby improving the stability and activity retention rate of the enzyme.

[0012] Step three: enzyme catalytic reaction, the immobilized enzyme carrier is put into the first reactor for catalytic reaction, and L-sugar oxidase catalyzes L-sugar to generate L-sugar acid; the pH of the first reactor is controlled at 7.0±0.2, and the reaction temperature is controlled at 37±2°C. It is suitable for L-sugar concentration of 10-100 mM, and the single batch reaction volume is 0.1-10 L.

[0013] Step four: chemical complexation reaction, in the second reactor, by adding magnesium chloride or magnesium sulfate, under the condition of pH 8.0±0.2, the reaction is carried out for 30-60 minutes to realize the complexation of L-sugar acid and magnesium ion to generate L-sugar acid magnesium.

[0014] Step five: magnetic separation and recovery, the carrier is separated and recovered from the reaction solution by an external magnetic field, and the magnetic separation adopts a magnetic field strength of 0.30±0.05T. After the carrier is separated and recovered by the magnetic field and washed by the buffer, it can be recycled for 6-10 times, the enzyme activity retention rate is not less than 75% at the 6th time of use, and the enzyme activity retention rate is not less than 65% at the 10th time of use.

[0015] The first reactor and the second reactor are connected in series to form a two-stage series reactor system, which adopts a continuous flow design, the volume ratio of each reactor is determined according to the optimal residence time ratio of reaction kinetics, 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 arranged in the reactor, and the rotating speed is 200-300 rpm. The magnetic separation device is arranged at the outlet of each reactor, which adopts a permanent magnet array structure, and the magnetic field gradient is 50T / m.

[0016] By setting pH control systems in the first and second reactors respectively, a gradient pH microenvironment is realized. A multi-parameter collaborative optimization control system is adopted, the pH values of each reactor are adjusted by a PID control system, the pH control accuracy is ±0.2, the system response time is less than 3 minutes; the parameters such as temperature, pH, magnetic field strength and flow rate are collaboratively controlled through a data bus.

[0017] The medical grade L-magnesium threonate product is obtained through recrystallization, activated carbon decolorization, vacuum drying and the like. The recrystallization solvent is an ethanol-water mixed solution with a volume ratio of 3:1; the activated carbon dosage is 2-5% of the product weight; and the vacuum drying conditions are 60 DEG C, -0.08 MPa and 12 hours. The glutaraldehyde residual amount is controlled to be below 1 ppm.

[0018] The L-magnesium threonate conversion rate of the method reaches 88±2%, the product purity reaches 99.0±0.3%, the stereoselectivity ee value of the product is not less than 95%, the production cycle of the method is 48-72 hours, the heavy metal content is not more than 5 ppm, and the microbial limit meets the relevant standards in the 2020 edition of the People's Republic of China Pharmacopoeia.

[0019] The iron ion leakage amount of the carrier during use is not more than 2 ppm, the carrier structural integrity retention rate is ≥95% after 10 times of continuous use, and the biocompatibility test meets the ISO10993 medical material biological evaluation standard.

[0020] Through the continuous flow reactor design, large-scale production can be realized, and the continuous production capacity is 1-50 kg / day.

[0021] The core technical innovation of the application is that: a three-layer structure design, the middle layer protects the magnetic core from chemical corrosion and provides a stable carrier base, the functional layer provides enzyme immobilization sites; the secondary series reactor design realizes step-by-step optimization of enzyme catalytic reaction and chemical complexation reaction, and through pH gradient regulation technology, each reaction is carried out under its optimum conditions, thereby significantly improving the overall conversion efficiency and product purity.

[0022] Advantages The application has the following advantages relative to the prior art: 1. The conversion rate is significantly improved: through the three-layer functional carrier immobilized enzyme catalytic system, the conversion rate of L-magnesium threonate is increased from 70-80% of the prior art to 88±2%, the raw material utilization rate is greatly improved, and the production cost is reduced.

[0023] 2. The product purity is greatly improved: the pH gradient regulation technology of the secondary series reactor is adopted, the product purity is increased from 90-95% of the prior art to 99.0±0.3%, the medical grade product standard is reached, and the stereoselectivity ee value is not less than 95%.

[0024] 3. The production efficiency is significantly improved: through the integrated process design of synergistic catalysis and purification, the production cycle is shortened from 7-10 days of the traditional method 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... 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 first adopts a composite immobilization method of cross-linking and covalent binding with glutaraldehyde, and then embedding with chitosan; the amount of the carrier used 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 coordinated control of parameters such as temperature, pH, magnetic field strength, and flow rate is realized through 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 48–72 hours; the heavy metal content does not exceed 5 ppm, and the microbial limits comply with the relevant standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China; it is applicable to L-threonate concentrations of 10–100 mM and single-batch reaction volumes of 0.1–10 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 the synthesis of magnesium L-threonate, 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, and the biocompatibility test meets the ISO10993 standard for biological evaluation of medical materials; 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.

10. The magnetic carrier according to claim 6, characterized in that, 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%.

Images