Enzyme fermentation method for producing alpha-ketoglutaric acid by using sodium glutamate

Abstract

The invention relates to the technical field of biology, in particular to an enzyme fermentation method for producing alpha-ketoglutaric acid by using sodium glutamate. The invention discloses an enzyme fermentation method for producing alpha-ketoglutaric acid by using sodium glutamate. The enzyme fermentation method comprises the following steps: step 1) pre-culturing a strain: culturing escherichia coli in an LB culture medium to a logarithmic production period; (2) induction culture: transferring the strain in the step (1) into a reaction tank, culturing with a fermentation culture medium until OD600 is equal to 12-16, and adding an inducer to continue induction culture for 2 hours; 3) substrate feeding: uniformly feeding a substrate within 12 hours, detecting the concentration of succinic acid, adding catalase to control the concentration of succinic acid to be 0.1-0.5 g / L, maintaining the pH to be 6.8-7.8, and reacting for 24 hours; and 4) determining the product. Compared with a conventional production method, the enzyme fermentation method provided by the invention has the advantages that additional steps of separation and reaction are reduced, the production period is effectively shortened, and the substrate conversion rate is effectively improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an enzyme fermentation method for producing alpha-ketoglutaric acid by utilizing sodium glutamate. Background Art

[0002] α-Ketoglutaric acid is an important organic acid, widely used in food, medicine, chemical industry and cosmetics.

[0003] In the process of producing α-ketoglutaric acid (α-KG), two steps are usually required: enzyme fermentation and enzyme catalysis. In the fermentation stage, microorganisms are used to produce the required enzymes, and the expression level of the enzymes is optimized through fermentation; in the catalytic stage, purified or partially purified enzymes are added to the reaction system to catalyze the conversion of the substrate sodium L-glutamate into the target product (α-KG). In addition to α-KG, the products of the L-glutamate oxidase-catalyzed reaction also produce ammonia (NH3) and hydrogen peroxide (H2O2). The accumulation of ammonia may have an adverse effect on subsequent separation processes and applications, and requires additional treatment. In this step, the separation and purification steps of the enzyme increase the production cost, and the fermentation and catalysis are operated separately. The process flow is long and requires additional equipment and time. This method limits its market prospects. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an enzyme fermentation method for producing α-ketoglutarate using sodium glutamate.

[0005] The present application provides an enzyme fermentation method for producing α-ketoglutarate using sodium glutamate, comprising the following steps: Step 1) Pre-cultivation of bacteria: culturing Escherichia coli in LB medium to the logarithmic production phase; Step 2) Induction culture: transfer the strain in step 1) to a reaction tank, cultivate it with fermentation medium until OD600=12-16, add inducer and continue induction culture for 2h; Step 3) Substrate addition: uniformly add the substrate within 12 hours, detect the concentration of succinic acid at the same time, add catalase to control the concentration of succinic acid at 0.1-0.5 g / L, maintain pH 6.8-7.8, and react for 24 hours; Step 4) Product determination.

[0006] In some embodiments of the present invention, in step 3), the substrate is sodium L-glutamate, the substrate concentration is maintained between 5-10 g / L, and the dissolved oxygen level is maintained above 50%.

[0007] In some embodiments of the present invention, the following condition is satisfied: substrate conversion rate ≥ 99%.

[0008] In some embodiments of the present invention, the inducing agent uses IPTG with a final concentration of 0.5 mM.

[0009] In some embodiments of the present invention, 0.01%-0.1% Triton X-100 is added in step 2).

[0010] In some embodiments of the present invention, 1%-2% glycerol or 1%-5% PEG4000 is added in step 2).

[0011] In some embodiments of the present invention, 1-5 mM ascorbic acid is added in step 2).

[0012] In some embodiments of the present invention, the induction culture conditions are: temperature set at 37° C., pH 7.0, stirring speed at 500 rpm, and dissolved oxygen level at 50%.

[0013] In some embodiments of the present invention, the LB medium is prepared by: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH is adjusted to 7.2 with NaOH.

[0014] In some embodiments of the present invention, the fermentation medium is prepared as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L glycerol, 3 g / L KH2PO4, 7 g / L K2HPO4, 1 g / L MgSO4•7H2O, 2-5 mL / L trace element solution, add purified water to 2 L, and adjust the pH to 7.0 with NaOH.

