A method for biosynthesizing high-purity L-threonate magnesium

Abstract

This invention discloses a method for the biosynthesis of high-purity L-threonate magnesium, belonging to the field of food fortification agents and biochemical synthesis technology. The invention uses L-xylorate calcium solution as a substrate, and obtains a purified L-threose solution through oxidative cleavage. Then, a complex dehydrogenase system composed of arabinose dehydrogenase, xylose dehydrogenase, and aldose dehydrogenase synergistically catalyzes the oxidation of L-threose to L-threonate. Finally, L-threonate magnesium is prepared by salt formation with magnesium hydroxide, crystallization, and drying. The purity of the obtained product is greater than 99.0%, the oxalic acid residue is less than 0.01%, the process is safe and environmentally friendly, and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to a method for biosynthesizing high-purity L-threonate magnesium, belonging to the fields of food fortification agents and biochemical synthesis technology. Background Technology

[0002] L-Threonate magnesium is a magnesium ion supplement. Studies have shown that L-Threonate magnesium has a high blood-brain barrier penetration rate, effectively increasing the concentration of magnesium ions in brain tissue. It shows potential application value in improving cognitive function and alleviating neurodegenerative diseases, thus possessing broad application prospects in the nutrition and medicine fields. As a nutritional supplement, it has stringent requirements for product purity, especially the control of harmful byproducts and heavy metal residues. Specifically, impurities with nephrotoxicity (such as oxalic acid) exceeding the standard may cause kidney stones or kidney damage with long-term intake; while heavy metal residues such as lead, arsenic, and cadmium may cause chronic toxic accumulation in the human body. Therefore, ensuring that L-Threonate magnesium meets strict purity and safety standards is an important prerequisite for its commercial application and long-term health benefits.

[0003] However, current industrial preparations of L-threonate magnesium mainly rely on chemical oxidation or ion exchange methods, which have limitations in terms of purity control and impurity separation. For example, patent CN106083567B discloses a preparation method using vitamin C as a raw material and magnesium peroxide as an oxidant; patent CN120004718A discloses a preparation method using hydrogen peroxide as an oxidant in ethanol. However, these processes use oxidants for oxidative chain scission, and the side reaction easily produces oxalic acid, which has the risk of causing kidney stones. Even if catalase is used to remove hydrogen peroxide or ethanol is used for recrystallization, it is still difficult to completely remove oxalic acid to below 0.01% at the industrial level.

[0004] Patent CN120097823A discloses a method for pyrolysis by adding ferrous salt to catalyze the generation of hydroxyl radicals in hydrogen peroxide. However, the Fenton pyrolysis process using D-glucose as a raw material is prone to causing sugar dehydration, producing impurities such as 5-hydroxymethylfurfural, and resulting in residual iron ions in the final product, increasing the difficulty and cost of subsequent purification. The displacement process using L-threonate calcium as a raw material (patent CN119504413A) actively introduces toxic reagents. If the displacement reaction is incomplete or the filtration process has errors, magnesium oxalate impurities can easily be mixed into the product, posing a food safety hazard. While enzymatic preparation has strong substrate specificity and fewer byproducts, the scarcity of natural catalytic enzymes for rare four-carbon sugars (such as L-threon) means that single-enzyme catalysis often suffers from poor substrate affinity and is easily limited by the mutarotation equilibrium of sugars in aqueous solution, resulting in low conversion rates (usually less than 30%), making it difficult to meet the demands of high yields for industrial applications.

[0005] Therefore, there is an urgent need to develop a synthesis process that does not actively introduce toxic reagents and has high process controllability. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for the biosynthesis of high-purity L-threonate magnesium. This invention uses L-xylorate calcium solution as a substrate, which undergoes an oxidative cleavage reaction to obtain a purified L-threose solution. Then, a composite dehydrogenase system consisting of arabinose dehydrogenase, xylose dehydrogenase, and aldose dehydrogenase synergistically catalyzes the oxidation of L-threose to L-threonate. Finally, L-threonate magnesium is prepared by salt formation with magnesium hydroxide, crystallization, and drying. This invention utilizes multi-enzyme synergistic catalysis to improve substrate conversion rate and product purity. Simultaneously, the in-situ precipitation of calcium oxalate during the process effectively controls oxalic acid residue. The resulting product has a purity greater than 99.0% and an oxalic acid residue content less than 0.01%. The process is safe and environmentally friendly, suitable for industrial production.

