A method for separating and purifying alpha-ketoglutaric acid from a bioconversion solution

Abstract

The application belongs to the technical field of bio-chemical industry and separation engineering, and discloses a method for separating and purifying alpha-ketoglutaric acid from bioconversion liquid. The application constructs a separation and purification process integrated by membrane separation and ion exchange, which sequentially comprises the following steps: membrane separation for removing bacteria and macromolecular impurities, integrated impurity removal treatment of heat denaturation combined with decolorization flocculation, nanofiltration selective interception and desalination, cation exchange fine desalination, and concentration and crystallization steps. In the process, the nanofiltration process effectively retains alpha-ketoglutaric acid while reducing the content of small-molecule inorganic salts, and the cation exchange further removes residual cations in the system, thereby providing a low-salt environment for subsequent crystallization. Through the synergistic integration of membrane separation and ion exchange, the application realizes efficient removal of impurities and significant reduction of salt load, significantly improves the purity and yield of alpha-ketoglutaric acid while ensuring the simplicity of the process, and has the advantages of simple process, low cost and suitability for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomanufacturing and separation and purification technology, specifically to a method for separating and purifying α-ketoglutaric acid from a conversion solution obtained from a whole-cell catalytic system or an enzyme catalytic system, belonging to the field of downstream processing technology of organic acid biomanufacturing. Background Technology

[0002] α-Ketoglutaric acid (α-KG) is a key intermediate metabolite in the tricarboxylic acid cycle and has wide applications in food additives, pharmaceutical intermediates, animal nutrition, and fine chemicals. With the development of metabolic engineering and enzyme catalysis, the production of α-ketoglutaric acid using whole-cell catalysis or enzyme catalysis systems has gradually become the mainstream technical route. In these biotransformation systems, α-ketoglutaric acid usually exists in its salt form, especially sodium α-ketoglutarate. The system also contains bacterial residues, proteins, peptides, small-molecule metabolic byproducts, and a high concentration of inorganic salts. This complex system significantly increases the difficulty of separating and purifying α-ketoglutaric acid, especially during subsequent concentration and crystallization. The high salt load in the system can easily cause inorganic salt co-crystallization or uncontrolled crystallization, leading to reduced product purity and yield. Therefore, developing efficient, simple, and controllable downstream separation and purification processes is of great significance.

[0003] Currently, the existing technologies for the separation and purification of α-ketoglutarate mainly include the following types: (1) Ion exchange resin method: This method usually uses anion or cation exchange resins to adsorb or desalinate α-ketoglutarate, which is a common industrial technology route. However, this method usually relies on a multi-stage resin treatment process, which has problems such as large resin consumption, frequent regeneration, high operating costs and complex process flow. In addition, in high-salt systems, the resin exchange efficiency is easily inhibited, affecting the treatment effect. (2) Acidification-extraction method: After converting α-ketoglutarate into free acid by strong acid, organic solvents (such as ethyl acetate) are used for extraction and separation. Although this method can obtain high-purity products, it has disadvantages such as large solvent consumption, heavy environmental burden and complex process. (3) Single membrane separation method: Including ultrafiltration, nanofiltration or electrodialysis, which are mainly used to remove bacteria, proteins or some salts. Membrane separation has the advantages of simple operation and low energy consumption, but a single membrane process often cannot simultaneously achieve the removal of macromolecular impurities and salt control, especially in high-salt systems, where the separation efficiency of nanofiltration or electrodialysis is limited, making it difficult to directly obtain a low-salt system suitable for crystallization. (4) Multi-step combined process: Combining multiple methods such as membrane separation, ion exchange and crystallization to improve purification efficiency. However, existing combined processes are usually long and redundant, and there is a lack of effective coupling between the unit operations, resulting in low overall efficiency.

