Co-immobilized three-enzyme system for preparing L-2-aminobutyric acid and preparation method of L-2-aminobutyric acid

Abstract

The invention discloses a co-immobilized three-enzyme system for preparing L-2-aminobutyric acid, which is obtained by co-immobilizing L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on a carrier silicon dioxide nanoflower, the mass ratio of the L-threonine deaminase to the L-leucine dehydrogenase to the formate dehydrogenase used for preparing the co-immobilized three-enzyme system is (0.1-2): 1: (1-10). According to the co-immobilized three-enzyme system, the L-threonine deaminase, the L-leucine dehydrogenase and the formate dehydrogenase are successfully and jointly immobilized on the same carrier, so that the stability and reusability of the three enzymes are improved, and the reaction yield of the L-2-aminobutyric acid prepared through catalysis of the co-immobilized three-enzyme system can reach 92% or above; meanwhile, the effective separation of the co-immobilized enzyme and the product, the recycling of the enzyme and the cyclic regeneration of the coenzyme are realized, so that the synthetic reaction step and the subsequent separation and purification step for producing the L-2-aminobutyric acid can be simplified, the production process is optimized, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of preparing L-2-aminobutyric acid by an immobilized enzyme method, and in particular to a co-immobilized three-enzyme system for preparing L-2-aminobutyric acid, as well as the preparation of the co-immobilized enzyme and a method for preparing L-2-aminobutyric acid by catalyzing a reaction using the co-immobilized three-enzyme system. Background Art

[0002] L-2-aminobutyric acid is a non-natural chiral amino acid that can inhibit the transmission of human neural information and promote brain cell metabolism. It is also an important chemical raw material and pharmaceutical intermediate, widely used in the fields of medicine, pesticides and food. In the field of medicine, it is widely used in the synthesis of a variety of chiral drugs. For example, L-2-aminobutyric acid is the main raw material for the synthesis of the new anti-epileptic drug levetiracetam, and is also the key chiral precursor for the synthesis of the antibacterial and anti-tuberculosis drug ethambutol hydrochloride.

[0003] At present, there are mainly chemical and biological methods for preparing L-2-aminobutyric acid. The traditional chemical preparation method mainly adopts ketobutyric acid reduction method, desulfurization reaction method, halogenated aminolysis method, chemical splitting method, etc. The disadvantage of the chemical method is that it is easy to form a racemate, which is not conducive to the synthesis of chiral compounds, and the reaction conditions are harsh, the environmental pollution is serious, and it does not conform to the industrial development trend. The biological preparation method mostly adopts the bioenzyme method, using aminoacylase, amino acid oxidase, nitrile hydratase, transaminase, dehydrogenase and other bioenzymes as catalysts. The reaction conditions are mild, the stereoselectivity is high, and it is green and environmentally friendly. It has the incomparable advantages of chemical synthesis, but the free enzyme used as a catalyst has the disadvantages of high catalyst cost, difficulty in recycling and reuse, poor enzyme activity stability, easy inactivation, and mixing with the product to increase the burden of separation and purification. For example, a bioenzyme method with a high conversion rate currently prepares L-2-aminobutyric acid by using three free enzymes of threonine deaminase, leucine dehydrogenase and formate dehydrogenase at the same time. These three free enzymes are difficult to recycle and reuse when used together, and the coenzyme regeneration efficiency is low, and the product purification is complicated. In order to solve the defects of free enzymes, people usually use the method of immobilized enzymes, but the mutual influence of multiple free enzymes brings technical difficulties to the co-immobilization system. Therefore, there is no report in the prior art on the successful co-immobilization of the three free enzymes of threonine deaminase, leucine dehydrogenase and formate dehydrogenase. Instead, by changing the type of free enzyme used as a catalyst, such as replacing leucine dehydrogenase with alcohol dehydrogenase, threonine aldehyde dehydrogenase and L-amino acid dehydrogenase, a co-immobilized multi-enzyme system and a co-immobilized coenzyme system are used to produce L-2-aminobutyric acid. For details, see Chinese patent application CN112522336A. Although the preparation method successfully adopts an immobilized enzyme system, it increases the types and costs of enzymes, complicates the reaction steps, and is not conducive to industrial production. Therefore, how to effectively co-immobilize the three free enzymes of threonine deaminase, leucine dehydrogenase and formate dehydrogenase to achieve a high conversion rate of the reaction and a high recycling rate of the immobilized enzyme has a great significance for the production of L-2-aminobutyric acid. Summary of the invention