[0007] The first objective of this invention is to provide a method for the biosynthesis of magnesium L-threonate, comprising the steps of: An enzyme mixture of 100-600 U / g L-threose, NAD+ at a final concentration of 0.1-0.5 mmol / L, and NOX at a final concentration of 0.05-0.1 mg / mL were added to a purified L-threose solution with a concentration of 50-150 g / L to obtain a reaction solution. The reaction solution was then subjected to ultrafiltration and adsorption, and the eluent was collected to obtain an L-threonic acid solution. L-threonic acid solution with a concentration of 50~150 g / L was mixed with magnesium hydroxide at a ratio of 800~1000 mL: 4.5~6.5 g, stirred, pH adjusted, concentrated, crystallized, and dried to obtain magnesium L-threonate; The enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 1~3:1~3:1~2; the UniProt accession number of AraDH is Q53TZ2, the UniProt accession number of CcXylDH is B8H1Z0, and the UniProt accession number of AldH is Q88JG9.

[0008] In one embodiment, the enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH at an enzyme activity ratio of 15~360 U: 15~360 U: 14~300 U.

[0009] In one embodiment, the L-threose purification solution is prepared as follows: (1) Inoculate the seed culture of Gluconobacterium oxidans into a medium containing 50-100 g / L xylose at an inoculation rate of 5-10% v / v and ferment. During fermentation, add calcium carbonate solution to maintain the pH of the fermentation broth. After fermentation, centrifuge to obtain L-xylose calcium solution. (2) Mix L-xylorate calcium solution with anhydrous ferric sulfate at a ratio of 1000~2000 mL: 0.5~2.0 g, and then add H2O2 solution dropwise to react. After the reaction is completed, adjust the pH and filter to obtain the filtrate. Add catalase with a final concentration of 0.05~0.2 mg / mL to the filtrate, stir, and filter to obtain the clear liquid. Purify the clear liquid to obtain L-threose purified solution.

[0010] In one embodiment, the accession number of *Glucosamine oxidans* is ATCC 621H.

[0011] In one embodiment, the concentration of the L-xylorate calcium solution is 50~150 g / L.

[0012] In one embodiment, the concentration of the calcium carbonate solution added is 0.02~0.05 g / mL.

[0013] In one embodiment, a calcium carbonate solution is added to maintain the pH of the fermentation broth at 5.5 to 6.5.

[0014] In one embodiment, the fermentation process in the preparation of L-xylorate calcium solution is carried out at 28~32°C, 300~500 rpm, and an aeration rate of 1.0~2.0 vvm for 36~48 h.

[0015] In one embodiment, the reaction in step (2) involves adding H2O2 solution dropwise to the reaction system at a rate of 2-5 mL / min, with a concentration of 25-30%; the reaction is carried out for 2-4 h. The pH adjustment is adjusted to 7.0-7.5.

[0016] In one embodiment, the reaction in step (2) involves adding H2O2 solution dropwise to the reaction system at a rate of 2-5 mL / min, with a concentration of 25-30%; the reaction continues until the reaction is complete, and the reaction lasts for 2-4 hours. The pH adjustment is to adjust the pH to 7.0-7.5.

[0017] In one embodiment, an enzyme mixture of 100-600 U / g L-threose, NAD+ with a final concentration of 0.1-0.5 mmol / L, and NOX with a final concentration of 0.05-0.1 mg / mL are added to an L-threose purification solution with a concentration of 50-150 g / L. The mixture is then reacted with sterile air at 30-35°C and 100-200 rpm for 4-6 h to obtain a reaction solution. After ultrafiltration and adsorption, the eluent from the reaction solution is collected to obtain an L-threonic acid solution.

[0018] In one embodiment, the pH adjustment is to adjust the pH to 6.5~7.5; the crystallization is to add 2~4 volumes of anhydrous ethanol dropwise at a flow rate of 2~5 mL / min to crystallize and collect the crystals; the drying is to dry at -0.08~-0.1 MPa and 40~50℃ for 10~14 h.

