Co-immobilized tri-enzyme system for preparing l-2-aminobutyric acid and method for preparing l-2-aminobutyric acid

Abstract

The application discloses a co-immobilized tri-enzyme system for preparing L-2-amino butyric acid, which is obtained by co-immobilizing L-threonine deaminase, L-leucine dehydrogenase and formic acid dehydrogenase on a carrier, namely, silica nanoflower, and the mass ratio of the L-threonine deaminase, the L-leucine dehydrogenase and the formic acid dehydrogenase used for preparing the co-immobilized tri-enzyme system is (0.1-2):1:(1-10). The L-threonine deaminase, the L-leucine dehydrogenase and the formic acid dehydrogenase are successfully co-immobilized on the same carrier, the stability and the reusability of the three enzymes are improved, the reaction yield of the co-immobilized tri-enzyme system for catalyzing preparation of L-2-amino butyric acid can reach more than 92%, and the effective separation of the co-immobilized enzyme and the product, the recycling of the enzyme and the recycling of the coenzyme are realized, so that the synthesis reaction steps and the subsequent separation and purification steps for producing L-2-amino butyric acid can be simplified, the production process is optimized, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the technical field of immobilized enzyme preparation of L-2-aminobutyric acid, and particularly to a co-immobilized three-enzyme system for preparing L-2-aminobutyric acid, as well as the preparation of the co-immobilized enzyme and the method for preparing L-2-aminobutyric acid by catalyzing the reaction using the co-immobilized three-enzyme system. Background Technology

[0002] L-2-Aminobutyric acid (ABA) is a non-natural chiral amino acid that can inhibit the transmission of nerve signals in the human body and promote brain cell metabolism. It is also an important chemical raw material and pharmaceutical intermediate, widely used in the pharmaceutical, pesticide, and food industries. In the pharmaceutical field, it is widely used in the synthesis of various chiral drugs. For example, L-2-Aminobutyric acid is the main raw material for the synthesis of the novel antiepileptic drug levetiracetam, and it is also a key chiral precursor for the synthesis of the antibacterial and antituberculosis drug ethambutol hydrochloride.

[0003] Currently, the preparation of L-2-aminobutyric acid (L-2-aminobutyric acid) mainly involves chemical and biological methods. Traditional chemical methods primarily employ ketobutyric acid reduction, desulfurization, halogenation ammonolysis, and chemical resolution. The disadvantages of chemical methods include the tendency to form racemic mixtures, which is detrimental to the synthesis of chiral compounds. Furthermore, the reaction conditions are stringent, resulting in severe environmental pollution, which is inconsistent with industrial development trends. Biological methods often utilize enzymatic methods, employing enzymes such as aminoacylases, amino acid oxidases, nitrile hydratases, transaminases, and dehydrogenases as catalysts. These methods offer mild reaction conditions, high stereoselectivity, and are environmentally friendly, possessing advantages unmatched by chemical synthesis. However, the free enzymes used as catalysts suffer from drawbacks such as high catalyst costs, difficulty in recycling and reuse, poor enzyme activity stability, easy inactivation, and increased separation and purification burden due to mixing with the product. For example, a currently high-conversion enzymatic method for L-2-aminobutyric acid preparation simultaneously uses three free enzymes: threonine deaminase, leucine dehydrogenase, and formate dehydrogenase. The combined use of these three free enzymes makes recycling and reuse difficult, and the coenzyme regeneration efficiency is low, leading to complex product purification. To address the shortcomings of free enzymes, immobilized enzymes are commonly employed. However, the interactions among multiple free enzymes present technical challenges to co-immobilization systems. Therefore, there are currently no reports of successful co-immobilization of threonine deaminase, leucine dehydrogenase, and formic acid dehydrogenase. Instead, methods have focused on altering the type of free enzyme used as a catalyst, such as replacing leucine dehydrogenase with alcohol dehydrogenase, threonine aldehyde dehydrogenase, or L-amino acid dehydrogenase. L-2-aminobutyric acid (L-2-aminobutyric acid) is produced using co-immobilized multi-enzyme and co-immobilized coenzyme systems (see Chinese patent application CN112522336A). While this method successfully utilizes an immobilized enzyme system, it increases the variety of enzymes and costs, complicates reaction steps, and hinders industrial production. Therefore, effectively co-immobilizing these three free enzymes—threonine deaminase, leucine dehydrogenase, and formic acid dehydrogenase—to achieve high conversion rates and high recycling rates of the immobilized enzymes would greatly promote the production of L-2-aminobutyric acid. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a co-immobilized three-enzyme system for the catalytic production of L-2-aminobutyric acid (GABA). This co-immobilized three-enzyme system successfully immobilizes L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase on the same carrier, porous silica nanoflowers, achieving efficient catalytic synthesis of L-2-aminobutyric acid, effective product separation, coenzyme recycling, and enzyme reuse.

