A method for high-throughput screening of leucine dehydrogenase and a leucine dehydrogenase

Abstract

The present invention discloses a method for high-throughput screening of leucine dehydrogenase and a leucine dehydrogenase. Using the method of the present invention, protein engineering modification is carried out on leucine dehydrogenase BtLDH, and a high-throughput screening method is used to culture recombinant strains and strain mutants containing leucine dehydrogenase BtLDH in 96-well plates, and subculture induction is carried out. Cells containing leucine dehydrogenase BtLDH of Bacillus thuringiensis are centrifuged and collected to transform 2-ketobutyric acid to produce L-2-aminobutyric acid, and after color development with 2,4-dinitrofluorobenzene (DNFB), the absorbance value is read at 500 nm, and the strains with large absorbance values are verified in shake flasks. Each leucine dehydrogenase is successfully screened within a short time. Among them, the optimal mutant A262S produces 9.22 g / L of L-2-aminobutyric acid, and the yield of L-2-aminobutyric acid is increased by 3.04 g / L compared with the wild type.

Description

Technical Field

[0001] The present invention relates to a method for high-throughput screening of leucine dehydrogenase and a leucine dehydrogenase, belonging to the technical field of bioengineering. Background Art

[0002] L-2-Aminobutyric acid (L-ABA) is an unnatural chiral amino acid, which has the functions of inhibiting human nerve information transmission, enhancing the activity of glucose phosphatase, and promoting brain cell metabolism. As an important chiral pharmaceutical intermediate, it is mainly used for synthesizing the drugs levetiracetam and brivaracetam for treating epilepsy, and ethambutol hydrochloride for treating tuberculosis.

[0003] Currently, the reported synthetic methods of L-ABA include chemical methods and biocatalytic methods. Chemical methods are prone to form racemates, which is not conducive to the synthesis of chiral compounds. Moreover, the reaction conditions are harsh, the reagent cost is high, the environmental pollution is serious, and it is not easy to carry out industrial production. Biocatalytic methods have received extensive attention due to their mild reaction conditions, high stereoselectivity, and environmental friendliness.

[0004] The preparation of L-2-aminobutyric acid by the microbial method can be further divided into fermentation method and enzyme conversion method. Using the fermentation method to produce fine chemicals has the significant advantages of wide raw material sources (such as glucose, glycerol, etc.) and low cost. Its relatively low substrate cost brings huge commercial benefits to large-scale industrialization. With the development of metabolic processes and synthetic biology, more opportunities will be available for the production of L-2-aminobutyric acid by the fermentation method. However, there are still huge challenges to achieve large-scale industrial production. Currently, the enzyme conversion method has more industrial application value due to its advantages such as high yield, high conversion rate, and short conversion cycle. The production of L-2-aminobutyric acid by the microbial method involves a key enzyme, leucine dehydrogenase (LDH, Leucine dehydrogenases, EC 1.4.1.9), which can catalyze 2-ketobutyric acid to deaminate at the α-position to form L-2-aminobutyric acid. It has high substrate specificity and a single product, and is more suitable for industrial production than other methods. However, there are still problems such as poor affinity and stability for non-natural substrates, easy product separation, but low key enzyme activity, complex process, and difficulty in industrialization. Summary of the Invention

[0005] To solve the above technical problems, the present invention first provides a high-throughput screening method for leucine dehydrogenase that is efficient, low-cost, and easy to scale up, saving the screening time of leucine dehydrogenase and providing a reference for realizing the industrial production of L-2-aminobutyric acid by the enzyme method. The present invention also provides a leucine dehydrogenase screened by the high-throughput screening method.

[0006] The first object of the present invention is to provide a method for high-throughput screening of leucine dehydrogenase, comprising the following steps:

[0007] S1. Inoculate the seed solutions of the wild strain and mutant strain expressing leucine dehydrogenase into a 96-well plate containing TBA fermentation medium for fermentation culture;

[0008] S2. After cultivation, collect whole cells, and add the whole cells into a conversion solution to convert the substrate 2-ketobutyric acid to produce L-2-aminobutyric acid;

[0009] S3. After the conversion is completed, centrifuge to obtain the supernatant, and the supernatant is colored by the 2,4-dinitrofluorobenzene colorimetric method, and screen for mutant strains with an absorbance value greater than that of the wild strain.