[0019] A second objective of this invention is to provide L-threonate magnesium prepared by any of the methods described above.

[0020] A third objective of this invention is to provide a method for biosynthesizing high-purity, high-yield, and low-oxalic acid-residue L-threonate magnesium, comprising the steps of: Add an enzyme mixture of 100-600 U / g L-threose and NAD+ to a final concentration of 0.1-0.5 mmol / L to a purified L-threose solution with a concentration of 50-150 g / L. + The reaction was carried out with 0.05~0.1 mg / mL NOX to obtain a reaction solution; the reaction solution was subjected to ultrafiltration and adsorption, and the eluent was collected to obtain L-threonic acid solution; L-threonic acid solution with a concentration of 50~150 g / L was mixed with magnesium hydroxide at a ratio of 800~1000 mL: 4.5~6.5 g, stirred, pH adjusted, concentrated, crystallized, and dried to obtain magnesium L-threonate; The enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 1~3:1~3:1~2; the UniProt accession number of AraDH is Q53TZ2, the UniProt accession number of CcXylDH is B8H1Z0, and the UniProt accession number of AldH is Q88JG9.

[0021] In one embodiment, the enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH at an enzyme activity ratio of 15~360 U: 15~360 U: 14~300 U.

[0022] In one embodiment, an enzyme mixture of 100-600 U / g L-threose, NAD+ with a final concentration of 0.1-0.5 mmol / L, and NOX with a final concentration of 0.05-0.1 mg / mL are added to an L-threose purification solution with a concentration of 50-150 g / L. The mixture is then reacted with sterile air at 30-35°C and 100-200 rpm for 4-6 h to obtain a reaction solution. After ultrafiltration and adsorption, the eluent from the reaction solution is collected to obtain an L-threonic acid solution.

[0023] In one embodiment, the L-threose purification solution is prepared as follows: (1) Inoculate the seed culture of Gluconobacterium oxidans into a medium containing 50-100 g / L xylose at an inoculation rate of 5-10% v / v and ferment. During fermentation, add calcium carbonate solution to maintain the pH of the fermentation broth. After fermentation, centrifuge to obtain L-xylose calcium solution. (2) Mix L-xylorate calcium solution with anhydrous ferric sulfate at a ratio of 1000~2000 mL: 0.5~2.0 g, and then add H2O2 solution dropwise to react. After the reaction is completed, adjust the pH and filter to obtain the filtrate. Add catalase with a final concentration of 0.05~0.2 mg / mL to the filtrate, stir, and filter to obtain the clear liquid. Purify the clear liquid to obtain L-threose purified solution.

[0024] In one embodiment, the accession number of *Glucosamine oxidans* is ATCC 621H.

[0025] In one embodiment, the concentration of the L-xylorate calcium solution is 50~150 g / L.

[0026] In one embodiment, the concentration of the calcium carbonate solution added is 0.02~0.05 g / mL.

[0027] In one embodiment, a calcium carbonate solution is added to maintain the pH of the fermentation broth at 5.5 to 6.5.

[0028] In one embodiment, the fermentation process in the preparation of L-xylorate calcium solution is carried out at 28~32°C, 300~500 rpm, and an aeration rate of 1.0~2.0 vvm for 36~48 h.

[0029] In one embodiment, the reaction in step (2) involves adding H2O2 solution dropwise to the reaction system at a rate of 2-5 mL / min, with a concentration of 25-30%; the reaction is carried out for 2-4 h. The pH adjustment is adjusted to 7.0-7.5.

[0030] In one embodiment, the reaction in step (2) involves adding H2O2 solution dropwise to the reaction system at a rate of 2-5 mL / min, with a concentration of 25-30%; the reaction continues until the reaction is complete, and the reaction lasts for 2-4 hours. The pH adjustment is to adjust the pH to 7.0-7.5.

[0031] In one embodiment, the pH adjustment is to adjust the pH to 6.5~7.5; the crystallization is to add 2~4 volumes of anhydrous ethanol dropwise at a flow rate of 2~5 mL / min to crystallize and collect the crystals; the drying is to dry at -0.08~-0.1 MPa and 40~50℃ for 10~14 h.