[0004] Furthermore, since α-ketoglutarate exists primarily as a salt in bioconversion solutions, direct concentration and crystallization without effectively reducing the salt load can easily lead to inorganic salt co-crystallization, affecting product purity and yield. Therefore, reducing the salt content and simplifying the separation process while ensuring the recovery rate of the target product is a critical technical problem that urgently needs to be solved in the downstream separation of α-ketoglutarate. Based on this, it is necessary to develop a separation and purification method with a clear structure, tightly coupled steps, and good industrial applicability. Specifically, this involves a rational combination of ceramic and ultrafiltration membranes for solid-liquid separation and macromolecular removal, along with an integrated impurity removal process combining thermal denaturation with decolorization and flocculation to achieve efficient removal of protein and pigment impurities. Further, nanofiltration is used to selectively retain α-ketoglutarate anions, while simultaneously reducing the content of small-molecule inorganic salts in the system. Finally, cation exchange resin is used to finely desalt the residual cations, creating a low-salt environment that provides favorable conditions for subsequent concentration and crystallization. Through the synergistic integration of these unit operations, it is expected that the purification efficiency and product quality of α-ketoglutarate can be significantly improved while simplifying the process. Summary of the Invention

[0005] Since α-ketoglutarate mainly exists in its salt form in whole-cell or enzyme biotransformation solutions, and the system is usually accompanied by impurities such as proteins, small molecule metabolic byproducts and inorganic salts, which makes crystallization and purification difficult, the purpose of this invention is to provide a method for separating and purifying α-ketoglutarate from whole-cell or enzyme biotransformation solutions, so as to solve the problems of complex process flow, low salt control efficiency and poor crystallization and purification effect in the existing technology, thereby achieving efficient and low-cost preparation of α-ketoglutarate.

[0006] In their research on the downstream separation of α-ketoglutaric acid prepared by biotransformation, the inventors discovered that α-ketoglutaric acid typically exists in its alkali metal salt form, particularly sodium α-ketoglutarate, in the conversion solution obtained from whole-cell catalytic or enzyme catalytic systems. The system also contains high concentrations of inorganic salts, proteins, polysaccharides, cell debris, and small-molecule metabolic byproducts. Among these, the high concentration of inorganic salts, especially Na+, is particularly significant. +The presence of impurities not only increases the subsequent separation load but also preferentially precipitates to form impurity salt crystals during concentration and crystallization, further inducing the embedding, entrainment, and co-crystallization of the target product. This leads to uncontrolled crystallization of α-ketoglutarate, ultimately resulting in decreased product purity and yield. Further research revealed that existing technologies using a single desalting process struggle to simultaneously achieve both desalting efficiency and target product retention. Specifically, when using ion exchange resin alone, the high salt load of the conversion solution leads to rapid depletion of the resin's exchange capacity, resulting in high resin consumption, frequent regeneration, and high operating costs. Conversely, when using membrane desalting technology alone, the residual cation content in the system remains high, making it difficult to effectively suppress the precipitation of impurities during subsequent crystallization stages. Therefore, relying solely on a single desalting unit is insufficient to construct a low-salt environment suitable for stable crystallization of α-ketoglutarate. The purpose of this invention is to provide a method for separating and purifying α-ketoglutarate from whole-cell or enzyme-based biotransformation solutions, addressing the problems of complex processes, low salt control efficiency, and poor crystallization purification effects in existing technologies, thereby achieving efficient and low-cost preparation of α-ketoglutarate.

[0007] Based on the above understanding, this invention proposes a method for the separation and purification of α-ketoglutarate based on synergistic desalting and controlled crystallization, establishing a synergistic process system of "front-end impurity removal and load reduction—nanofiltration selective desalting—cation exchange fine desalting—low-salt environment controlled crystallization". Specifically, firstly, membrane separation combined with integrated thermal denaturation-decolorization-flocculation treatment is used to remove bacterial cells, proteins, and colloidal impurities, reducing the complexity of the system; then, the selective retention of α-ketoglutarate anions by the nanofiltration membrane is used to achieve enrichment of the target product and initial reduction of the system's salt load; further, cation exchange resin is used to remove residual cations after nanofiltration, performing fine desalting treatment on the system, thereby establishing a low-salt crystallization environment; finally, concentration and cooling crystallization are carried out under controlled conditions to achieve efficient separation and purification of α-ketoglutarate.

