Leucine dehydrogenase mutant and application thereof

Abstract

The invention provides a leucine dehydrogenase mutant and application thereof, and belongs to the technical field of bioengineering. The leucine dehydrogenase mutant provided by the invention can maintain higher catalytic stability in a wider pH value and temperature range, and the adaptability and durability of the leucine dehydrogenase mutant in an industrial reaction environment are remarkably improved. The improvement directly improves the efficiency and reliability of the process for catalytically synthesizing L-2-aminobutyric acid by using the enzyme, so that the reaction process is easier to control, and the cost loss caused by enzyme inactivation is potentially reduced. Finally, the mutant provides a catalyst with excellent performance for green, efficient and large-scale production of L-2-aminobutyric acid with high optical purity, so that a preparation process of chiral amino acid is simplified; and moreover, the method has important significance in reducing the production cost of key intermediates of downstream chiral drugs (such as certain antiepileptic drugs and antituberculosis drugs) and improving the product quality, and shows a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a leucine dehydrogenase mutant and its applications. Background Technology

[0002] Leucine dehydrogenase is an important nicotinamide adenine dinucleotide-dependent oxidoreductase widely found in microorganisms. It catalyzes the reversible deamination of various branched-chain amino acids, and has applications in microbial nitrogen metabolism, industrial biocatalysis, and biosensing. Utilizing its reductive amination activity, it can catalyze the synthesis of corresponding chiral amino acids from α-keto acid substrates, providing a possibility for the green and efficient preparation of optically pure amino acids. L-2-aminobutyric acid (GABA), as an important non-natural chiral amino acid, is a key chiral building block in the synthesis of many high-value drugs, playing an irreplaceable role in the production of antiepileptic and antituberculosis drugs.

[0003] Currently, the main methods for preparing L-2-aminobutyric acid (GABA) include chemical synthesis and enzymatic catalysis. Chemical synthesis often suffers from drawbacks such as cumbersome steps, the use of expensive chiral catalysts, significant environmental pollution, and difficulty in controlling optical purity. In contrast, enzymatic catalysis has attracted widespread attention due to its mild conditions, high stereoselectivity, and environmental friendliness. However, in practical industrial applications, naturally derived leucine dehydrogenases often face problems such as low catalytic efficiency and insufficient stability under reaction conditions (e.g., tolerance to acids, bases, and temperatures). This directly affects the economics and feasibility of the catalytic process, limiting its large-scale industrial application. Therefore, developing leucine dehydrogenase mutants with higher catalytic activity and stronger stability is of great significance for promoting the industrialization of efficient and green enzymatic synthesis of L-2-aminobutyric acid and related chiral drug intermediates. Summary of the Invention

[0004] The purpose of this invention is to provide a leucine dehydrogenase mutant and its application, which solves the technical problem that the wild-type leucine dehydrogenase has insufficient catalytic stability under industrial application conditions (such as specific pH and temperature ranges), resulting in limited efficiency and applicability in the catalytic synthesis of L-2-aminobutyric acid.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a leucine dehydrogenase mutant derived from Bacillus cereus. Compared with the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the leucine dehydrogenase mutant includes the substitution of tyrosine at position 282 with leucine.

[0007] Preferably, the leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO.4.

[0008] The present invention also provides an isolated nucleic acid molecule that encodes the above-mentioned leucine dehydrogenase mutant.

[0009] Preferably, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.3.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0011] Preferably, the recombinant vector is a bacterial plasmid, bacteriophage, yeast plasmid, or retroviral packaging plasmid.

[0012] The present invention also provides a host cell comprising the above-mentioned nucleic acid molecules or recombinant vectors.

[0013] Preferably, the host cell is a prokaryotic cell or a lower eukaryotic cell.

[0014] The present invention also provides an enzyme system comprising the above-described leucine dehydrogenase mutant.

[0015] The present invention also provides a method for preparing L-2-aminobutyric acid, the method comprising the step of using the above-mentioned leucine dehydrogenase mutant or enzyme system to catalyze the production of L-2-aminobutyric acid from L-threonine in the presence of NAD or NADH;

[0016] The leucine dehydrogenase mutant is used in the form of crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme or whole cell.

[0017] The catalytic reaction is carried out in a buffer solution with a pH of 6.0 to 7.5;

[0018] The catalytic reaction is carried out at a temperature of 20°C to 50°C;

[0019] The concentration of L-threonine in the method is from 0.1 mM to 500 mM.

[0020] The beneficial effects of this invention are:

[0021] The leucine dehydrogenase mutant provided by this invention maintains higher catalytic stability over a wider range of pH and temperature compared to the wild-type enzyme, significantly improving its adaptability and durability in industrial reaction environments. This improvement directly enhances the process efficiency and reliability of synthesizing L-2-aminobutyric acid using this enzyme, making the reaction process easier to control and potentially reducing cost losses due to enzyme inactivation. Ultimately, this mutant provides a high-performance catalyst for the green, efficient, and large-scale production of high optical purity L-2-aminobutyric acid. It not only simplifies the preparation process of chiral amino acids but also plays a significant role in reducing the production cost of key intermediates for downstream chiral drugs (such as certain antiepileptic and antituberculosis drugs) and improving product quality, demonstrating promising prospects for industrial application. Attached Figure Description

[0022] Figure 1 The pH and stability of the leucine dehydrogenase mutant Y282L compared to the wild type are shown in the figure.