[0032] A fourth object of the present invention is to provide the application of any of the methods described above in the preparation of magnesium L-threonate.

[0033] Beneficial effects Compared with existing technologies, the L-threonate magnesium prepared by this invention and its process have the following technical advantages: (1) Synergistic catalysis by a complex dehydrogenase. This invention uses L-threose as a substrate and AraDH, XylDH, and AldH in an enzyme activity ratio of 2:2:1 to synergistically catalyze L-threose, improving the problem of incomplete substrate conversion in single-enzyme catalysis. This process utilizes the specificity of enzymes to oxidize C1 aldehyde groups, reducing skeletal side reactions and controlling the generation of HMF impurities and the introduction of heavy metals.

[0034] (2) Purifying the impurity profile to eliminate toxic residues from a mechanistic perspective. Although some existing pure chemical synthesis processes have high apparent conversion rates, their reaction systems are violent, and the "impurity profile" of the product inevitably contains nephrotoxic oxalic acid and excessively oxidized polymers. Even after multiple recrystallizations to achieve a purity of 99%, there is still a significant food safety hazard. This process abandons the strong oxidizing chain-breaking line, not only utilizing the specificity of the complex enzyme to eliminate side reactions, but also introducing endogenous calcium ions through fermentation, which precipitate in situ during the carbon chain degradation stage, thus cutting off the path of impurities migrating to the later stages in the early separation steps. This ensures that the oxalic acid residue in the final product is strictly controlled below the instrument detection limit (<0.01%), achieving a fundamental leap in product safety.

[0035] (3) Industrial Implementation and Environmental Benefits. The reaction conditions throughout the process of this invention are relatively mild, avoiding the need for multi-stage ion exchange resin purification systems introduced in conventional strong chemical methods to remove heavy metals and oxalic acid residues. From the perspective of overall process calculation, it reduces the difficulty of waste liquid treatment and the cost of purification. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0037] Raw materials involved in the examples: L-xylose was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The preservation number for *Glucobacterium oxidans* is ATCC 621H. Calcium carbonate was purchased from Sinopharm Chemical Reagent Co., Ltd. Anhydrous ferric sulfate was purchased from Sinopharm Chemical Reagent Co., Ltd. 30% H2O2 solution was purchased from Sinopharm Chemical Reagent Co., Ltd. Catalase was purchased from Sigma-Aldrich; shelf number C9322, enzyme activity ≥ 2,000,000 U / g; NAD+ was purchased from Sigma Aldrich; NOX was purchased from Sigma-Aldrich, catalog number N1288, with an enzyme activity of ≥35 U / mg; Magnesium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. The UniProt accession number for arabinose 1-dehydrogenase (AraDH) is Q53TZ2. It was sent to Nanjing Genscript Biotech Co., Ltd. for customized recombinant expression and purification, and the enzyme activity was ≥15 U / mg. The xylose dehydrogenase (CcXylDH) from *Stylosus crescentis* has the UniProt accession number B8H1Z0. It was sent to Nanjing GenScript Biotech Co., Ltd. for customized recombinant expression and purification, and the enzyme activity was ≥20 U / mg. Aldose dehydrogenase (AldH) with UniProt accession number Q88JG9 was sent to Nanjing GenScript Biotech Co., Ltd. for customized recombinant expression and purification, and the enzyme activity was ≥10 U / mg. Glucose dehydrogenase (GDH) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., model number 19359, with an enzyme activity of ≥250 U / mg; Galactose dehydrogenase (GalDH) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., model number 10662046001, with an enzyme activity of ≥10 U / mg; The xylose dehydrogenase (TrXylDH) from Trichoderma reesei, with UniProt accession number A8BT09, was sent to Sangon Biotech (Shanghai) Co., Ltd. for customized recombinant expression and purification, and the enzyme activity was ≥15 U / mg.

[0038] Culture media involved in the examples: YPM medium formulation: yeast extract 5 g / L, peptone 3 g / L, mannitol 25 g / L, pH natural.