[0008] The core of this invention is not a simple superposition of unit operations, but rather the establishment of a synergistic mechanism between nanofiltration pre-desalination and cation exchange fine desalination: the nanofiltration step prioritizes reducing the overall salt load of the system, thereby reducing the pressure on subsequent resin exchange; the cation exchange step further removes residual Na+. + The presence of isocations inhibits co-precipitation of mixed salts at the source; the synergistic effect of the two significantly improves the crystallization system environment, enabling the transformation from a high-salt system to a low-salt crystallization system, thereby effectively regulating the crystallization behavior of α-ketoglutarate and improving product purity and yield.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for separating and purifying α-ketoglutarate from whole-cell conversion solution or enzyme conversion solution, wherein α-ketoglutarate exists mainly in its salt form in the conversion solution, the method comprising the following steps: First, the conversion solution is subjected to membrane separation treatment, preferably using ceramic membrane filtration to remove bacterial cells and suspended impurities, and further removed by ultrafiltration to remove proteins and other macromolecular impurities, thereby obtaining a clear filtrate containing α-ketoglutarate; Second, the filtrate is subjected to heat treatment to denature the proteins in the system, and combined with decolorizing agent and flocculant for integrated impurity removal treatment to achieve efficient removal of protein residues, pigments and colloidal impurities. The system is first purified to reduce its complexity. Then, the purified solution is subjected to nanofiltration. During nanofiltration, α-ketoglutarate is selectively retained in its anionic form, while small-molecule inorganic salts permeate through the membrane, thereby enriching the target product and reducing the salt load of the system. Further, the nanofiltration-treated solution is treated with a cation exchange resin to remove residual cations, further reducing the salt content and improving subsequent crystallization conditions. Finally, the cation exchange-treated solution is concentrated under reduced pressure and cooled under controlled conditions to obtain the α-ketoglutarate product. Preferably, the mother liquor from crystallization is recovered to further improve the product yield.

[0010] This invention provides a method for separating and purifying α-ketoglutarate from whole-cell conversion solution or enzyme conversion solution, comprising the following steps:

[0011] a. The conversion solution is subjected to membrane filtration with a pore size of no more than 0.2 μm to remove bacterial cells or suspended impurities;

[0012] b. Perform ultrafiltration on the solution obtained in step a to remove proteins and macromolecular impurities;

[0013] c. Heat the solution obtained in step b, and add a decolorizing agent and a flocculant to remove impurities;

[0014] d. Perform nanofiltration on the solution obtained in step c to retain α-ketoglutarate in anionic form, while reducing the content of small molecule inorganic salts;

[0015] e. The solution obtained in step d is treated with a cation exchange resin to remove cations from the system; the cation exchange resin is a strong acid type cation exchange resin.

[0016] f. The solution obtained in step e is concentrated and cooled to crystallize, yielding α-ketoglutaric acid product; the concentration temperature is 50–80°C, and the crystallization temperature is 0–25°C.

[0017] The whole-cell conversion solution refers to a reaction solution containing α-ketoglutaric acid obtained through whole-cell catalytic reaction using microbial cells capable of producing α-ketoglutaric acid as the catalytic host. The whole-cell catalytic reaction includes the process of directly catalyzing the conversion of a substrate into α-ketoglutaric acid using intact microbial cells (such as whole-cell catalysts obtained from fermentation broth). Preferably, the α-ketoglutaric acid exists primarily in its salt form, and more preferably, sodium α-ketoglutarate.

[0018] The enzyme conversion solution refers to a reaction solution obtained by using free enzyme, immobilized enzyme, or crude enzyme solution as the catalytic host to generate α-ketoglutarate through an enzyme-catalyzed reaction. Preferably, the enzyme-catalyzed reaction includes a process of converting L-glutamic acid or its salt into α-ketoglutarate under the action of glutamic acid oxidase, wherein the α-ketoglutarate is preferably in the form of its salt, and more preferably sodium α-ketoglutarate.

[0019] In a specific embodiment, the membrane pore size in the membrane filtration is 0.2 μm, the operating pressure is controlled at 0.12–0.18 MPa, the temperature is maintained at 28–32°C, and the circulation flow rate is approximately 2.0–2.8 m / s.

[0020] In a specific embodiment, the membrane filtration is a tubular ceramic membrane filtration, which filters to a clear filtrate with a transmittance of not less than 95% at 600 nm.

[0021] In a specific embodiment, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 5 to 50 kDa.

[0022] Preferably, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 10 kDa and is an organic spiral wound ultrafiltration membrane, which operates under a pressure of 0.20–0.30 MPa and an operating temperature controlled at 24°C–26°C.

[0023] In a specific embodiment, the heating treatment temperature is 70-100℃ and the time is 30-60 min. After natural cooling to 30-40℃, a decolorizing agent is added, and a flocculant is added after decolorization.