[0005] The co-immobilized three-enzyme system for preparing L-2-aminobutyric acid of the present invention is obtained by immobilizing L-threonine deaminase (LTD), L-leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) on porous silica nanoflowers. The mass ratio of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase used in preparing the co-immobilized three-enzyme system is (0.1-2):1:(1-10).

[0006] By utilizing a specific porous support, silica nanoflowers, the co-immobilization of three enzymes—LTD, LDH, and FDH—effectively improved the substrate tolerance of LTD to 2-ketobutyric acid, the stability of the three enzymes, and their reusability. Using this co-immobilized three-enzyme system to catalyze the production of L-2-aminobutyric acid not only resulted in high reaction yields but also facilitated product separation, simplifying synthesis and subsequent purification steps and effectively reducing production costs.

[0007] As a limitation of the above technical solution, L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase are immobilized on the carrier silica nanoflower in any of the following manner:

[0008] (1) L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase were immobilized on silica nanoflowers in sequence, and denoted as LTD+LDH+FDH.

[0009] (2) L-leucine dehydrogenase, L-threonine deaminase and formate dehydrogenase were immobilized on the carrier silica nanoflower in sequence, and denoted as LDH+LTD+FDH.

[0010] (3) Simultaneously immobilize L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on the carrier silica nanoflower, denoted as (LTD+LDH+FDH).

[0011] (4) First, L-threonine deaminase was immobilized on the carrier silica nanoflower, and then L-leucine dehydrogenase and formate dehydrogenase were simultaneously immobilized, denoted as LTD+(LDH+FDH).

[0012] (5) First, L-leucine dehydrogenase was immobilized on the carrier silica nanoflower, and then L-threonine deaminase and formate dehydrogenase were simultaneously immobilized, denoted as LDH+(LTD+FDH).

[0013] As a limitation of the above technical solution, the mass ratio of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase used in the preparation of the co-immobilized three-enzyme system is (0.5-1):1:(1-10).

[0014] As a limitation of the above technical solution, the carrier silica nanoflower is prepared by stirring and reacting at 60-80°C with hexadecyltrimethylammonium bromide as template, tetraethyl silicate as silicon source, n-butanol and cyclohexane as organic phase, water as solvent, and urea as surfactant, followed by drying and calcination.

[0015] As a limitation of the above technical solution, L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase are mixed with the carrier and immobilized by full shaking under the conditions of 25-35℃ and pH 7.0-9.0.

[0016] Further improve the conditions for co-immobilized enzymes to create a suitable catalytic environment for the enzymes, thereby optimizing the performance of immobilized enzymes and giving them advantages such as high catalytic efficiency, high stability, easy recovery and reuse.

[0017] Meanwhile, the present invention also provides a method for preparing the co-immobilized three-enzyme system as described above, comprising the following steps: a porous carrier silica nanoflower activated with glutaraldehyde is mixed with PBS buffer solution and three enzymes, L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase, and the resulting suspension is fully shaken and immobilized at 25-35°C and pH 7.0-9.0 to obtain the co-immobilized enzyme system.

[0018] As a limitation of the above technical solution, the mass ratio of the immobilization process carrier to the total enzyme amount is 1:0.6 to 1.2.