[0004] In order to solve the defects of the prior art, the present invention provides a co-immobilized three-enzyme system for catalytic production of L-2-aminobutyric acid. The co-immobilized three-enzyme system of the present invention successfully immobilizes three enzymes, L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase, on the same carrier, i.e., porous silica nanoflowers, to achieve efficient catalytic synthesis of L-2-aminobutyric acid, effective separation of products, recycling of coenzymes and reuse of enzymes.

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

[0006] The three enzymes LTD, LDH and FDH were co-immobilized by using a specific porous carrier silica nanoflower, which effectively improved the substrate tolerance of LTD to 2-ketobutyric acid, the stability of the three enzymes and their reusability. The use of this co-immobilized three-enzyme system to catalyze the production of L-2-aminobutyric acid not only has a high reaction yield, but also makes the product easy to separate, which can simplify the synthesis and subsequent separation and purification steps, and effectively reduce 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 ways:

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

[0009] (2) L-leucine dehydrogenase, L-threonine deaminase and formate dehydrogenase were immobilized on the carrier silica nanoflowers in order, which was recorded as LDH+LTD+FDH;

[0010] (3) L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase were simultaneously immobilized on the carrier silica nanoflowers, denoted as (LTD+LDH+FDH);

[0011] (4) First, L-threonine deaminase was immobilized on the carrier silica nanoflowers, and then L-leucine dehydrogenase and formate dehydrogenase were immobilized at the same time, which was recorded as LTD+(LDH+FDH);

[0012] (5) First, L-leucine dehydrogenase was immobilized on the carrier silica nanoflowers, and then L-threonine deaminase and formate dehydrogenase were immobilized at the same time, which was recorded 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 to prepare 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 nanoflowers are made of hexadecyltrimethylammonium bromide as a template, tetraethyl silicate as a silicon source, n-butanol and cyclohexane as an organic phase, water as a solvent, and urea as a surfactant, and are prepared by stirring and reacting at 60-80°C, followed by drying and calcining.

[0015] As a limitation of the above technical solution, after L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase are mixed with the carrier, they are fully shaken at 25-35° C. and pH 7.0-9.0 for immobilization.

[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 the advantages of high catalytic rate, high stability, easy recovery and reuse.

[0017] At the same time, the present invention also provides a preparation method of the co-immobilized three-enzyme system as described above, comprising the following steps: mixing the porous carrier silica nanoflowers activated by glutaraldehyde with a PBS buffer solution and three enzymes, L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase, and fully shaking the resulting suspension at 25 to 35° C. and pH 7.0 to 9.0 for immobilization to obtain a co-immobilized enzyme system.

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

[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 the 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 synthetic reaction catalyst for preparing L-2-aminobutyric acid from L-threonine.

[0021] As a limitation of the above technical solution, the synthetic reaction conditions are to react 100-500 mM L-threonine, 0.4-2 M sodium formate, 0.25-2.5% NADH by mass concentration, a buffer solution with a pH of 7.0-9.0 and an appropriate amount of co-immobilized enzyme at 25-35° C. for 12-20 hours.

[0022] The co-immobilized enzyme of the present invention is used to catalyze the synthesis of L-2-aminobutyric acid, with a yield of more than 92%, and can achieve effective separation of products after the reaction, recovery and reuse of the enzyme, and recycling of the coenzyme, which can effectively simplify the synthesis steps and subsequent separation and purification steps and reduce production costs.