[0015] The beneficial effects of the present invention are: Compared with the existing method for preparing α-ketoglutaric acid, the present invention directly adds sodium glutamate as a substrate during the fermentation process to produce α-ketoglutaric acid, and the enzyme-producing microorganism simultaneously completes the expression and catalytic reaction of the enzyme to achieve the production of α-ketoglutaric acid, without the need for an additional enzyme purification step, and directly utilizes the enzyme catalytic reaction in the fermentation liquid, thereby reducing the production cost, and the production and conversion are carried out simultaneously, reducing the additional steps of separation and reaction, shortening the production cycle, and realizing the integration of the production process; and the microorganisms can synergistically utilize the substrate during the fermentation process to improve the conversion efficiency, without the need to use a large amount of organic solvents or auxiliary reagents, which is in line with the concept of green chemical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0017] Figure 1 HPLC diagram of the product when the reaction is terminated in Example 1; Figure 2 HPLC diagram of the product when the reaction is terminated in Example 2; Figure 3 HPLC diagram of the product when the reaction is terminated in Example 3; Figure 4 HPLC diagram of the product when the reaction is terminated in Example 4; Figure 5 HPLC diagram of the product when the reaction is terminated in Example 5; Figure 6 HPLC diagram of the product when the reaction is terminated in Example 6; Figure 7 HPLC diagram of the product when the reaction is terminated in Example 7; Figure 8 HPLC chart of the product when the reaction was terminated in Comparative Example 1; Fig. 9 The HPLC chart of the product when the reaction was terminated in Comparative Example 2; Fig.10 This is the HPLC chart of the product when the reaction is terminated in the blank sample. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0019] As used herein, "and / or" includes the term of any and all combinations of one or more associated listed items. The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. As used herein, the singular forms "a", "an", "an" and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It is further understood that "including", when used in this specification, specifies the stated features, integers, steps, operations, elements and / or compositions, but does not exclude the existence or addition of one or more other features, integers, steps, operations, elements, compositions and / or combinations thereof.

[0020] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It is further understood that terms, such as defined in commonly used dictionaries, are interpreted in accordance with their meanings in the context of the relevant art and are not idealized or overly formal meanings unless explicitly defined as such herein.

[0021] The exemplary invention described herein may appropriately lack any one or more element limitations that are not specifically disclosed herein. Therefore, the terms "comprise", "include", "contain", etc. should be understood broadly and non-restrictively. In addition, the terminology used herein is used as a description, not a limitation, and it is unintentional to use these terminology expressions that do not include any equivalent characteristics, but only describe some of their characteristics, but various modifications are possible within the scope of the present invention according to the rights. Therefore, although the present invention has been specifically disclosed by preferred embodiments and optional features, the modifications disclosed herein to embody the changes of the present invention may be recorded by those skilled in the art, and such modifications and changes will be considered to be within the scope of the present invention.

[0022] Terminology explanation: Substrate conversion rate: Substrate conversion rate refers to the ratio of the reaction substrate to the product in a chemical reaction. It reflects the speed of chemical changes in the reaction system. The calculation formula is:

[0023] HPLC detection: HPLC is a commonly used analytical technique that can be used to quantitatively analyze the concentrations of substrates and products in enzyme reactions, thereby calculating substrate conversion rates. The specific steps are as follows: (1) Sample preparation Take a certain amount of enzyme reaction mixture and remove impurities (such as by centrifugation or filtration); derivatize the substrate and product in the reaction system (such as using pre-column derivatization method) to improve detection sensitivity.

[0024] (2) HPLC analysis Use appropriate chromatographic column and mobile phase conditions, select the appropriate detection wavelength according to the chemical properties of the substrate and product; inject the sample, record the chromatograph, and analyze the peak areas of the substrate and product.

[0025] The mobile phase used in this application is: 5mmol / L sulfuric acid aqueous solution. 1ml of sulfuric acid is pipetted into 3750ml of water, filtered and sonicated.

[0026] The detection parameters are: chromatographic column: 5um Fortis H2o 250*4.6mm; flow rate: 0.6ml / min; column temperature: 50℃; wavelength: 210nm; injection volume: 10μL.