[0010] Further, in step S1, the fermentation culture is carried out until the OD 600 reaches 0.6 - 1.0, then cool down to 24 - 26 °C to induce the cells to produce leucine dehydrogenase BtLDH, and the induction time should be 14 - 15 h.

[0011] Further, the conditions of the fermentation culture are 35 - 38 °C, 750 - 900 rpm.

[0012] Further, in step S1, the seed solution is obtained by culturing at 35 - 38 °C, 750 - 900 rpm for 12 h.

[0013] Further, in step S2, the content of 2-ketobutyric acid in the conversion solution is 40 - 60 mmol / L.

[0014] Further, in step S2, the conversion conditions are conversion at 35 - 38 °C, 750 - 900 rpm for 10 - 15 h.

[0015] Further, in step S3, the 2,4-dinitrofluorobenzene colorimetric method comprises the following steps: The operation process of 2,4-dinitrofluorobenzene color development is as follows: Take 15 - 25 μL of the supernatant, add 15 - 25 μL of 0.4 - 0.6 mol sodium bicarbonate solution, 5 - 15 μL of 0.8 - 1.2% 2,4-dinitrofluorobenzene solution, mix evenly, react at 55 - 65 °C in the dark for 55 - 65 min, after the reaction is completed, add 200 - 300 μL of PBS buffer solution, and detect the absorbance value of the solution at 500 nm.

[0016] Further, the method further includes verifying the screened mutant strains by using shake flasks. Specifically, the mutant strains are fermented and cultured in shake flasks, the cells are centrifuged and collected for converting 2-ketobutyric acid to produce L-2-aminobutyric acid, after the conversion, the supernatant is centrifuged and taken, and the yield of L-2-aminobutyric acid is detected by high performance liquid chromatography.

[0017] Furthermore, during shake flask fermentation, inoculate into TB medium at an inoculum size of 1-3% and culture until the OD 600 reaches 0.6-1.0, then add IPTG with a final concentration of 0.08-0.12 g / L to induce the expression of the enzyme. The induction temperature is 24-26 °C and the induction time is 14-15 h.

[0018] Furthermore, during transformation, the transformation conditions are 24-26 °C, reaction at 150-250 rpm for 10-15 h. The transformation system has a cell mass of 15-25 g / L, and the feeding amount of 2-ketobutyric acid is 5-15 g / L.

[0019] The second object of the present invention is to provide a leucine dehydrogenase mutant, wherein the leucine dehydrogenase mutant is obtained by mutating the aspartic acid at position 144 of the leucine dehydrogenase parent with the amino acid sequence shown in SEQ ID NO.1 to leucine.

[0020] The third object of the present invention is to provide a nucleic acid encoding the leucine dehydrogenase mutant.

[0021] The fourth object of the present invention is to provide an application of the leucine dehydrogenase mutant in the preparation of L-2-aminobutyric acid.

[0022] The beneficial effects of the present invention are as follows:

[0023] Using the method of the present invention, protein engineering modification is carried out on the leucine dehydrogenase BtLDH derived from Bacillus thuringiensis, and a high-throughput screening method is used to culture recombinant strains and strain mutants containing the leucine dehydrogenase BtLDH of Bacillus thuringiensis in 96-well plates, and then transfer and induce. Centrifuge to collect the cells containing the leucine dehydrogenase BtLDH of Bacillus thuringiensis to transform 2-ketobutyric acid to produce L-2-aminobutyric acid. After color development with 2,4-dinitrofluorobenzene (DNFB), read the absorbance value at 500 nm, and verify the strains with large absorbance values in shake flasks. Each leucine dehydrogenase is successfully screened out in a short time. Among them, the optimal mutant A262S produces 9.22 g / L of L-2-aminobutyric acid, and the yield of L-2-aminobutyric acid is increased by 3.04 g / L compared with the wild type. Description of the Drawings

[0024] Figure 1 It is the schematic diagram of the color development principle of L-2-aminobutyric acid and 2,4-dinitrofluorobenzene and the standard curve of the product.