[0023] In summary, the present invention successfully co-immobilizes L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on a porous carrier silica nanometer, and the co-immobilized three-enzyme system formed improves the substrate tolerance of L-leucine dehydrogenase to 2-ketobutyric acid, the stability and storage of the three natural enzymes, and the reusability. The co-immobilized three-enzyme system of the present invention is used to catalyze the synthesis of L-2-aminobutyric acid, which can realize the recycling and reuse of enzymes and the recycling of coenzymes, simplify the synthesis steps and subsequent separation and purification steps, optimize the process, and reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 , is the yield of enzymes immobilized on different carriers in Example 1;

[0025] Figure 2 , is the effect of the dosage ratio of the three enzymes LTD, LDH and FDH on the yield in Example 3;

[0026] Figure 3 , is the substrate tolerance of free LDH and LDH in the co-immobilized enzyme in Example 4-1;

[0027] Figure 4 , is the reaction progress curve of the co-immobilized enzyme in Example 4-2;

[0028] Figure 5 , is the storage stability of the free enzyme and the co-immobilized enzyme in Example 4-3;

[0029] Figure 6 , is the reusability of the co-immobilized enzyme in Example 4-4;

[0030] Figure 7 , L-threonine standard curve;

[0031] Figure 8 , L-2-ABA standard curve. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0034] Example 1

[0035] This example involves the effects of different carriers on the co-immobilized three-enzyme system, and the specific experiment is as follows.

[0036] Example 1-1: The amino resin LX-1000EPHA was placed in a 50mM, pH 8.0 PBS solution for incubation for 2 hours, washed with a PBS solution (the PBS solution used in the following washing, resuspension, and immobilization system was 50mM, pH 8.0), and filtered with a Buchner funnel. 100 mg of the amino resin LX-1000EPHA was soaked in a 2% (v / v) glutaraldehyde solution and activated by shaking for 2 hours, washed with a PBS solution to remove the residual glutaraldehyde, and dried to obtain a carrier material.

[0037] Take 20 mg of the carrier material, shake it thoroughly and resuspend it in a 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 (the volume is adjusted with PBS solution, the same below) and a total enzyme addition amount of 20 mg. Immobilize at 25°C and 180 rpm to obtain the immobilized enzyme of the carrier LX-1000EPHA.

[0038] Example 1-2: Epoxy resins LX-109S and LX-1000HFA were placed in 200mM, pH 9.0 PBS solution for incubation for 2h, washed with PBS solution (the PBS solution used for the following washing, resuspension, and immobilization system was 50mM, pH 8.0), and filtered with a Buchner funnel. 100mg of epoxy resins LX-109S and LX-1000HFA were soaked and activated with 2% (v / v) glutaraldehyde for 2h, washed with PBS solution to remove residual glutaraldehyde, and dried to obtain a carrier material.

[0039] Take 20 mg of the carrier material, shake it thoroughly and resuspend it 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 addition amount of 20 mg. Immobilization was carried out at 25°C and 180 rpm to obtain immobilized enzymes of carriers LX-109S and LX-1000HFA, respectively.

[0040] Example 1-3: Preparation of silica nanoflowers DSN, namely, in a 250mL single-necked flask, add 3g CTAB, 100g urea aqueous solution (0.4M), ultrasonicate until the solution becomes clear, then add 3.5g n-butanol and 20g cyclohexane, ultrasonicate at room temperature for 30min until the solution becomes a uniform milky white solution; the flask is placed in a water bath, and during the stirring process, 3g tetraethyl silicate (TEOS) is slowly added dropwise using a syringe, reacted at room temperature for 30min, and then the temperature was raised to 70°C for 24h. After the reaction, the reaction solution was centrifuged to obtain a white precipitate, which was washed with water and ethanol three times respectively, dried at 60°C, placed in a calcining furnace at 550°C, calcined for 5h, and CTAB was removed to obtain DSN.