[0027] (3) Data processing The peak areas were converted into the concentrations of substrates and products by standard curve method or external standard method; the substrate conversion rate was calculated.

[0028] The existing process of producing α-ketoglutaric acid (α-KG) is usually divided into two steps: enzyme fermentation and enzyme catalysis. In the enzyme fermentation stage, microorganisms are used to produce the required enzymes, and the expression level of the enzymes is optimized through fermentation; in the enzyme catalysis stage: purified or partially purified enzymes are added to the reaction system to catalytically convert the substrate sodium L-glutamate into the target product (α-KG). In the catalytic conversion process, the products of the L-glutamate oxidase-catalyzed reaction include ammonia (NH3) and hydrogen peroxide (H2O2) in addition to α-KG. The accumulation of ammonia in the reaction system may have an adverse effect on the subsequent separation process and application, requiring additional treatment. Based on this, in order to reduce the extra steps of separation and reaction, shorten the production cycle, and realize the integration of the production process, the present invention optimizes the production process, by directly adding the substrate to the fermentation system, the enzyme production microorganism simultaneously completes the enzyme expression and catalytic reaction, and adds additives during the reaction process to promote the reaction, so as to achieve high conversion rate production of α-ketoglutaric acid. The present application provides an enzyme fermentation method for producing α-ketoglutaric acid using sodium glutamate, and the enzyme fermentation method comprises the following steps: Step 1) Pre-cultivation of bacteria: culturing Escherichia coli in LB medium to the logarithmic production phase; Step 2) Induction culture: transfer the strain in step 1) to a reaction tank, cultivate it with fermentation medium until OD600=12-16, add inducer and continue induction culture for 2h; Step 3) Substrate addition: uniformly add the substrate within 12 hours, detect the concentration of succinic acid at the same time, add catalase, maintain pH 6.8-7.8, and react for 24 hours; Step 4) Product determination.

[0029] In some specific embodiments, the substrate added in step 3) is sodium L-glutamate, and during the addition process, the substrate concentration is maintained between 5-10 g / L, and the dissolved oxygen level is maintained above 50%.

[0030] In some specific embodiments, the concentration threshold of succinic acid is controlled to be 0.5 g / L. When the system detects that the content of succinic acid exceeds the set threshold of 0.5 g / L, the feeding of catalase is started to ensure that the succinic acid content does not affect the conversion rate of α-ketoglutaric acid, and the content of catalase does not inhibit the activity of L-glutamate oxidase, resulting in a decrease in the conversion rate of α-ketoglutaric acid.

[0031] Preparation of LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, adjust the pH to 7.2 with NaOH.

[0032] Preparation of fermentation medium: 10g / L tryptone, 5g / L yeast extract, 10g / L glycerol, 3g / L KH2PO4, 7g / L K2HPO4, 1 g / L MgSO4•7H2O, 2-5 mL / L trace element solution, add purified water to 2L, and adjust the pH to 7.0 with NaOH.

[0033] By using the method of the present application to produce α-ketoglutaric acid, the substrate conversion rate reaches more than 99%, and the reaction process is effectively shortened, thereby effectively improving the production efficiency.

[0034] Sodium glutamate is a common amino acid derivative in industrial production, with stable supply, low price, good water solubility, and sodium glutamate is easily soluble in water. L-sodium glutamate is selected as the fermentation substrate, which can quickly enter the fermentation liquid and fully contact with the enzyme and the bacteria, which is conducive to improving the reaction efficiency. In addition, L-glutamate oxidase has a high specificity for sodium glutamate, which can ensure that the substrate is directed to be converted into α-ketoglutaric acid with few by-products. Directly adding the substrate for catalysis during the fermentation process can avoid the complex operation of the intermediate steps and achieve rapid and efficient product accumulation. In the enzyme fermentation process, the substrate is added in a uniform flow-addition manner, and the amount of sodium glutamate added is controlled, which can accurately regulate the reaction rate and product concentration, and avoid substrate inhibition caused by excessive substrate.