[0025] Figure 2 For the substrate 2-ketobutyric acid ( Figure 2 a) and L-2-aminobutyric acid ( Figure 2 b) high performance liquid chromatography diagrams;

[0026] Figure 3 This is the flask verification L-2-amino butyric acid production graph of the screening strain involved in the present invention. Specific embodiments

[0027] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0028] The experimental materials used in the following implementation cases are all available from commercial channels, and the experimental methods not specifically described are conventional experimental methods in the art. A genetic engineering strain of leucine dehydrogenase BtLDH derived from Bacillus thuringiensis was constructed. The host used was Escherichia coli BL21(DE3), the vector was pET-28a, and the resistance was kanamycin resistance.

[0029] Sample pretreatment: Centrifuge the conversion solution at 12000 rpm for 10 min, and collect the supernatant. Using L-2-amino butyric acid (as the standard product, prepare a standard solution. After moderately diluting the supernatant and the standard solution, filter them through a 0.22 μm microporous filter membrane respectively to obtain the filtrate. The filtrate is used as the sample to be tested, and the content of L-2-amino butyric acid (and the remaining amount of the substrate) is determined by high performance liquid chromatography.

[0030] Determination of the remaining amount of 2-ketobutyric acid by high performance liquid chromatography: Use a Dionex high performance liquid chromatograph, a Bio-Rad Aminex HPX-87H chromatographic column (300×7.8 mm, 9 μm), and the mobile phase is 5 mM dilute sulfuric acid after ultrasonic degassing treatment. The detection conditions are set as follows: injection volume 10 μL, flow rate 0.6 mL / min, column temperature 35 °C, ultraviolet detection wavelength 210 nm, and the injection time of the sample is set to 20 min.

[0031] Determination of the content of L-2-amino butyric acid by high performance liquid chromatography: The yield of the product L-2-amino butyric acid in the conversion solution was determined by pre-column derivation with 2,4-dinitrofluorobenzene (DNFB) and reverse-phase HPLC. Specifically, a Dionex high performance liquid chromatograph and an Agilent ZORBAX SB-C18 chromatographic column (250×4.6 mm, 5 μm) were used. Mobile phase A: 50% acetonitrile, which was filtered through an organic membrane and degassed by ultrasonic for 15 min; Mobile phase B: Weighed 4.1 g of anhydrous sodium acetate and dissolved it in 800 mL of ultrapure water, adjusted the pH to 6.4 with glacial acetic acid, and then made up the volume to 1 L with ultrapure water; The mobile phase was filtered through a 0.22 μm filter membrane and degassed by ultrasonic. The sample to be tested was also filtered through a 0.22 μm filter membrane; Sample treatment method: Take 1 mL of the fermentation broth in a 1.5 mL EP tube, centrifuge at 12000 rpm for 1 min, take the supernatant and dilute it 10 times with ultrapure water, then take 100 μL in a 1.5 mL EP tube, add 100 μL of 0.5 mol / L sodium bicarbonate solution, and then add 50 μL of the derivatization reagent (1% 2,4-dinitrofluorobenzene-acetonitrile solution), react at 60 °C in the dark for 60 min, then add 750 μL of phosphate buffer (10.9251 g of disodium hydrogen phosphate dodecahydrate and 3.043 g of sodium dihydrogen phosphate dihydrate were dissolved in 250 mL of ddH2O, pH = 7, 0.2 mol / L) to make up the volume to 1 mL, filter through a 0.22 μm organic filter membrane and detect by high performance liquid chromatography (HPLC). The gradient elution program shown in Table 1 was used for elution; The column temperature was 33 °C, the flow rate was 0.5 mL·min -1 , the injection volume was 10 μL, the chromatographic retention time was 30 min, and the detection wavelength was 360 nm.

[0032] Table 1 Gradient elution table of L-2-amino butyric acid

[0033]

[0034] The seed medium used in high-throughput screening was LB medium, and the formula was as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L. The sterilization conditions of the seed medium were 121 °C for 15 - 20 min.

[0035] The fermentation medium used in high-throughput screening was TBA medium. The formula of the fermentation medium is shown in Table 2. The sterilization conditions of the fermentation medium were 115 °C for 15 - 20 min. Deionized water was used to make up the volume when preparing the seed medium and the fermentation medium.

[0036] 96-well plates used in high-throughput screening: They need to be wrapped with kraft paper (newspaper) and then sterilized by high-pressure steam moist heat. The sterilization conditions are 121 °C for 20 - 30 minutes. The operation of dispensing the sterilized medium should be carried out in a laminar flow hood. Kanamycin should be added before dispensing the medium, and the addition amount of kanamycin should be 50 μg / mL. The loading volume of the seed medium in each well of the 96-well plate is 400 - 600 μL, and the loading volume of the fermentation medium in each well of the 96-well plate is 600 - 800 μL.