[0041] 100 mg of DSN was soaked in 2% (v / v) glutaraldehyde and activated by shaking for 2 h, then washed with PBS solution (PBS solution used in the following washing, resuspension and immobilization system was 50 mM, pH 8.0) to remove residual glutaraldehyde and dried.

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

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

[0044] The catalytic reaction of the above-mentioned immobilized enzyme is the preparation reaction of L-2-aminobutyric acid, and 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 by mass concentration, ammonium chloride-ammonia buffer solution with a pH of 7.0-9.0 and an appropriate amount of co-immobilized enzyme, reacting at 25-35° C. and 180 rpm shaking conditions for 12-20 hours.

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

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

[0047] Table 1 Gradient elution program

[0048]

[0049] The results of the immobilized enzyme catalytic reactions are shown in 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 example involves the effects of different immobilization methods on the co-immobilized three-enzyme system, and the specific experiment is as follows.

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

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

[0054] (2) Performance evaluation of the co-immobilized three-enzyme system 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 those in Example 1. The results of each catalytic reaction are shown in Table 2 below.

[0056] Table 2 Effect of immobilization method on yield

[0057]

[0058]

[0059] From the results in Table 2, it can be seen that 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 example involves optimizing the ratio of the three enzymes in the co-immobilized three-enzyme system.

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

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

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

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

[0066] Example 4

[0067] This example involves the performance measurement of the co-immobilized three-enzyme system

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

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

[0070] Take 20 mg of 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 make 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 free LDH and LDH in co-immobilized enzyme

[0072] LDH enzyme activity determination method: add 2-ketobutyric acid (2-OBA) to NH4Cl-NH3·H2O (900mM, pH 9.5), then add enzyme, mix well and then add NADH to start the reaction. The concentration of 2-OBA in the reaction system is required to be 1-60mM, the enzyme concentration is 0.05mg / mL, and the NADH concentration is 2.5mg / mL. The absorbance change value is measured at 340nm. The enzyme activity is defined as: 1μmol NADH is consumed per minute to generate 1μmol NAD + The amount of enzyme required is 1U.

[0073] Take free enzyme LDH and co-immobilized enzyme with the same enzyme activity, add different amounts of 2-OBA (corresponding to concentrations of 1 to 60 mM) according to the LDH enzyme activity determination method, react for 3 minutes, and determine the enzyme activity. The results are shown in Figure 3 .

[0074] Depend on Figure 3 It can be seen that the downward trend of the inhibition curve of the co-immobilized enzyme is slower than that of the free enzyme, and its half-inhibitory concentration (half-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 enzyme: The specific preparation operation is the same as that in Example 4-1.

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

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

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

[0080] (1) Preparation of co-immobilized enzyme: The specific preparation operation is the same as that in Example 4-1.

[0081] (2) Study on the storage stability of co-immobilized enzyme

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

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

[0084] LTD enzyme activity determination method: 50mM potassium phosphate buffer (pH 7.5), 10μM PLP, 50mM L-threonine, add LTD enzyme solution (enzyme final concentration is 0.05mg / mL) and mix to a volume of 1mL, and measure the absorbance change of the product 2-OBA at 230nm at 30℃. The enzyme activity is defined as: the amount of enzyme required to catalyze the production of 1μmol 2-OBA in 1min is 1U.

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

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

[0087] Example 4-4: Reusability of immobilized enzyme

[0088] (1) Preparation of co-immobilized enzyme: The specific preparation operation is the same as that in Example 4-1.

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

[0090] 500 mM L-threonine was used as substrate, 40 mg of co-immobilized enzyme was added, and the mixture was reacted at 30°C for 16 h and then centrifuged. The supernatant was determined for yield by HPLC (derivation and detection operations were the same as in Example 1). The catalyst co-immobilized enzyme obtained by centrifugation was washed and put into the next batch of catalytic reaction. The above experiment was repeated to determine the reusability of the immobilized enzyme. The experimental results are shown in FIG. 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 that cannot be reused, the co-immobilized enzyme can effectively prevent the reduction of enzyme activity.