[0035] As a fermentation substrate, sodium glutamate is converted into α-ketoglutarate by L-glutamate oxidase expressed by microorganisms during the fermentation process. In this process, microorganisms not only act as enzyme production factories, but also realize substrate conversion through metabolic pathways, forming a comprehensive biocatalytic system. During the fermentation process, sodium glutamate undergoes the following key reactions: Oxidation reaction: Under the action of L-glutamate oxidase (GOX), L-glutamate is oxidized to produce α-ketoglutaric acid, with ammonia (NH3) and hydrogen peroxide (H2O2) as byproducts. The reaction equation is:

[0036] Since the α-ketoglutaric acid generated during the reaction is easily decarboxylated to produce succinic acid under the action of hydrogen peroxide, catalase needs to be added to the reaction system to hydrolyze the hydrogen peroxide generated by the reaction into water and oxygen to prevent the α-ketoglutaric acid from being oxidized. The specific reaction equation is as follows:

[0037] The experiment found that the conversion rate of α-ketoglutaric acid was relatively high when the succinic acid content in the system was controlled at 0.1-1.0 g / L. If the succinic acid content was higher than 1.0 g / L, it indicated that the residual hydrogen peroxide was high, which not only had an adverse effect on the enzyme activity, but also was not conducive to the stability of α-ketoglutaric acid.

[0038] During the reaction, byproducts ammonia (NH3) and hydrogen peroxide (H2O2) may accumulate in the system, affecting the reaction efficiency and stability. However, by using enzymes to generate ketoglutarate by feeding the substrate L-glutamate sodium in the fermentation process, the NH3 generated by the reaction can be used by microorganisms and re-enter the nitrogen metabolism cycle, further improving the efficiency of the system. In the reaction, sodium glutamate is a cheap and easily accessible substrate. Its high water solubility and bioavailability make it very suitable for the fermentation system. By optimizing the fermentation system and enzyme catalytic conditions, sodium glutamate can be efficiently converted into α-ketoglutarate, and the target product can be accumulated with high yield, low cost and high purity.

[0039] In some specific embodiments, during the process of adding the substrate, 0.01%-0.1% Triton X-100 is added simultaneously.

[0040] In one embodiment, during the addition of the substrate, 0.01% Triton X-100 is added simultaneously.

[0041] Alternatively, 0.05% Triton X-100 was added simultaneously during the addition of substrate.

[0042] Alternatively, 0.1% Triton X-100 was added during the addition of substrate.

[0043] Triton X-100 is a non-ionic surfactant that can dissolve lipids in cell membranes, thereby increasing the permeability of cell membranes, effectively improving the contact efficiency between substrates and enzymes, and thus improving the catalytic efficiency of enzymes.

[0044] In some specific embodiments, during the feeding process, 1%-2% glycerol or 1%-5% PEG4000 is added simultaneously.

[0045] In one embodiment, 1% glycerol is added simultaneously during the feeding process.

[0046] Alternatively, 1% PEG4000 was added simultaneously during the feeding process.

[0047] Alternatively, 1.5% glycerol was added during the feeding process.

[0048] Alternatively, 2.5% PEG4000 was added simultaneously during the feeding process.

[0049] Alternatively, 2% glycerol was added simultaneously during the feeding process.

[0050] Alternatively, 5% PEG4000 was added simultaneously during the feeding process.

[0051] Adding an osmotic balancer during enzyme catalysis can help maintain the balance of osmotic pressure inside and outside the cell, promote the discharge of products, and reduce the restriction of enzyme reaction. For example, adding a certain amount of glycerol can optimize the catalytic efficiency of the enzyme by changing the dielectric constant and viscosity of the solvent; adding a certain amount of PEG4000 can improve the catalytic selectivity and efficiency of the enzyme by reducing the nonspecific binding between the enzyme and the substrate. In addition, PEG4000, as a high molecular polymer, can change the viscosity and surface tension of the reaction system, thereby optimizing the interaction between the enzyme and the substrate.

[0052] In some specific embodiments, during the feeding process, 1-5 mM ascorbic acid is added simultaneously.

[0053] In one embodiment, during the feeding process, 1 mM ascorbic acid is added simultaneously.

[0054] Alternatively, during the feeding process, 2.5 mM ascorbic acid was added simultaneously.

[0055] Alternatively, during the addition process, 5 mM ascorbic acid was added simultaneously.