[0039] The methods involved in the embodiments: 1. Determination of purity and conversion rate of magnesium L-threonate High-performance liquid chromatography (HPLC) was used for detection. Chromatographic conditions: an amino column (4.6 mm × 250 mm, 5 μm), a mobile phase of acetonitrile-water (75:25 v / v), a flow rate of 1.0 mL / min, a column temperature of 35 °C, and a differential refractive index detector were used.

[0040] Conversion rate = (C0 - C)t ) / C0×100% in: C0: Initial molar concentration (mmol / L) of the substrate (such as L-xylose or L-threose) in the reaction system. C t : The molar concentration of the remaining substrate in the system at the end of the reaction (mmol / L); The concentration was calculated by substituting the peak area obtained by HPLC into the standard curve equation.

[0041] 2. Determination of oxalic acid residue Trace oxalate ions were determined by ion chromatography. Chromatographic conditions: anion exchange column was used with gradient elution of potassium hydroxide solution as the eluent; the flow rate was 1.2 mL / min; and a conductivity detector was used. The limit of detection was 0.001%.

[0042] 3. Determination of residual iron ions Inductively coupled plasma mass spectrometry was used to quantitatively determine the iron content in the sample digestion solution (refer to GB5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food").

[0043] Example 1: A method for biosynthesizing high-purity L-threonate magnesium A method for biosynthesizing high-purity L-threonate magnesium, comprising the following steps: (1) Select a single colony of Gluconobacterium oxysporum (ATCC 621H) and inoculate it into YPM medium. Ferment at 30℃ for 24h to activate the seed culture. Inoculate the seed culture into YPM medium containing 50 g / L L-xylose at an inoculation rate of 10% v / v. Ferment at 30℃, 400 rpm and 1.5 vvm for 48h. During the fermentation, add calcium carbonate solution with a concentration of 0.03 g / mL to maintain the pH of the system at 6 to ensure the normal metabolism of Gluconobacterium oxysporum. After the fermentation is completed, centrifuge at 8000 rpm for 15min to remove the cells and obtain a calcium L-xylose solution with a concentration of 80 g / L. (2) After the temperature of the L-xylorate calcium solution drops to 15℃ (pre-cooling can prevent local overheating when adding H2O2), add 0.8 g of anhydrous ferric sulfate to 1000 mL of L-xylorate calcium solution, and use a constant flow pump to add 30% H2O2 solution (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a rate of 3 mL / min until the reaction is complete. The reaction time is 3 hours, and the temperature is controlled at around 35℃ during the reaction. After the reaction is complete, adjust the pH to 7.2 to convert the iron ions into ferric hydroxide precipitate, remove it by filtration, and obtain the filtrate. Add catalase (purchased from Sigma-Aldrich, shelf number C9322) with a final concentration of 0.1 mg / mL to the filtrate, and stir until no bubbles are generated. Remove the precipitated calcium oxalate by filtering through a 0.45 μm filter membrane to obtain the clear liquid. Purify the clear liquid through a weak anion exchange resin at a flow rate of 2 BV / h and collect the L-threose purified solution. (3) The pH of the L-threose purification solution was adjusted to 7.5 using phosphate buffer and the concentration of the L-threose purification solution was adjusted to 80 g / L. An enzyme mixture with an enzyme dosage of 250 U / g L-threose, a final concentration of 0.2 mmol / L NAD+ (purchased from Sigma-Aldrich), and 0.05 mg / mL NOX (purchased from Sigma-Aldrich) were added to 1000 mL of L-threose purification solution and reacted with microporous aeration (sterile air) for 5 h at 32℃ and 150 rpm to obtain the reaction solution. After the protein was removed by ultrafiltration through a 10 kDa ultrafiltration tube, the reaction solution was passed through a strongly basic anion exchange chromatography system for adsorption, and the eluent was the L-threonine solution. The enzyme mixture was prepared by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 2:2:1 (100 U:100 U:50 U). (4) Mix L-threonic acid solution with magnesium hydroxide at a ratio of 1000 mL: 5.8 g and stir, then adjust the pH to 7.0; concentrate the solution under reduced pressure at 50℃ and -0.08 MPa to form a syrup; crystallize the solution by adding 3 times the volume of anhydrous ethanol at 5 mL / min while stirring at 300 rpm; collect the crystals and dry them in a vacuum drying oven (-0.09 MPa, 45℃) for 12 hours to obtain magnesium L-threonate.