[0019] The preparation of the co-immobilized three-enzyme system is simple and convenient, and the conditions are mild, which is conducive to the application of this co-immobilized enzyme in the production of L-2-aminobutyric acid.

[0020] In addition, the present invention also provides a method for preparing L-2-aminobutyric acid, which uses the above-mentioned co-immobilized three-enzyme system as a catalyst for the synthetic reaction of preparing L-2-aminobutyric acid from L-threonine.

[0021] As a limitation of the above technical solution, the synthesis reaction conditions are as follows: 100-500 mM L-threonine, 0.4-2 M sodium formate, 0.25-2.5% NADH, a buffer solution with pH 7.0-9.0, and an appropriate amount of co-immobilized enzyme are reacted with shaking at 25-35°C for 12-20 h.

[0022] Using the co-immobilized enzyme of this invention to catalyze the synthesis of L-2-aminobutyric acid, the yield reaches over 92%, and it can achieve effective separation of the reaction products, enzyme recovery and reuse, and coenzyme recycling and regeneration. This can effectively simplify the synthesis steps and subsequent separation and purification steps, and reduce production costs.

[0023] In summary, this invention successfully immobilizes L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase on porous silica nanoparticles. The resulting co-immobilized three-enzyme system improves the substrate tolerance of L-leucine dehydrogenase to 2-ketobutyric acid, as well as the stability, storability, and reusability of the three natural enzymes. Utilizing this co-immobilized three-enzyme system to catalyze the synthesis of L-2-aminobutyric acid allows for enzyme recovery and reuse, coenzyme recycling, simplifies synthesis and subsequent separation and purification steps, optimizes the process, and reduces production costs. Attached Figure Description

[0024] Figure 1 The yields of enzymes immobilized on different carriers in Example 1 are shown below.

[0025] Figure 2 Example 3 illustrates the effect of the ratio of LTD, LDH, and FDH dosages of the three enzymes on the yield.

[0026] Figure 3 Example 4-1 illustrates the substrate tolerance of LDH in free LDH and co-immobilized enzymes.

[0027] Figure 4 The curves show the reaction progress of the co-immobilized enzyme in Example 4-2.

[0028] Figure 5 Storage stability of free enzymes and co-immobilized enzymes in Examples 4-3;

[0029] Figure 6 The reusability of the co-immobilized enzymes in Examples 4-4;

[0030] Figure 7 L-threonine standard curve;

[0031] Figure 8 L-2-ABA standard curve. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0034] Example 1

[0035] This embodiment involves the effects of different carriers on the co-immobilized three-enzyme system, and the specific experiments are as follows.

[0036] Example 1-1: Amino resin LX-1000EPHA was incubated in 50mM, pH 8.0 PBS solution for 2 hours. The mixture was then washed with PBS solution (the PBS solution used in the washing, resuspension, and immobilization systems described below was 50mM, pH 8.0) and filtered through a Buchner funnel. 100mg of amino resin LX-1000EPHA was soaked in 2% (v / v) glutaraldehyde solution and activated by shaking for 2 hours. The residue was removed by washing with PBS solution and the mixture was dried to obtain the carrier material.

[0037] Take 20 mg of the carrier material, shake and resuspend it thoroughly in PBS solution, add a mixed enzyme with a mass ratio of LTD:LDH:FDH of 1:1:1, and prepare an immobilization system with a total volume of 10 mL (adjust the volume with PBS solution, the same below) and a total enzyme addition of 20 mg. Immobilize at 25 °C and 180 rpm to obtain the immobilized enzyme of the carrier LX-1000EPHA.

[0038] Examples 1-2: Epoxy resins LX-109S and LX-1000HFA were incubated in 200mM, pH 9.0 PBS solution for 2 hours, washed with PBS solution (the PBS solution used for washing, resuspension, and immobilization systems below was 50mM, pH 8.0), and filtered through a Buchner funnel. 100mg of each of the epoxy resins LX-109S and LX-1000HFA were soaked in 2% (v / v) glutaraldehyde, shaken, and activated for 2 hours. The residue was removed by washing with PBS solution and the resins were dried to obtain the carrier materials.