[0056] Ascorbic acid is a powerful antioxidant that can protect the active center of the enzyme from damage by reactive oxygen species (ROS). ROS such as hydrogen peroxide (H2O2) and superoxide anion (O2⁻) may cause enzyme inactivation or degradation, while ascorbic acid can protect the structure and activity of the enzyme by reducing these oxidizing substances and prevent the enzyme from inactivating during the fermentation process.

[0057] In a certain embodiment, during the feeding process, 0.01% Triton X-100, 1% PEG4000, and 3 mM ascorbic acid were simultaneously added.

[0058] Alternatively, during the addition process, 0.05% Triton X-100 and 2.5% PEG4000 were added simultaneously.

[0059] Alternatively, during the addition process, 0.05% Triton X-100 and 1 mM ascorbic acid were added simultaneously.

[0060] Alternatively, during the addition process, 2.5% PEG4000 and 5 mM ascorbic acid were added simultaneously.

[0061] Furthermore, in the induction culture stage, the inducer added was IPTG with a final concentration of 0.5 mM. The induction culture conditions were: temperature set at 37° C., pH 7.0, stirring speed at 500 rpm, and dissolved oxygen level at 50%.

[0062] IPTG is a synthetic inducer commonly used in E. coli expression systems to induce the expression of recombinant proteins containing lac or tac promoters. In the absence of IPTG, the lac operon is in a repressed state, and the repressor protein binds to the operator gene, preventing RNA polymerase from binding to the promoter, thereby inhibiting transcription. When IPTG is added, IPTG binds to the repressor protein, causing the repressor protein to change its conformation and dissociate from the operator gene, thereby enabling RNA polymerase to bind to the promoter and initiate transcription, thereby achieving the expression of the target protein.

[0063] The fermentation method for producing α-ketoglutaric acid using sodium glutamate of the present invention is further described below in conjunction with specific embodiments and comparative examples: Embodiment 1: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0064] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 0.01% Triton X-100, 1% PEG4000, and 3mM ascorbic acid were added at the same time; and the flow acceleration of sodium L-glutamate was controlled, and 360g sodium L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by the automatic pH control device.

[0065] After 24 hours of reaction, the reaction volume of the system was 2850 ml. The total concentration of α-ketoglutaric acid was measured to be 272.448 g, and the total concentration of sodium L-glutamate was 1.18 g by HPLC detection method.

[0066] According to the conversion rate calculation formula, the substrate conversion rate was 99.67%.

[0067] Embodiment 2: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0068] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 0.01% Triton X-100, 1% PEG4000, and 3mM ascorbic acid were added at the same time; and the flow acceleration of sodium L-glutamate was controlled, and 280g sodium L-glutamate (about 23g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by an automatic pH control device.

[0069] After 24 hours of reaction, the reaction volume of the system was 2363 ml. The total concentration of α-ketoglutaric acid was 216.7 g and the total concentration of sodium L-glutamate was 0.66 g as measured by HPLC.

[0070] The substrate conversion rate was calculated according to the conversion rate calculation formula to be 99.8%.

[0071] Embodiment 3: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0072] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 0.05% Triton X-100, 3% PEG4000, and 1mM ascorbic acid were added at the same time; and the flow acceleration of sodium L-glutamate was controlled, and 360g sodium L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by the automatic pH control device.

[0073] After 24 hours of reaction, the reaction volume of the system was 2795 ml. The total concentration of α-ketoglutaric acid was measured to be 278.64 g, and the total concentration of sodium L-glutamate was 1.01 g by HPLC detection method.

[0074] According to the conversion rate calculation formula, the substrate conversion rate was 99.72%.

[0075] Embodiment 4: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0076] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 0.1% Triton X-100, 5% PEG4000, and 5mM ascorbic acid were added at the same time; and the flow rate of sodium L-glutamate was controlled, and 360g sodium L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by an automatic pH control device.

[0077] After 24 hours of reaction, the reaction volume of the system was 2674 ml. The total concentration of α-ketoglutaric acid was 281.5 g and the total concentration of sodium L-glutamate was 1.05 g as measured by HPLC.

[0078] According to the conversion rate calculation formula, the substrate conversion rate was 99.71%.

[0079] Embodiment 5: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0080] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 1% PEG4000 and 3mM ascorbic acid were added at the same time; and the flow acceleration of sodium L-glutamate was controlled, and 360g sodium L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by an automatic pH control device.