[0044] Example 2: Changing the enzyme activity ratio of AraDH:CcXylDH:AldH The specific implementation method is the same as in Example 1, except that the enzyme mixture in step (3) is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 1:1:1, and the remaining steps are kept the same to prepare L-threonate magnesium.

[0045] Example 3: Changing the enzyme activity ratio of AraDH:CcXylDH:AldH The specific implementation method is the same as in Example 1, except that the enzyme mixture in step (3) is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 3:1:1, while the other steps remain the same, and L-threonate magnesium is prepared.

[0046] Example 4: Scale-up Experiment The specific implementation method is the same as in Example 1, except that in step (3), the L-threose purification solution is placed in a 5 L reaction system for reaction; that is, an enzyme mixture with an enzyme dosage of 250 U / g L-threose, a final concentration of 0.2 mmol / L NAD+ (purchased from Sigma-Aldrich) and 0.05 mg / mL NOX (purchased from Sigma-Aldrich) are added to 5000 mL of L-threose purification solution, and the reaction is carried out under microporous aeration (sterile air) conditions of 32℃ and 150 rpm for 5 h, and the remaining steps are kept the same to prepare L-threose magnesium.

[0047] Comparative Example 1: Preparation by Chemical Oxidation Reference patent CN120004718A: 0.1 mol of vitamin C was dissolved in 500 mL of water, and 0.06 mol of magnesium hydroxide was added and stirred to neutralize the solution. Then, 0.25 mol of 30% hydrogen peroxide was added dropwise. The oxidation reaction was carried out at 50 °C for 3.5 h. After the reaction was completed, catalase was added to decompose the hydrogen peroxide. The solution was concentrated and ethanol was added dropwise to crystallize the product. After filtration and drying, the total yield was 31.5%, the product purity was 95.2%, and the oxalic acid residue was 1.85%.

[0048] Comparative Example 2: Using only xylose dehydrogenase (CcXylDH) The specific implementation method is the same as in Example 1, except that only CcXylDH is used for catalysis in step (3), while the other steps remain the same to prepare L-threonate magnesium.

[0049] Comparative Example 3: Using only L-arabinose 1-dehydrogenase (AraDH) The specific implementation method is the same as in Example 1, except that only AraDH is used for catalysis in step (3), while the other steps remain the same to prepare L-threonate magnesium.

[0050] Comparative Example 4: Using only aldose dehydrogenase (AldH) The specific implementation method is the same as in Example 1, except that only AldH is used for catalysis in step (3), while the other steps remain the same to prepare L-threonate magnesium.

[0051] Comparative Example 5: Xylose dehydrogenase (CcXylDH) was replaced with glucose dehydrogenase (GDH). The specific implementation method is the same as in Example 1, except that the xylose dehydrogenase (CcXylDH) in step (3) is replaced with an equal amount of glucose dehydrogenase (GDH), while the other steps remain the same, and L-threonate magnesium is prepared.

[0052] Comparative Example 6: L-arabinose 1-dehydrogenase (AraDH) was replaced with galactose dehydrogenase (GalDH). The specific implementation method is the same as in Example 1, except that L-arabinose 1-dehydrogenase (AraDH) in step (3) is replaced with an equal amount of galactose dehydrogenase (GalDH), while the other steps remain the same, to prepare L-threonate magnesium.

[0053] Comparative Example 7: No aldose dehydrogenase (AldH) added. The specific implementation method is the same as in Example 1, except that aldose dehydrogenase (AldH) is not added to the enzyme mixture in step (3); that is, AraDH and CcXylDH are mixed at an enzyme activity ratio of 1:1 to obtain an enzyme mixture, and the remaining steps are kept the same to prepare L-threonate magnesium.

[0054] Comparative Example 8: Replacing the xylose dehydrogenase (CcXylDH) from *Stipa crescentis* with isoenzymes from other species. The specific implementation method is the same as in Example 1, except that the xylose dehydrogenase CcXylDH from Bacillus crescentis in step (3) is replaced with xylose dehydrogenase TrXylDH from Trichoderma reesei, while the other steps remain the same, and L-threonate magnesium is prepared.