[0039] Take 20 mg of the carrier material, shake and resuspend it thoroughly in PBS solution, add a mixed enzyme with a mass ratio of LTD:LDH:FDH of 1:1:1, and prepare an immobilization system with a total volume of 10 mL and a total enzyme amount of 20 mg. Immobilize at 25 °C and 180 rpm to obtain immobilized enzymes of carriers LX-109S and LX-1000HFA, respectively.

[0040] Examples 1-3: Preparation of silica nanoflower DSN: In a 250mL single-necked flask, 3g of CTAB and 100g of urea aqueous solution (0.4M) were added. The solution was sonicated until clear. Then, 3.5g of n-butanol and 20g of cyclohexane were added, and the mixture was sonicated at room temperature for 30min until a homogeneous milky white solution was obtained. The flask was placed in a water bath, and while stirring, 3g of tetraethyl orthosilicate (TEOS) was slowly added dropwise using a syringe. After reacting at room temperature for 30min, the temperature was increased to 70℃ and reacted for 24h. After the reaction was completed, the reaction solution was centrifuged to obtain a white precipitate. The precipitate was washed three times with water and three times with ethanol, dried at 60℃, and then calcined in a furnace at 550℃ for 5h. After removing CTAB, DSN was obtained.

[0041] Take 100 mg of DSN, soak it in 2% (v / v) glutaraldehyde, shake and activate it for 2 h, then wash it with PBS solution (the PBS solution used in the washing, resuspension and immobilization systems below is 50 mM, pH 8.0) to remove residual glutaraldehyde and dry it.

[0042] 20 mg of the carrier material was thoroughly shaken and resuspended in PBS solution. A mixed enzyme with a mass ratio of LTD:LDH:FDH of 1:1:1 was added to prepare an immobilization system with a total volume of 10 mL and a total enzyme addition of 20 mg. The immobilization was carried out at 25 °C and 180 rpm to obtain the immobilized enzyme of the carrier DSN.

[0043] Catalytic reaction using immobilized enzyme: 500 mM L-threonine, 2 M sodium formate, and 0.6% (w / w) NADH were added to a 900 mM, pH 9.0 ammonium chloride-ammonia buffer solution. 20 mg of co-immobilized enzyme was added, and the reaction temperature was controlled at 30℃. After reacting at 180 rpm for 16 h, the sample was placed in a 100℃ water bath for 5 min to inactivate the enzyme. The supernatant was collected by centrifugation at 12000 rpm for 2 min, and the yield of the supernatant was determined by HPLC.

[0044] The catalytic reaction of the immobilized enzyme described above is the preparation reaction of L-2-aminobutyric acid. The preparation conditions can be adjusted within a certain range, namely 100-500 mM L-threonine, 0.4-2 M sodium formate, 0.25-2.5% NADH, ammonium chloride-ammonia buffer solution with pH 7.0-9.0, and an appropriate amount of co-immobilized enzyme, and reacted at 25-35℃ and 180 rpm for 12-20 h.

[0045] Detection of reaction results: Take the supernatant of each reaction and perform derivatization treatment (the principle of derivatization is that under alkaline conditions, the free terminal NH2 of amino acids undergoes a nucleophilic aromatic ring substitution reaction with 2,4-dinitrofluorobenzene to generate a yellow dinitrophenyl amino acid derivative, which can be quantitatively determined by HPLC). Specifically, take 100 μL of reaction supernatant, 100 μL of sodium bicarbonate (0.5 M) and 50 μL of 2,4-dinitrofluorobenzene-acetonitrile solution (1%), mix well, and react at 60℃ in the dark for 1 h.

[0046] The conversion rate and product yield of the reductive amination reaction (i.e., the immobilized enzyme-catalyzed reaction) were determined using HPLC. The conversion rate was based on... Figure 7 The L-threonine standard curve shown is obtained, and the yield is based on... Figure 8 The L-2-ABA standard curve shown was converted to the chromatographic conditions: Agilent ZORBAX Eclipse Plus C18 column (4.6*250mm), column temperature 30℃, flow rate 1mL / min. Gradient elution was used, and the detection wavelength was 360nm. The gradient elution program was as shown in Table 1 below.