[0091] In summary, the present invention successfully achieves the co-immobilization of three enzymes, L-threonine deaminase, L-leucine dehydrogenase, and formate dehydrogenase, on the same carrier silica nanoflower, thereby improving the stability and reusability of the three enzymes. The reaction yield of preparing L-2-aminobutyric acid using the co-immobilized three-enzyme system can reach more than 92%. At the same time, the effective separation of the co-immobilized enzyme and the product, the recovery and reuse of the enzyme, and the recycling of the coenzyme are achieved, which simplifies the synthetic reaction steps and subsequent separation and purification steps for producing L-2-aminobutyric acid, optimizes the production process, and reduces the production cost.

Claims

1. A co-immobilized three-enzyme system for preparing L-2-aminobutyric acid, characterized in that: The co-immobilized three-enzyme system is obtained by co-immobilizing L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on a porous carrier silica nanoflower. The mass ratio of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase used to prepare the co-immobilized three-enzyme system is (0.1-2):1:(1-10).

2. The co-immobilized three-enzyme system for preparing L-2-aminobutyric acid according to claim 1, characterized in that: L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase are immobilized on the carrier silica nanoflowers in any of the following ways: (1) Immobilizing L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on the carrier silica nanoflowers in order; (2) Immobilizing L-leucine dehydrogenase, L-threonine deaminase and formate dehydrogenase on the carrier silica nanoflowers in order; (3) Immobilizing L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase on the carrier silica nanoflowers at the same time; (4) First, L-threonine deaminase is immobilized on the carrier silica nanoflowers, and then L-leucine dehydrogenase and formate dehydrogenase are immobilized simultaneously; (5) First, L-leucine dehydrogenase is immobilized on the carrier silica nanoflowers, and then L-threonine deaminase and formate dehydrogenase are immobilized at the same time.

3. The co-immobilized three-enzyme system for preparing L-2-aminobutyric acid according to claim 1, characterized in that: The mass ratio of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase used to prepare the co-immobilized three-enzyme system is (0.5-1):1:(1-10).

4. The co-immobilized three-enzyme system for preparing L-2-aminobutyric acid according to claim 1, characterized in that: The carrier silica nanoflower is prepared by using hexadecyltrimethylammonium bromide as a template, tetraethyl silicate as a silicon source, n-butanol and cyclohexane as an organic phase, water as a solvent, and urea as a surfactant, and then drying and calcining after stirring and reacting at 60-80°C.

5. The co-immobilized three-enzyme system for preparing L-2-aminobutyric acid according to claim 1, characterized in that: After L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase are mixed with the carrier, they are fully shaken at 25-35° C. and pH 7.0-9.0 for immobilization.

6. The method for preparing the co-immobilized three-enzyme system according to any one of claims 1 to 5, characterized in that: The steps include: The porous carrier silica nanoflowers activated by glutaraldehyde were mixed with PBS buffer solution and three enzymes: L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase. The resulting suspension was fully shaken at 25-35°C and pH 7.0-9.0 for immobilization to obtain a co-immobilized enzyme system.

7. The method for preparing the co-immobilized three-enzyme system according to claim 6, characterized in that: The mass ratio of the carrier to the total enzyme in the immobilization process is 1:0.6-1.

2.

8. A method for preparing L-2-aminobutyric acid, characterized in that: The co-immobilized three-enzyme system according to any one of claims 1 to 5 is used as a catalyst for the synthesis reaction of preparing L-2-aminobutyric acid from L-threonine.

9. The method for preparing L-2-aminobutyric acid according to claim 7, characterized in that: The synthetic 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 a pH of 7.0-9.0 and a co-immobilized enzyme are shaken and reacted at 25-35° C. for 12-20 hours.

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