[0037] Table 2 TBA Fermentation Medium Formulation Table

[0038]

[0039] During high-throughput screening, using the TBA fermentation medium can make the operation more simple and fast. Compared with the commonly used TB fermentation medium, the TBA medium has richer components. Glucose is added to supplement the energy required for cell growth; adding lactose can reduce the number of times of opening the 96-well plate while acting as an inducer, that is, it is equivalent to reducing the risk of contamination during cell culture. It only needs to reduce the temperature to induce the production of leucine dehydrogenase BtLDH within 2 - 4 hours after subculture. This operation is more convenient. Therefore, the fermentation medium used in high-throughput screening is the TBA medium.

[0040] The parental strain E. coli-pET-28a-BtLDH used in high-throughput screening: Using Escherichia coli BL21(DE3) as the host and pET-28a as the expression vector to express the leucine dehydrogenase BtLDH encoded by Proteus mirabilis. The amino acid sequence of the BtLDH parental strain derived from Bacillus thuringiensis is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2.

[0041] SEQ ID NO.1:

[0042] MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGGTRMWTYDSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAVGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSANNQLKENRHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR

[0043] SEQ ID NO.2:

[0044]

[0045] Example 1: High-throughput screening

[0046] Based on the structural characteristics of the crystallized leucine dehydrogenase, 5 amino acid sites on the surface of the NADH nicotinamide ring were screened for mutation and high-throughput screening was carried out.

[0047] 1. Construction of strains expressing the leucine dehydrogenase BtLDH mutant

[0048] Primer design: The 5 amino acid sites are T152, V186, S261, A262, and N263 respectively. Saturation mutation primers were designed for these 5 sites, and the nucleic acid sequences of the primers are shown in Table 3.

[0049] Table 3 High-throughput screening mutant primer sequences

[0050]

[0051] Construction of mutants by whole plasmid PCR: The construction reaction PCR amplification system uses the KOD system. Using pET-28a-BtLDH as the template, PCR was carried out using the primers shown in Table 3. The KOD system table is shown in Table 4. The PCR reaction conditions are as follows: ① 94°C for 2 min; ② 98°C for 30 s; ③ 55°C for 30 s; ④ 68°C for 3.5 min; ⑤ Repeat steps ② - ④ 29 times; ⑥ 68°C for 5 min; ⑦ Incubate at 12°C. The entire PCR time is 2 h 39 min.

[0052] Table 4 KOD system table

[0053]

[0054] Transformation: After the PCR reaction is completed, the above PCR reaction system is incubated at 37°C for 3 - 4 h to digest the plasmid template (the digestion system is: 0.5 μL of DpnI, 45 μL of the above reaction PCR product, 5 μL of 10×T Buffer). The digestion product obtained after digestion is introduced into Escherichia coli BL21 competent cells by chemical transformation method. The specific steps of the chemical transformation method are as follows:

[0055] (1) Introduce 10 μL of the homologous recombination product into 100 μL of BL21 competent cells;

[0056] (2) Incubate on ice for 15 - 30 min;

[0057] (3) Heat in a 42°C water bath for 90 s, take out and quickly place it in ice and let it stand in the ice bath for 3 - 5 min;

[0058] (4) Add 800 μL of antibiotic-free LB medium and mix well, and culture at 37°C and 200 rpm for 45 min - 1 h;

[0059] (5) Centrifuge at 5000 rpm for 2 min to collect bacteria.

[0060] (6) Remove the supernatant, and resuspend the remaining 100 - 200 μL by pipetting up and down, then spread it evenly on a kanamycin-resistant plate containing 0.05 mg / mL kanamycin, and incubate at 37 °C for about 12 h.

[0061] (7) Pick monoclonal colonies and transfer them into a 96-well plate containing LB medium with 0.05 mg / mL kanamycin, and incubate at 750 - 900 rpm and 37 °C for 12 h to obtain the seed plate for high-throughput screening.