[0047] Table 1 Gradient elution program

[0048]

[0049] The results of the catalytic reactions of each immobilized enzyme are shown in the figure. Figure 1 Subsequently, the carrier with the highest reaction yield, namely silica nanoflower DSN, was selected for co-immobilization of the three-enzyme system.

[0050] Example 2

[0051] This embodiment relates to the effects of different immobilization methods on the co-immobilized three-enzyme system, and the specific experiments are as follows.

[0052] (1) Preparation of co-immobilized three-enzyme system

[0053] Take 20 mg of carrier DSN, weigh the three enzymes at a mass ratio of LTD:LDH:FDH = 1:1:5, add them to PBS buffer (50 mM, pH 8.0) to prepare an immobilization system with a total volume of 10 mL and a total enzyme addition of 20 mg. Shake at 25 °C and 180 rpm for 3 h. Prepare various forms of immobilized enzymes under controlled immobilization mode (specifically, the order of addition of the three enzymes during the immobilization process is different, see Table 2 below for details). Centrifuge to obtain the precipitate, wash the precipitate with PBS buffer and centrifuge again. Repeat the washing and centrifugation 3 times to obtain co-immobilized three-enzyme systems LTD&LDH&FDH@DSN with different immobilization methods. Store at 4 °C for later use.

[0054] (2) Performance evaluation of co-immobilized three-enzyme systems with different immobilization methods

[0055] Different co-immobilized three-enzyme systems were subjected to catalytic reactions, and the reaction results were detected. The catalytic reaction and detection conditions were the same as in Example 1. The results of each catalytic reaction are shown in Table 2 below.

[0056] Table 2. Impact of Immobilization Method on Yield

[0057]

[0058]

[0059] As can be seen from the results in Table 2, the different immobilization modes have a significant impact on the conversion rate and yield of the co-immobilized three-enzyme system in the catalytic reaction.

[0060] Example 3

[0061] This embodiment relates to the optimization of the proportions of the three enzymes in a co-immobilized three-enzyme system.

[0062] (1) Preparation of co-immobilized enzymes

[0063] The three enzymes LTD, LDH and FDH are arranged in order. Figure 2 Weigh out the proportions and add 20 mg of the carrier DSN to a 50 mL centrifuge tube. Add 20 mg of the mixed enzyme in different proportions and add PBS (50 mM, pH 8.0) buffer solution to prepare a 10 mL immobilization system. Shake thoroughly at 180 rpm at room temperature, centrifuge, and wash with PBS to obtain three co-immobilized enzymes LTD&LDH&FDH@DSN with different enzyme proportions.

[0064] (2) Effect of the ratio of the three enzymes on the reaction yield

[0065] The co-immobilized three-enzyme systems with different enzyme ratios were subjected to catalytic reactions, and the reaction results were detected. The catalytic reaction and detection conditions were the same as in Example 1. The effect of the three-enzyme ratio on the reaction yield was investigated, and the results are shown in [Figure 1]. Figure 2 The optimal ratio of the three enzymes was found to be LTD / LDH / FDH = 0.5:1:1.5.

[0066] Example 4

[0067] This embodiment involves the performance determination of a co-immobilized three-enzyme system.

[0068] Example 4-1: Substrate tolerance test of leucine dehydrogenase LDH

[0069] (1) Preparation of co-immobilized enzymes

[0070] Take 20 mg of the carrier DSN, add 20 mg of mixed enzyme (the mass ratio of the three enzymes is LTD / LDH / FDH = 0.5:1:1.5), add PBS buffer (50 mM, pH 8.0) to prepare a 10 mL immobilization system, shake at 25 °C and 180 rpm for 3 h, then centrifuge the co-immobilized enzyme and wash with PBS buffer to obtain LTD&LDH&FDH@DSN.