[0024] In a specific embodiment, the decolorizing agent is powdered activated carbon; the flocculant is chitosan or an inorganic flocculant; the amount of powdered activated carbon added is 1.0-1.5% of the solution mass, and the mixture is stirred for 10-30 minutes; before the decolorization is completed, chitosan is added as a flocculant at an amount of 0.2-0.3‰, and the mixture is stirred for another 6-12 minutes. Subsequently, solid-liquid separation is performed using plate and frame filtration to obtain a clarified liquid.

[0025] In a specific embodiment, the nanofiltration membrane used in the nanofiltration process has a molecular weight cutoff of 100 to 500 Da, and the nanofiltration operating pressure is 0.5 to 3.0 MPa.

[0026] Preferably, a nanofiltration membrane with a molecular weight cutoff of approximately 200 Da is selected, and the membrane is operated within a pressure range of 1.2–1.8 MPa and a temperature controlled at 24–26°C.

[0027] First, concentrate the liquid obtained in the previous step to 2 / 5 to 2 / 7 of its original volume, then add an equal volume of deionized water to dilute it, perform nanofiltration, and repeat the above operation twice.

[0028] In the specific implementation, in step e, 732 strong acid cation exchange resin (a strong acid cation exchange material with 7% crosslinking degree of styrene-divinylbenzene copolymer as the backbone and sulfonic acid groups (-SO3H) is selected. It is manufactured in sodium form and belongs to industrial-grade polymer materials. It is a commonly used strong acid cation exchange resin) with an operating flow rate of 1.8 BV / h. The resin pretreatment involves activation with 1 mol / L hydrochloric acid followed by washing with deionized water until neutral.

[0029] In a specific embodiment, step f involves concentration using reduced pressure evaporation, with the temperature controlled at 58–62°C and a vacuum degree of -0.085 MPa. The concentrate is then transferred to a crystallization container and cooled to 10°C at a rate of 1°C / h under slow stirring, while maintaining stillness to complete the crystallization process. After crystallization, wet crystals and mother liquor are obtained by centrifugation. The obtained wet crystals are washed with anhydrous ethanol, stirred at room temperature, and then centrifuged again. The washed crystals are placed in a vacuum drying oven and dried at 50–70°C and -0.06–0.10 MPa for 4–8 h, finally yielding a white powdered α-ketoglutaric acid product.

[0030] Beneficial effects of the invention

[0031] Compared with existing technologies, this invention addresses the problem of uncontrolled α-ketoglutarate crystallization caused by high-salt systems in biotransformation solutions. It proposes and establishes a separation and purification technology system based on synergistic desalination to control crystallization. Through the organic coupling of nanofiltration pre-desalination and cation exchange fine desalination, an integrated synergistic approach is achieved for impurity removal, salt load regulation, and crystallization optimization. Specifically, the membrane separation step, through the combined application of ceramic and ultrafiltration membranes, effectively removes large molecular impurities such as bacterial cells, suspended particles, and proteins, providing a stable clarified system for subsequent processing. The integrated impurity removal treatment, through heating denaturation combined with the synergistic effect of decolorizing agents and flocculants, integrates the traditional dispersed protein removal and decolorization steps, significantly improving impurity removal efficiency and simplifying the operation process. The nanofiltration step, based on the selective retention of multivalent organic anions, effectively retains α-ketoglutarate anions while allowing small-molecule inorganic salts to permeate the membrane, reducing the system's salt load from the source. Further fine desalination of residual cations using cation exchange resin synergizes with the nanofiltration step, significantly optimizing the solution system and creating a low-salt environment suitable for subsequent crystallization. Based on the synergistic effect of the above processes, this invention effectively shortens the process flow, reduces repetitive functions between unit operations, lowers energy consumption and operating costs, and significantly improves the crystallization behavior of α-ketoglutaric acid while ensuring processing efficiency, thereby improving product purity and yield, and has good prospects for industrial application.

[0032] Preferably, after treatment by the separation and purification method described in this invention, the purity of the α-ketoglutaric acid product obtained from the whole-cell transformation solution is approximately 98.8%, with an overall yield of 80.3%; the purity of the α-ketoglutaric acid product obtained from the enzyme-catalyzed transformation solution is approximately 99.2%, with an overall yield of 85.4%. During crystallization, the system operates stably, and the co-precipitation of impurities is effectively suppressed, thereby improving crystallization quality and product purity, and enhancing the controllability and repeatability of the crystallization process. The above results indicate that the method of this invention exhibits good applicability in different types of biotransformation systems, possessing strong process versatility and stability. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, so that the technical solution and its beneficial effects of the present invention are clearer and more complete. It should be noted that the following embodiments are only for illustrating the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and substance of the present invention, and all such modifications or substitutions should fall within the scope of protection of the present invention.