[0081] After 24 hours of reaction, the reaction volume of the system was 2842 ml. The total concentration of α-ketoglutaric acid was 261.2 g and the total concentration of sodium L-glutamate was 3.32 g as measured by HPLC.

[0082] According to the conversion rate calculation formula, the substrate conversion rate was 99.07%.

[0083] Embodiment 6: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0084] After two hours of induction culture, the sodium glutamate feeding device was started to feed L-glutamate, and 0.01% Triton X-100 and 3mM ascorbic acid were added at the same time; and the flow rate of L-glutamate was controlled, and 360g L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by the automatic pH control device.

[0085] After 24 hours of reaction, the reaction volume of the system was 2795 ml. The total concentration of α-ketoglutaric acid was 259.97 g and the total concentration of sodium L-glutamate was 3.51 g as measured by HPLC.

[0086] According to the conversion rate calculation formula, the substrate conversion rate was 99.02%.

[0087] Embodiment 7: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0088] After two hours of induction culture, the sodium glutamate feeding device was started to feed sodium L-glutamate, and 0.01% Triton X-100 and 1% PEG4000 were added at the same time; and the flow acceleration of sodium L-glutamate was controlled, and 360g sodium L-glutamate (about 30g / h) was evenly fed within 12 hours to ensure that the substrate concentration was maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth was monitored online. When the succinic acid concentration exceeded the set threshold (such as 0.5g / L), the catalase feeding was started. During the reaction, the dissolved oxygen level was maintained above 50%. If the dissolved oxygen was insufficient, it could be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH was maintained at 6.8-7.2 by the automatic pH control device.

[0089] After 24 hours of reaction, the reaction volume of the system was 2804 ml. The total concentration of α-ketoglutaric acid was measured to be 265.32 g, and the total concentration of sodium L-glutamate was 2.71 g by HPLC detection method.

[0090] According to the conversion rate calculation formula, the substrate conversion rate was 99.24%.

[0091] Blank sample: The modified Escherichia coli seeds were cultured in LB medium in a shake flask to the logarithmic cycle (OD600=0.8-1.0), transferred to a 5L reaction tank, the initial fermentation medium volume in the reaction tank was adjusted to 2L, the temperature was set to 37°C, the pH was adjusted to 7.0, the stirring speed was 500rpm, the dissolved oxygen was initially set to 50%, the basic culture nutrition was maintained, the strain was cultivated to OD600=12-16, and a final concentration of 0.5mM IPTG was added to promote the enzyme expression of Escherichia coli for induced culture.

[0092] After two hours of induction culture, the sodium glutamate feeding device is started to feed L-sodium glutamate, and the flow acceleration of L-sodium glutamate is controlled. 360g L-sodium glutamate (about 30g / h) is evenly fed within 12 hours to ensure that the substrate concentration is maintained between 5-10g / h to avoid excessive substrate inhibition. At the same time, the concentration of succinic acid in the fermentation broth is monitored online. When the succinic acid concentration exceeds the set threshold (such as 0.5g / L), the catalase feeding is started. During the reaction, the dissolved oxygen level is maintained above 50%. If the dissolved oxygen is insufficient, it can be improved by increasing the stirring speed or supplementing the air flow (0.5-1.0vvw); and the pH is maintained at 6.8-7.2 through an automatic pH control device.

[0093] After 24 hours of reaction, the reaction volume of the system was 2674 ml. The total concentration of α-ketoglutaric acid was 255.13 g and the total concentration of sodium L-glutamate was 6.92 g as measured by HPLC.

[0094] According to the conversion rate calculation formula, the substrate conversion rate was 98.1%.

[0095] Comparative Example 1: In a 5L reactor, add a certain amount of bacterial sludge, adjust the volume to 2L, and evenly add 360g of L-glutamate (about 30g / h) within 12 hours to ensure that the substrate concentration is maintained between 5-10g / L to avoid excessive substrate inhibition. During the addition process, the concentration of byproduct succinic acid in the fermentation broth is monitored online. When the succinic acid concentration exceeds the set threshold (such as 0.5g / L), start the catalase feed. During the reaction, the dissolved oxygen level is maintained above 50%; if the dissolved oxygen is insufficient, it can be improved by increasing the stirring rate or supplementing the air flow (0.5-1.0 vvm); and the system pH is maintained at 6.8-7.2 through an automatic pH adjustment device.