[0071] (2) Determination of substrate tolerance of LDH in free LDH and co-immobilized enzymes

[0072] LDH enzyme activity assay: 2-Ketobutyric acid (2-OBA) is added to NH4Cl-NH3·H2O (900mM, pH 9.5), followed by the enzyme. After mixing, NADH is added to initiate the reaction. The required concentrations of 2-OBA in the reaction system are 1–60 mM, the enzyme concentration is 0.05 mg / mL, and the NADH concentration is 2.5 mg / mL. The absorbance change is measured at 340 nm. Enzyme activity is defined as: the production of 1 μmol NAD per minute from the consumption of 1 μmol NADH. + The required amount of enzyme is 1 U.

[0073] Free LDH and co-immobilized enzymes with the same enzyme activity were taken, and different amounts of 2-OBA (corresponding concentrations of 1–60 mM) were added according to the LDH enzyme activity assay method. The reactions were carried out for 3 minutes, and the enzyme activities were measured. The results are shown in [Figure number missing]. Figure 3 .

[0074] Depend on Figure 3 It is evident that the inhibition curve of the co-immobilized enzyme decreases more slowly than that of the free enzyme, and its half-maximal inhibitory concentration (half-maximal inhibitory concentration is defined as the concentration required for a substance to inhibit enzyme activity to 50%) is significantly higher than that of the free enzyme.

[0075] Example 4-2: Immobilized enzyme reaction process

[0076] (1) Preparation of co-immobilized enzymes, the specific preparation operation is the same as in Example 4-1.

[0077] (2) Progress curve of co-immobilized enzyme catalytic reaction

[0078] In a 900 mM ammonium chloride-ammonia buffer solution at pH 9.0, 500 mM L-threonine, 2 M sodium formate, and 0.6% (w / w) NADH were added, along with 40 mg of co-immobilized enzyme. The reaction temperature was controlled at 30°C, and the reaction was carried out at 180 rpm for a certain period of time. Samples were taken out at regular intervals and placed in a 100°C water bath for 5 min to inactivate the enzyme. After centrifugation at 12000 rpm for 2 min, the supernatant was collected. The supernatant was derivatized, and the yield of L-2-aminobutyric acid was determined by HPLC (derivatization and detection procedures were the same as in Example 1). The results are as follows: Figure 4As shown, the conversion rate and yield increase over time, reaching their maximum at 16 hours, with a maximum yield of 92%.

[0079] Example 4-3: Determination of storage stability of free enzymes and co-immobilized enzymes

[0080] (1) Preparation of co-immobilized enzymes, the specific preparation operation is the same as in Example 4-1.

[0081] (2) Storage stability study of co-immobilized enzymes

[0082] The three free enzymes (LTD, LDH, and FDH) and co-immobilized enzyme samples were stored at 4°C, and samples were taken out at regular intervals to determine their enzyme activity.

[0083] LDH enzyme activity assay method: same as in Example 4-1.

[0084] LTD enzyme activity assay: 50 mM potassium phosphate buffer (pH 7.5), 10 μM PLP, 50 mM L-threonine, and LTD enzyme solution (final enzyme concentration 0.05 mg / mL) are added and mixed to a volume of 1 mL. The absorbance change of the product 2-OBA at 230 nm is measured at 30 °C. Enzyme activity is defined as: the amount of enzyme required to catalyze the generation of 1 μmol 2-OBA in 1 min is 1 U.

[0085] FDH enzyme activity assay method: 100mM PBS buffer (pH 7.0), 100mM sodium formate, 1mM NAD + Mix an appropriate amount of FDH enzyme solution (final enzyme concentration 0.05 mg / mL) to a volume of 1 mL, and measure the change in absorbance of NADH at 340 nm for 1 minute at 30℃. Enzyme activity is defined as the amount of NADH consumed per minute (1 μmol NAD). + The amount of enzyme required to generate 1 μmol NADH is 1 U.

[0086] The results are as follows Figure 5 As shown, the storage stability of the co-immobilized enzyme is significantly improved compared to that of the free enzyme.