[0055] Test Example 1: Product Indicators and Yield Determination Magnesium L-threonate prepared in Examples 1-4 and Comparative Examples 1-8 were tested, and the results are shown in Table 1.

[0056] Table 1

[0057] The results showed that when AraDH, CcXylDH or AldH were used as catalysts alone, the reaction was incomplete, and the highest overall yield of the four-step cascade process was only 28.6%.

[0058] The yield and purity of magnesium L-threonate were significantly improved when AraDH, CcXylDH and AldH were synergistically catalyzed. Furthermore, when the enzyme activity ratio of AraDH, CcXylDH and AldH was 2:2:1, the total yield reached the highest level of 48.2%, achieving a relatively obvious synergistic catalytic effect.

[0059] Comparisons of data from Examples 5 and 6 show that replacing the specific enzyme in the complex system with a non-specific common dehydrogenase (such as GDH or GalDH) fails to achieve perfect conformational dislocation capture of rare four-carbon sugars, resulting in a significant drop in yield to below 25%. In Example 7, removing AldH prevented the free sweep of open-chain trace conformations in aqueous solution, causing the overall process yield to be capped at 30.5%. Particularly noteworthy is Example 8, which demonstrates that even with xylose dehydrogenases, replacing them with isozymes derived from conventional fungi instead of the specific, highly conformationally tolerant sequence selected according to this invention still fails to achieve good catalytic complementarity with the complex system, leading to a significant drop in the overall yield of the multi-enzyme system to 32.4%. This further confirms the synergistic necessity and non-obviousness of the specific dehydrogenase combination defined in this invention in overcoming substrate conversion bottlenecks.

[0060] Furthermore, it should be noted that although traditional pure chemical oxidation methods (such as Comparative Example 1 or the vitamin C oxidation process in the prior art) may achieve high apparent yields, achieving high purity often relies on extremely energy-intensive multiple recrystallizations or multi-stage resin purification, accompanied by the generation of large amounts of high-salt wastewater and trace amounts of toxic byproducts. The 48.2% yield of this invention is a "green yield" achieved in a four-step cascade, entirely mild biological system. This invention successfully overcomes the kinetic bottleneck of conformational limitation in the biocatalysis of rare four-carbon sugars, achieving excellent product purity (99.65%) and extremely low impurity residues with a more environmentally friendly and safer process route while avoiding the introduction of toxic reagents. Therefore, this invention achieves a technological advancement that is difficult to match by existing chemical purification processes in terms of the non-obviousness of the technical concept and the food safety of the final product.

[0061] In step 2 of the process of this invention, an iron catalyst is introduced. By controlling the amount added (0.05%~0.15%) and cooperating with the precipitation separation process, the final Fe residue is controlled below 1.0 ppm (0.58 ppm in Example 4), which meets the heavy metal limit requirements.

[0062] Compared with the vitamin C oxidation method (Comparative Example 1), the cascade process of the present invention uses the calcium ion in-situ binding mechanism to convert oxalic acid into precipitate separation in the early stage of the reaction, so that the oxalic acid residue in the final crystallized product is kept below the detection limit (<0.01%).

[0063] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for biosynthesizing magnesium L-threonate, characterized in that, Including the following steps: An enzyme mixture of 100-600 U / g L-threose, NAD+ at a final concentration of 0.1-0.5 mmol / L, and NOX at a final concentration of 0.05-0.1 mg / mL were added to a purified L-threose solution with a concentration of 50-150 g / L to obtain a reaction solution. The reaction solution was then subjected to ultrafiltration and adsorption, and the eluent was collected to obtain an L-threonic acid solution. L-threonic acid solution with a concentration of 50~150 g / L was mixed with magnesium hydroxide at a ratio of 800~1000 mL: 4.5~6.5 g, stirred, pH adjusted, concentrated, crystallized, and dried to obtain magnesium L-threonate; The enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 1~3:1~3:1~2; the UniProt accession number of AraDH is Q53TZ2, the UniProt accession number of CcXylDH is B8H1Z0, and the UniProt accession number of AldH is Q88JG9.