[0034] Example 1: Extraction and purification of α-ketoglutarate from whole-cell transformation medium

[0035] α-ketoglutarate conversion solution was obtained by whole-cell catalytic reaction using L-glutamate as a substrate.

[0036] Using L-glutamate as a substrate, a whole-cell transformation solution containing α-ketoglutarate was prepared by employing recombinant Corynebacterium glutamicum expressing glutamate oxidase as a whole-cell catalyst for biotransformation. The recombinant Corynebacterium glutamicum heterologously expresses a glutamate oxidase gene derived from *Streptomyces mobaraensis*, which catalyzes the oxidative deamination of L-glutamate to produce α-ketoglutarate, ammonia, and hydrogen peroxide. Specifically, after fermenting the recombinant Corynebacterium glutamicum expressing glutamate oxidase, the cells were collected by centrifugation, washed 2-3 times with distilled water, resuspended, and the cell concentration adjusted to OD=25 for use as the whole-cell catalyst. The whole-cell catalytic reaction was carried out in a fermenter using monosodium glutamate (MSG) as a substrate. Preferably, the initial concentration of MSG in the whole-cell transformation system was 270 g / L, with 0.5% (v / v) Triton X-100 added to improve substrate transfer efficiency. The catalytic temperature was 32 °C, and the reaction time was 40 h. After catalysis, a whole-cell transformation solution containing α-ketoglutarate was obtained. In this solution, α-ketoglutarate mainly exists in its sodium salt form. The system also contains unreacted substrate, small amounts of byproducts, organic acids, proteins, cell debris, and inorganic salts. No further purification was performed after the reaction; the transformation solution was directly used as a raw material for subsequent separation and purification steps. The total volume of the transformation solution was 10 L, with the sodium α-ketoglutarate concentration calculated based on the total α-ketoglutarate concentration in the transformation solution being 182.6 g / L (not the concentration of the sodium α-ketoglutarate itself). The system also contained small amounts of byproducts such as pyruvate (approximately 12.4 g / L), soluble proteins (approximately 4.2 g / L), bacterial cell debris, and polysaccharides, as well as Na+. + The system is mainly composed of inorganic salts (total salt concentration of about 70-80 g / L), with an initial pH of about 7.6. The system is obviously turbid and has typical characteristics of a high-salt, high-organic-load biotransformation solution.

[0037] First, the conversion solution was subjected to solid-liquid separation. Cross-flow filtration was performed using a tubular ceramic membrane with a pore size of 0.2 μm. The operating pressure was controlled at 0.16 MPa, the temperature was maintained at 30℃, and the circulation flow rate was approximately 2.5 m / s. During filtration, the transmembrane pressure differential was kept stable to prevent further membrane fouling. After approximately 40 minutes of treatment, a clear filtrate was obtained, with the transmittance (600 nm) increasing from less than 10% to over 97%. Bacterial cells and suspended particles were essentially completely removed, and the filtrate volume was approximately 9.6 L.

[0038] The filtrate was then subjected to ultrafiltration to remove proteins and other macromolecular impurities. An organic spiral-wound ultrafiltration membrane with a molecular weight cutoff of 10 kDa was selected and operated at a pressure of 0.25 MPa with the operating temperature controlled at approximately 25°C. Partial reflux was used to maintain a stable flux during ultrafiltration, and the permeate was obtained after approximately 60 minutes. Analysis showed that the permeate volume was approximately 9.2 L, and the protein content in the system decreased from 4.2 g / L to below 0.18 g / L, with a removal rate exceeding 95%. The loss rate of α-ketoglutarate was controlled within 2%, indicating that this step had minimal impact on the target product.