[0096] After 24 hours of reaction, the reaction volume of the system was 2790 ml. The total concentration of α-ketoglutaric acid was 253.87 g and the total concentration of sodium L-glutamate was 7.2 g as determined by HPLC.

[0097] The substrate conversion rate was calculated according to the conversion rate calculation formula to be 98%.

[0098] Comparative Example 2: In a 5L reactor, add a certain amount of bacterial sludge, adjust the volume to 2L, and evenly add 280g of L-sodium glutamate (about 30g / h) within 12 hours to ensure that the substrate concentration is maintained between 5-10g / L to avoid excessive substrate inhibition. During the addition process, the concentration of byproduct succinic acid in the fermentation broth is monitored online. When the succinic acid concentration exceeds the set threshold (such as 0.5g / L), start the catalase feed. During the reaction, the dissolved oxygen level is maintained above 50%; if the dissolved oxygen is insufficient, it can be improved by increasing the stirring rate or supplementing the air flow (0.5-1.0 vvm); and the system pH is maintained at 6.8-7.2 through an automatic pH adjustment device.

[0099] After 24 hours of reaction, the reaction volume of the system was 2400 ml, and the total concentration of α-ketoglutaric acid was 192.64 g and the total concentration of sodium L-glutamate was 5.04 g as determined by HPLC.

[0100] The substrate conversion rate was calculated according to the conversion rate calculation formula to be 98.6%. The raw materials or reagents used in the examples and comparative examples of the present invention are all purchased from mainstream manufacturers in the market. Those without indicating the manufacturer or concentration are all analytically pure raw materials or reagents that can be routinely obtained. As long as they can play the expected role, there is no special limitation.

[0101] The instruments and equipment used in this embodiment are all purchased from major manufacturers in the market, and there is no particular limitation as long as they can play the expected role. In this embodiment, if no specific technology or conditions are specified, the technology or conditions described in the literature in this field or the product instructions are used. The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. An enzyme fermentation method for producing α-ketoglutarate using sodium glutamate, characterized in that: The following steps are involved: Step 1) Pre-cultivation of bacteria: culturing Escherichia coli in LB medium to the logarithmic production phase; Step 2) Induction culture: transfer the strain in step 1) to a reaction tank, cultivate it with fermentation medium until OD600=12-16, add inducer and continue induction culture for 2h; Step 3) Substrate addition: uniformly add the substrate within 12 hours, detect the concentration of succinic acid at the same time, add catalase to control the concentration of succinic acid at 0.1-0.5 g / L, maintain pH 6.8-7.8, and react for 24 hours; Step 4) Product determination.

2. The enzyme fermentation method according to claim 1, characterized in that In step 3), the substrate is sodium L-glutamate, and the substrate concentration is maintained between 5-10 g / L, and the dissolved oxygen level is maintained above 50%.

3. The enzyme fermentation method according to claim 1, characterized in that Meet: Substrate conversion rate ≥ 99%.

4. The enzyme fermentation method according to claim 1, characterized in that: The inducing agent used was IPTG with a final concentration of 0.5 mM.

5. The enzyme fermentation method according to claim 1, characterized in that: In the step 2), 0.01%-0.1% Triton X-100 is added.

6. The enzyme fermentation method according to claim 1, characterized in that: In the step 2), 1%-2% glycerol or 1%-5% PEG4000 is added.

7. The enzyme fermentation method according to claim 1, characterized in that: In the step 2), 1-5 mM ascorbic acid is added.

8. The enzyme fermentation method according to claim 1, characterized in that The induction culture conditions are: temperature set at 37° C., pH 7.0, stirring speed 500 rpm, and dissolved oxygen level 50%.

9. The enzyme fermentation method according to claim 1, characterized in that: The LB medium was prepared by: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH was adjusted to 7.2 with NaOH.

10. The enzyme fermentation method according to claim 1, characterized in that: The fermentation medium is prepared by: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L glycerol, 3 g / L KH2PO4, 7 g / L K2HPO4, 1 g / L MgSO4•7H2O, 2-5 mL / L trace element solution, adding purified water to 2 L, and adjusting the pH to 7.0 with NaOH.

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