2. The method according to claim 1, characterized in that, The L-threose purification solution is prepared as follows: (1) Inoculate the seed culture of Gluconobacterium oxidans into a medium containing 50-100 g / L xylose at an inoculation rate of 5-10% v / v and ferment. During fermentation, add calcium carbonate solution to maintain the pH of the fermentation broth. After fermentation, centrifuge to obtain L-xylose calcium solution. (2) Mix L-xylorate calcium solution with anhydrous ferric sulfate at a ratio of 1000~2000 mL: 0.5~2.0 g, and then add H2O2 solution dropwise to react. After the reaction is completed, adjust the pH and filter to obtain the filtrate. Add catalase with a final concentration of 0.05~0.2 mg / mL to the filtrate, stir, and filter to obtain the clear liquid. Purify the clear liquid to obtain L-threose purified solution.

3. The method according to claim 2, characterized in that, In step (2), the H2O2 solution is added dropwise to the reaction system at a rate of 2-5 mL / min to carry out the reaction; the reaction is carried out for 2-4 h; the pH is adjusted to 7.0-7.

5.

4. The method according to claim 1, characterized in that, The pH adjustment is to adjust the pH to 6.5~7.5; the crystallization is to add 2~4 volumes of anhydrous ethanol dropwise at a flow rate of 2~5 mL / min to crystallize and collect the crystals; the drying is to dry at -0.08~-0.1 MPa and 40~50℃ for 10~14 h.

5. Magnesium L-threonate prepared by any one of the methods described in claims 1 to 4.

6. A method for biosynthesizing high-purity, high-yield, and low-oxalic acid residue L-threonate magnesium, characterized in that, Including the following steps: An enzyme mixture of 100-600 U / g L-threose, NAD+ at a final concentration of 0.1-0.5 mmol / L, and NOX at a final concentration of 0.05-0.1 mg / mL were added to a purified L-threose solution with a concentration of 50-150 g / L to obtain a reaction solution. The reaction solution was then subjected to ultrafiltration and adsorption, and the eluent was collected to obtain an L-threonic acid solution. L-threonic acid solution with a concentration of 50~150 g / L was mixed with magnesium hydroxide at a ratio of 800~1000 mL: 4.5~6.5 g, stirred, pH adjusted, concentrated, crystallized, and dried to obtain magnesium L-threonate; The enzyme mixture is obtained by mixing AraDH, CcXylDH and AldH in an enzyme activity ratio of 1~3:1~3:1~2; the UniProt accession number of AraDH is Q53TZ2, the UniProt accession number of CcXylDH is B8H1Z0, and the UniProt accession number of AldH is Q88JG9.

7. The method according to claim 6, characterized in that, The L-threose purification solution is prepared as follows: (1) Inoculate the seed culture of Gluconobacterium oxidans into a medium containing 50-100 g / L xylose at an inoculation rate of 5-10% v / v and ferment. During fermentation, add calcium carbonate solution to maintain the pH of the fermentation broth. After fermentation, centrifuge to obtain L-xylose calcium solution. (2) Mix L-xylorate calcium solution with anhydrous ferric sulfate at a ratio of 1000~2000 mL: 0.5~2.0 g, and then add H2O2 solution dropwise to react. After the reaction is completed, adjust the pH and filter to obtain the filtrate. Add catalase with a final concentration of 0.05~0.2 mg / mL to the filtrate, stir, and filter to obtain the clear liquid. Purify the clear liquid to obtain L-threose purified solution.

8. The method according to claim 7, characterized in that, In step (2), the H2O2 solution is added dropwise to the reaction system at a rate of 2-5 mL / min to carry out the reaction; the reaction is carried out for 2-4 h; the pH is adjusted to 7.0-7.

5.

9. The method according to claim 6, characterized in that, The pH adjustment is to adjust the pH to 6.5~7.5; the crystallization is to add 2~4 volumes of anhydrous ethanol dropwise at a flow rate of 2~5 mL / min to crystallize and collect the crystals; the drying is to dry at -0.08~-0.1 MPa and 40~50℃ for 10~14 h.

10. The use of the method according to any one of claims 6 to 9 in the preparation of magnesium L-threonate.

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