[0039] After membrane separation, the ultrafiltrate underwent integrated impurity removal. The resulting solution was heated to 80°C and held for 30–45 min to cause thermal denaturation of residual proteins and the formation of flocculent precipitates. It was then allowed to cool naturally to approximately 35°C, and powdered activated carbon was added for decolorization. The addition amount was typically 1.0–1.5% of the solution mass; in this example, 1.2% was added. The mixture was stirred at 150 rpm for 20 min. Before the decolorization was complete, chitosan was added as a flocculant, typically at 0.2–0.3‰; in this example, 0.25‰ was added. Stirring continued for approximately 8–10 min to promote the aggregation and growth of fine particles. Solid-liquid separation was then performed using plate and frame filtration to obtain a clarified solution with a volume of approximately 8.8 L. Measurements showed that the color of the treated solution decreased by more than 90%, the residual protein content was below 0.12 g / L, and the system stability was significantly improved.

[0040] Based on the above, the treated solution was subjected to nanofiltration desalination and enrichment. A nanofiltration membrane with a molecular weight cutoff of approximately 200 Da was selected, and the operation was carried out within a pressure range of 1.8 MPa and a temperature control of 25℃. A "concentration-dilution-reconcentration" dialysis washing method was used during the operation to enhance the desalination effect: first, the system was concentrated to approximately 1 / 3 of its original volume, then an equal volume of deionized water was added for dilution, and this operation was repeated twice. The final nanofiltration retentate volume was approximately 3.2 L. The results showed that the retention rate of α-ketoglutaric acid during the nanofiltration process reached over 95%, while the removal rate of inorganic salts was approximately 73%, and the system conductivity decreased significantly.

[0041] To further reduce the residual cation content in the system, the nanofiltration retentate was subjected to cation exchange desalting treatment. The inventors screened and compared 001×7MB strong acid cation exchange resin, D113 weak acid cation exchange resin, and 732 strong acid cation exchange resin for the high-salt biotransformation system of α-ketoglutaric acid. The results showed that although the 001×7 resin had a high exchange capacity, with a system conductivity of approximately 9.4 mS / cm after treatment, it exhibited some retention of the target product, with an α-ketoglutaric acid recovery rate of approximately 87.5%. The D113 weak acid resin showed good retention of Na+.+ The exchange capacity is relatively weak, and the conductivity of the system after treatment remains at a high level, approximately 11.2 mS / cm, indicating limited desalination effect and difficulty in meeting the requirements for subsequent low-salt crystallization. In contrast, 732 strong acid cation exchange resin combines high exchange capacity with low adsorption characteristics of the target product, maintaining stable desalination performance even in high-salt systems. Therefore, it is the preferred desalination resin. In this example, 732 strong acid cation exchange resin was used for treatment, with a column packing volume of 1.2 L and an operating flow rate of 1.8 BV / h. The volume of the resulting solution after treatment was approximately 3.6 L. α-ketoglutaric acid was essentially not adsorbed, and the recovery rate reached 98.5%. Na + The removal rate reached over 93%, and the system conductivity was further reduced from approximately 26 mS / cm after nanofiltration to approximately 5 mS / cm. Further research revealed that the upstream nanofiltration step preferentially reduced the overall salt load of the system, while the 732 resin further removed residual Na+. + The presence of isocations and their synergistic effect reduces the ionic strength gradient of the system, creating a low-salt environment suitable for the stable crystallization of α-ketoglutarate. This effectively suppresses the co-precipitation of impurities and crystal inclusion during subsequent concentration and crystallization, creating key conditions for the preparation of high-purity α-ketoglutarate.

[0042] The desalted solution proceeded to the concentration and crystallization steps. Concentration was achieved using vacuum evaporation at 60°C and a vacuum of -0.085 MPa, concentrating the system to approximately 2.5 L. At this point, the α-ketoglutaric acid concentration was approximately 580 g / L, and the system exhibited high viscosity. The concentrate was then transferred to a crystallization vessel and cooled to 10°C at a rate of approximately 1°C / h with slow stirring, and allowed to stand for 48 h to complete the crystallization process. After crystallization, centrifugation (8000 rpm, 10 min) was used to obtain wet crystals and mother liquor. The obtained wet crystals were washed with anhydrous ethanol (approximately 1 / 3 of the crystal volume), stirred at room temperature for 10 min, and then centrifuged again. The washed crystals were placed in a vacuum drying oven and dried at 60°C and -0.08 MPa for 6 h, finally yielding approximately 1.51 kg of white powdered α-ketoglutaric acid.