[0087] Example 4-4: Reusability of Immobilized Enzymes

[0088] (1) Preparation of co-immobilized enzymes, the specific preparation operation is the same as in Example 4-1.

[0089] (2) Study on the reusability of immobilized enzymes

[0090] Using 500 mM L-threonine as a substrate, 40 mg of co-immobilized enzyme was added. After reacting at 30 °C for 16 h, the mixture was centrifuged, and the yield of the supernatant was determined by HPLC (derivation and detection procedures were the same as in Example 1). The co-immobilized enzyme obtained by centrifugation was washed and added to the next batch of catalytic reaction. The above experiment was repeated to determine the reusability of the immobilized enzyme. The experimental results are as follows: Figure 6 As shown, after being reused 8 times, the catalytic yield of the co-immobilized enzyme can still be maintained at 54%. Compared with the free enzyme, which cannot be reused, the co-immobilized enzyme can effectively prevent the reduction of enzyme activity.

[0091] In summary, this invention successfully immobilizes three enzymes—L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase—on the same silica nanoflower carrier, improving the stability and reusability of the three enzymes. Furthermore, the reaction yield for preparing L-2-aminobutyric acid (GABA) using this co-immobilized three-enzyme system can reach over 92%. Simultaneously, it achieves effective separation of the co-immobilized enzymes and products, enzyme recovery and reuse, and coenzyme recycling, simplifying the synthesis reaction steps and subsequent separation and purification steps for L-2-aminobutyric acid production, optimizing the production process, and reducing production costs.

Claims

1. A co-immobilized three-enzyme system for preparing L-2-aminobutyric acid, characterized in that: The co-immobilized three-enzyme system was obtained by immobilizing L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase together on a porous support silica nanoflower. The mass ratio of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase used in the preparation of the co-immobilized three-enzyme system was (0.5~1):1:(1~10). L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase are immobilized on silica nanoflowers using any of the following methods: (1) L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase were immobilized on the carrier silica nanoflower in sequence. (2) L-leucine dehydrogenase, L-threonine deaminase and formate dehydrogenase were immobilized on the carrier silica nanoflower in sequence. (3) Simultaneously immobilize L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on the carrier silica nanoflower; (4) First, L-threonine deaminase was immobilized on the carrier silica nanoflower, and then L-leucine dehydrogenase and formate dehydrogenase were simultaneously immobilized. (5) First, L-leucine dehydrogenase was immobilized on the carrier silica nanoflower, and then L-threonine deaminase and formate dehydrogenase were simultaneously immobilized. The carrier silica nanoflowers were prepared by stirring and reacting at 60-80°C with hexadecyltrimethylammonium bromide as template, tetraethyl silicate as silicon source, n-butanol and cyclohexane as organic phase, water as solvent, and urea as surfactant, followed by drying and calcination. Porous silica nanoflowers activated with glutaraldehyde were mixed with PBS buffer solution and three enzymes: L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase. The resulting suspension was immobilized by thorough shaking at 25–35°C and pH 7.0–9.0 to obtain a co-immobilized enzyme system. The mass ratio of carrier to total enzyme during immobilization was 1:0.6–1.

2. This co-immobilized three-enzyme system is used as a catalyst for the preparation of L-2-aminobutyric acid from L-threonine. The reaction system only requires the addition of L-threonine, sodium formate, NADH, a pH 7.0~9.0 buffer solution, and the co-immobilized enzyme.

2. A method for preparing L-2-aminobutyric acid, characterized in that: The co-immobilized three-enzyme system described in claim 1 is used as a catalyst for the synthetic reaction of L-2-aminobutyric acid from L-threonine.

3. The method for preparing L-2-aminobutyric acid according to claim 2, characterized in that: The synthesis reaction conditions were as follows: 100-500 mM L-threonine, 0.4-2 M sodium formate, 0.25-2.5% NADH, a buffer solution with pH 7.0-9.0, and the co-immobilized enzyme were reacted with shaking at 25-35 °C for 12-20 h.

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