[0062] 2. Fermentation culture and collection of the strain expressing the BtLDH mutant of leucine dehydrogenase

[0063] Ferment BtLDH of leucine dehydrogenase. Different strains expressing BtLDH of leucine dehydrogenase used for fermentation should be the wild-type strain of BtLDH of leucine dehydrogenase, E.coli-pET-28a-BtLDH(WT), and mutant strains (the mutant strains are directly numbered in the 96-well plate). Inoculate 4 - 8 strains of the wild-type strain in the 12th column of the 96-well plate, and inoculate the mutant strains of BtLDH of leucine dehydrogenase in the remaining wells. The layout of the entire 96-well plate is shown in Table 5:

[0064] Table 5 Layout of the 96-well plate for inoculating BtLDH strains of leucine dehydrogenase (seed plate)

[0065]

[0066] After the seed liquid is cultured at 37 °C and 750 - 900 rpm for 12 h, transfer it to the same position in a 96-well plate (fermentation plate) containing TBA fermentation medium with kanamycin in the laminar flow hood (well A1 still corresponds to well A1). The remaining seed liquid needs to be added with 30% glycerol of the same volume as the remaining seed liquid for preservation. The 96-well plate for preservation should be stored in a -80 °C refrigerator. The fermentation culture should be carried out at 37 °C and 750 - 900 rpm. When OD 600 reaches 0.6 - 1.0, lower the temperature to 25 °C to induce the cells to produce BtLDH of leucine dehydrogenase, and the induction time should be 14 - 15 h. After the induction is completed, centrifuge to collect the cells: the centrifugation conditions should be 4 °C and 3700 rpm for 10 - 15 min. Discard all the culture medium and collect the cells for the conversion of 2-ketobutyric acid to L-2-aminobutyric acid by BtLDH of leucine dehydrogenase.

[0067] 3. Screen mutants from the perspective of the conversion of 2-ketobutyric acid to L-2-aminobutyric acid by BtLDH of leucine dehydrogenase

[0068] The reaction buffer used in high-throughput screening is PBS buffer at pH 7.0. The preparation method of PBS buffer at pH 7.0 is as follows: Weigh 11.41 g of dipotassium hydrogen phosphate trihydrate and dissolve it in a certain amount of deionized water. After it is completely dissolved, make up the volume to 1 L with deionized water; Weigh 6.8 g of potassium dihydrogen phosphate and dissolve it in a certain amount of deionized water. After it is completely dissolved, make up the volume to 1 L with deionized water. Then add 615 mL of the dipotassium hydrogen phosphate trihydrate solution and 385 mL of the potassium dihydrogen phosphate solution. At this time, the pH of the solution is 7.0. For the specific formula, please refer to "Industrial Microbiology Experimental Technology Manual" (edited by Zhuge Jian, published by China Light Industry Press).

[0069] Fully mix the bacterial cells collected in step 2 with the prepared reaction solution and carry out transformation on a high-throughput shaker. The transformation conditions are 37 °C, 750 - 900 rpm, and 12 h. After the transformation is completed, centrifuge at 4 °C and 3700 rpm for 10 - 15 min using a swing-out centrifuge. Dilute the supernatant by an appropriate multiple and carry out a color reaction with 2,4-dinitrofluorobenzene. Then use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance at 500 nm. The darker the color, the greater the absorbance value, indicating a higher yield of L-2-aminobutyric acid.

[0070] The specific operation of the color reaction of the product with L-2-aminobutyric acid is as follows: ① Preparation of the color reagent: The color reagent used for color development is 1% 2,4-dinitrofluorobenzene (DNFB). The preparation method of the 1% 2,4-dinitrofluorobenzene (DNFB) color reagent is as follows: Pipette 100 μL of 2,4-dinitrofluorobenzene (DNFB) and dissolve it in 10 mL of acetonitrile, and store it in the dark. ② Color reaction of L-2-aminobutyric acid: The color development operation process of 2,4-dinitrofluorobenzene (DNFB) is as follows: Take 20 μL of the sample solution, add 20 μL of 0.5 mol sodium bicarbonate solution and 10 μL of 1% DNFB solution, mix well (react at 60 °C in the dark for 60 min). After the reaction is completed, add 250 μL of 0.2 mol / L PBS buffer at pH 7.0 and detect the absorbance of the solution at 500 nm.