[0043] High-performance liquid chromatography (HPLC) analysis showed that the purity of the obtained product was 98.5%–99.2%, with an average of 98.8%. Based on the total amount of α-ketoglutaric acid in the initial conversion solution, the overall yield was 76.3%–82.7%, with an average of approximately 80.3%. In summary, this embodiment demonstrates that solid and macromolecular impurities are removed through ceramic membrane filtration and ultrafiltration; integrated impurity removal is achieved through thermal denaturation combined with decolorization and flocculation; and nanofiltration selective retention and cation exchange desalting significantly reduce the salt load of the system, ultimately achieving efficient crystallization and separation of α-ketoglutaric acid. This method can still obtain a high-purity product (≥99%) and maintain a reasonable yield (≥80%) even under high initial concentrations (≥180 g / L). The obtained product has high purity and stable yield, and the process does not require the use of organic solvents, showing good prospects for industrial application.

[0044] Example 2: Extraction and purification of α-ketoglutarate from enzyme catalytic solution

[0045] In this embodiment, to further verify the applicability of the method of the present invention to systems from different sources, an enzyme-catalyzed system was used to prepare α-ketoglutarate conversion solution. Specifically, L-glutamate sodium was used as a substrate, and the conversion reaction was carried out in an enzyme-catalyzed system containing L-glutamate oxidase (LGOX) and catalase (CAT). L-glutamate oxidase catalyzes the oxidation and deamination of L-glutamate to α-ketoglutarate, while catalase decomposes the hydrogen peroxide generated during the reaction, thereby reducing its inhibitory effect on enzyme activity and improving catalytic efficiency. The enzyme-catalyzed reaction is preferably carried out in a fermenter, with the liquid volume controlled below 60% of the effective volume of the fermenter; the reaction temperature is controlled at 35 °C, the reaction pH is controlled at 6.5, the stirring speed is 400 rpm, the aeration rate is 1.0 vvm, and the dissolved oxygen is maintained at 50% air saturation. As the reaction proceeds, α-ketoglutarate gradually accumulates in the system. When the substrate is essentially completely converted or the product concentration tends to stabilize, the reaction is terminated, yielding the enzyme-catalyzed conversion solution. Compared to the whole-cell conversion system, this enzyme-catalyzed system does not contain intact bacterial cells or large granular cell structures, and the system is relatively clearer. However, it still contains a certain amount of enzyme proteins, coenzymes, peptides, and inorganic salts introduced by the buffer system and pH adjuster, among other impurities.

[0046] The enzyme conversion solution had a total volume of 10 L, with an α-ketoglutarate (sodium salt) concentration of 60.2 g / L. The system contained approximately 5.6 g / L of soluble protein (mainly enzyme protein), and approximately 2–4 g / L of polypeptides and low-molecular-weight organic byproducts (including residual substrates and organic acids). Inorganic salts were mainly present as sodium salts, with a total salt concentration of approximately 25–35 g / L. The initial pH was approximately 7.5. The system was generally clear, but still exhibited a high protein content and a certain salt load.

[0047] The enzyme-catalyzed conversion solution was separated and purified according to the method described in Example 1, including sequential membrane filtration to remove trace suspended impurities, ultrafiltration to remove proteins and macromolecular components, integrated impurity removal treatment combining heating with decolorization and flocculation, nanofiltration desalting, and cation exchange fine desalting. Subsequently, it was concentrated under reduced pressure and crystallized at low temperature, and then washed with ethanol and dried to obtain the final product. The operating conditions for each step were basically the same as in Example 1, and will not be repeated here.

[0048] After the above processing, the purity of the obtained α-ketoglutaric acid product was 99.2% as determined by HPLC, and the total yield was 85.4% based on the total amount of α-ketoglutaric acid in the initial conversion solution. The results indicate that even in an enzyme-catalyzed system with a low initial concentration (approximately 60 g / L), the separation and purification method described in this invention can still achieve efficient desalting and crystallization separation, obtaining a high-purity product with a high yield.

[0049] In summary, this embodiment demonstrates that the method of the present invention is applicable not only to high-concentration whole-cell conversion fluid systems but also to enzyme-catalyzed conversion fluid systems, exhibiting good process versatility and stability.

Claims

1. A method for separating and purifying α-ketoglutaric acid from whole-cell conversion solution or enzyme conversion solution, characterized in that, Includes the following steps: a. The conversion solution is subjected to membrane filtration with a pore size of no more than 0.2 μm to remove bacterial cells or suspended impurities, and the filtrate is filtered until it is clear with a transmittance of no less than 95% at 600 nm; b. The solution obtained in step a is subjected to ultrafiltration to remove proteins and macromolecular impurities; the ultrafiltration membrane used for ultrafiltration has a molecular weight cutoff of 5 to 50 kDa. c. The solution obtained in step b is heated and a decolorizing agent and a flocculant are added to remove impurities. The heating temperature is 70-100℃ and the time is 30-60 min. After natural cooling to 30-40℃, the decolorizing agent is added. After decolorization, the flocculant is added. Then, solid-liquid separation is carried out by plate and frame filtration to obtain a clarified solution. d. The solution obtained in step c is subjected to nanofiltration to retain α-ketoglutarate in anionic form and reduce the content of small molecule inorganic salts. The nanofiltration membrane used in the nanofiltration has a molecular weight cutoff of 100-500 Da, and the nanofiltration operating pressure is 0.5-3.0 MPa. During the operation, the solution obtained in step c is first concentrated to 2 / 5 to 2 / 7 of its original volume, then diluted with an equal volume of deionized water, and nanofiltration is performed. The above operation is repeated twice. e. The solution obtained in step d is treated with a cation exchange resin to remove cations from the system; the cation exchange resin is 732 strong acid type cation exchange resin. f. The solution obtained in step e is concentrated and cooled to crystallize, yielding α-ketoglutaric acid product; the concentration temperature is 50–80°C, and the crystallization temperature is 0–25°C. The whole-cell conversion solution is obtained through whole-cell catalytic reactions, including the direct catalytic conversion of substrates into a reaction solution containing α-ketoglutaric acid using intact microbial cells. The enzyme conversion solution is obtained by a catalytic reaction that produces α-ketoglutaric acid as the target product.

2. The method according to claim 1, characterized in that, The membrane filtration system has a membrane pore size of 0.2 μm, an operating pressure controlled between 0.12 and 0.18 MPa, a temperature maintained between 28 and 32 °C, and a circulation flow rate of 2.0 to 2.8 m / s.

3. The method according to claim 1, characterized in that, The membrane filtration is a tubular ceramic membrane filtration.

4. The method according to claim 1, characterized in that, The ultrafiltration membrane selected is an organic spiral wound ultrafiltration membrane with a molecular weight cutoff of 10 kDa.

5. The method according to claim 4, characterized in that, The ultrafiltration treatment is performed under the following conditions: pressure of 0.20–0.30 MPa and operating temperature of 24°C–26°C.

6. The method according to claim 1, characterized in that, The heating treatment temperature is 80℃ and the time is 30-45 minutes. After natural cooling to 35℃, a decolorizing agent is added, and after decolorization, a flocculant is added.

7. The method according to claim 1, characterized in that, The decolorizing agent is powdered activated carbon; the flocculant is chitosan; the amount of powdered activated carbon added is 1.0-1.5% of the solution mass, and the mixture is stirred for 10-30 minutes; before the decolorization is completed, chitosan is added as a flocculant at an amount of 0.2-0.3‰, and the mixture is stirred for another 6-12 minutes. Subsequently, solid-liquid separation is performed by plate and frame filtration to obtain a clarified liquid.

8. The method according to claim 1, characterized in that, A nanofiltration membrane with a molecular weight cutoff of 200 Da was selected and operated within a pressure range of 1.2–1.8 MPa, with the temperature controlled at 24–26 °C.

9. The method according to claim 1, characterized in that, In step e, 732 strong acid cation exchange resin is selected, and the operating flow rate is 1.8 BV / h; the resin pretreatment involves activation with 1 mol / L hydrochloric acid followed by washing with deionized water until neutral.

10. The method according to claim 9, characterized in that: In step f, the solution is concentrated by vacuum evaporation, with the temperature controlled at 58–62°C and the vacuum degree at -0.085 MPa. The concentrate is then transferred to a crystallization container and cooled to 10°C at a rate of 1°C / h under slow stirring, and kept still to complete the crystallization process. After crystallization, wet crystals and mother liquor are obtained by centrifugation. The obtained wet crystals are washed with anhydrous ethanol, stirred at room temperature, and then centrifuged again. The washed crystals are placed in a vacuum drying oven and dried at 50–70°C and -0.06–0.10 MPa for 4–8 hours to finally obtain a white powdery α-ketoglutaric acid product.