[0071] 4. Shake flask verification

[0072] Pick the strains with high absorbance values for shake flask verification. That is, thaw the 96-well plate used for preservation during high-throughput screening, streak it on a kanamycin-resistant plate containing 0.05 mg / mL kanamycin, incubate it at 37 °C for about 12 h, and pick the strains into a 100 mL conical flask containing 30 mL of LB medium (containing 0.05 mg / mL kanamycin) and incubate at 37 °C and 200 rpm for 8 - 12 h to obtain the seed liquid for shake flask verification. Transfer it to a 500 mL conical flask containing 150 mL of TB medium (containing 0.05 mg / mL kanamycin) for cultivation at an inoculation amount of 2%. Considering that the TB medium has a simpler formula than the TBA medium and is more suitable for industrial production requirements, and the sterilization conditions are the same as those of the LB medium (121 °C, 15 - 20 min), the medium used for shake flask verification is the TB fermentation medium, and its formula is shown in Table 7. The cultivation conditions are 37 °C and 200 rpm. When the OD 600 reaches 0.6 - 1.0, add IPTG with a final concentration of 0.1 g / L to induce the expression of the enzyme, the induction temperature is 25 °C, and the induction time is 14 - 15 h to obtain the fermentation broth. Centrifuge the fermentation broth at 4 °C and 6000 rpm for 10 - 15 min to collect the cells. (The medium of the shake flask verification seed liquid is the same as the medium used during high-throughput screening. When preparing the solid LB medium, 2% agar powder needs to be added).

[0073] Table 7 Formulation table of TB fermentation medium for shake flask verification

[0074]

[0075] Use the collected cells for transformation experiments. The transformation conditions during shake flask verification are: react at 37 °C and 200 rpm for 12 h, the transformation system is 10 mL, the system includes 20 g / L of cell mass, the feeding amount of 2-ketobutyric acid is 10 g / L, the buffer used is PBS with a pH of 7.0 (the same as the buffer used during high-throughput transformation). After transformation for 12 h, centrifuge and take the supernatant, dilute it by an appropriate multiple, and use high-performance liquid chromatography to detect L-2-aminobutyric acid, and calculate the yield of L-2-aminobutyric acid according to the standard curve. The transformation results of shake flask verification are shown in Figure 3 , and the mutant strains in the figure are named after the sequenced strain names.

[0076] Results: A total of 480 BtLDH mutant strains of leucine dehydrogenase from Bacillus thuringiensis were screened. The optimal mutant A262S had a 3.04 g / L increase in the yield of L-2-aminobutyric acid compared with the wild type.

[0077] Example 2: Determination of BtLDH enzyme activity

[0078] 1. Method for determining BtLDH enzyme activity

[0079] Enzyme activity detection of leucine dehydrogenase (BtLDH): Add pure leucine dehydrogenase enzyme with a final concentration of 1 mg / mL to a 200 μL reaction system (reaction system: dissolve 1 mM NADH and 20 mmol of 2-ketobutyric acid in 50 mmol / L PBS buffer solution with pH = 7.0), and detect the change in absorbance value of NADH at 340 nm within 1 min in a constant temperature microplate reader at 30 °C. The enzyme activity unit is defined as the amount of enzyme required to consume 1 μmol of NADH within 1 min.

[0080] Specific enzyme activity determination method: Measure the enzyme activity of BtLDH using the above method. The enzyme activity of 1 unit of BtLDH is defined as the amount of enzyme required to consume 1 μmol of NADH within 1 min (U). The enzyme activity can be calculated by measuring the content of BtLDH.

[0081] Specific enzyme activity is defined as the number of enzyme activity units per milligram of protein (U / mg protein).

[0082]

[0083] 2. Enzyme activity determination of beneficial mutants

[0084] Determine the enzyme activity of the beneficial mutants obtained in Example 1, and the results are shown in Table 8.

[0085] Table 8 Enzyme activity determination of beneficial mutants

[0086]

[0087] The above-mentioned embodiments are only preferred embodiments cited to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A leucine dehydrogenase mutant, characterized in that, The leucine dehydrogenase mutant described above is obtained by mutating alanine at position 262 of the leucine dehydrogenase parent with the amino acid sequence shown in SE ID NO.1 to serine.

2. A nucleic acid encoding the leucine dehydrogenase mutant according to claim 1.

3. Use of the leucine dehydrogenase mutant according to claim 1 in the preparation of L-2-aminobutyric acid.

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