[0023] Figure 2 The peak diagrams for L-threonine and L-2-aminobutyric acid standards are shown in the liquid chromatography peak diagram.

[0024] Figure 3 Liquid chromatography peak diagram of the whole-cell catalysis of L-threonine to L-2-aminobutyric acid by the leucine dehydrogenase mutant Y282L. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Key sequences involved in the embodiments:

[0027] The nucleic acid sequence of leucine dehydrogenase is shown in SEQ ID NO.1:

[0028]

[0029] The amino acid sequence of leucine dehydrogenase is shown in SEQ ID NO.2:

[0030] MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGTRMWTYDSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGV GNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR

[0031] The nucleic acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO.3:

[0032]

[0033] The amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO.4:

[0034] MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGTRMWTYDSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGV GNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSANNQLKEDRHGDIIHEMGIVLAPDYVINAGGVINVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR

[0035] Example 1: Construction of engineered strains containing leucine dehydrogenase and its mutant strains

[0036] BamHI and HindIII restriction enzyme sites were added to both ends of the leucine dehydrogenase coding gene to synthesize the whole gene (codon optimization was performed using *E. coli* as the host as needed). The plasmid containing the leucine dehydrogenase gene and the pETDuet-1 plasmid were digested with restriction endonucleases BamHI and HindIII, respectively. The digestion system consisted of 30 μL of leucine dehydrogenase gene plasmid or pETDuet-1 plasmid, 1 μL of BamHI, 1 μL of HindIII, and 5 μL of 10× buffer, with the volume brought to 50 μL with ultrapure water. The mixture was incubated at 37°C for 3 h. Ligation was performed using T4 ligase. The ligation system consisted of 38 ng of the leucine dehydrogenase gene fragment, 50 ng of the pETDuet-1 plasmid, 2 μL of 10×T4 buffer, and 1 μL of T4 ligase, with the volume brought to 20 μL with ultrapure water. Ligation was performed overnight at 16°C.

[0037] The ligation product was mixed with E. coli BL21(DE3) competent cells on ice, heat-shocked at 42°C for 90 seconds, and then immediately placed on ice for 2 minutes. 600 μL of LB medium was added, and the cells were reviveed on a shaker at 37°C and 200 rpm for 40 minutes. The cells were then spread on LB plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. The resulting transformants were the engineered bacterial strains containing leucine dehydrogenase.

[0038] PCR was performed using TransFast® Taq DNA Polymerase (3 U, TransGen Biotech, China), a dNTP mixture (0.20 mM), each primer (0.2 μM), and a plasmid containing leucine dehydrogenase (10 ng). Primers F: ATGGGTATCGTTCTCGCTCCAGACTAT, as shown in SEQ ID NO.5; R: TCACATAGTCTGGAGCGAGAACGATAC, as shown in SEQ ID NO.6. The PCR program was (95°C 2 min, 1 cycle; 95°C 20 s / 56°C 20 s / 72°C 1.5 min, 30 cycles; 72°C 5 min, 1 cycle). After PCR, QuickCut™ Dpn I (1 μL; Takara, Japan) was added, and the mixture was incubated at 37°C for 15 min. The mixture was then transformed into Escherichia coli BL21 competent cells, and the transformants were engineered bacterial strains containing leucine dehydrogenase mutants.

[0039] The leucine dehydrogenases and their mutants involved in this embodiment are detailed in Table 1.

[0040] Table 1. Leucine dehydrogenases and their mutants

[0041]

[0042] Example 2: Expression of leucine dehydrogenase mutant Y282L and preparation of crude enzyme, crude enzyme lyophilized powder, and pure enzyme.

[0043] The recombinant expression vector of the leucine dehydrogenase mutant Y282L constructed in Example 1 was transformed into *E. coli* BL21(DE3) competent cells and cultured at 37°C for 12–16 h. After transformants emerged, a single transformant was inoculated into 5 mL of LB medium containing ampicillin (100 μg / mL) and cultured overnight at 37°C and 200 rpm for 12–16 h. Then, it was inoculated into LB medium at a ratio of 1% (volume percentage) and cultured at 37°C and 200 rpm until OD (dose retardation). 600The pH value was approximately 0.6-0.8. IPTG was added to a final concentration of 0.4 mM, and the cells were induced and cultured at 28°C for 14 h. The cells were then collected by centrifugation at 12000 rpm for 2 min at 4°C. The collected cells were whole cells. The cells were resuspended once in 50 mM NH4Cl-NH3•H2O buffer (pH 7.0) and washed. The cells were then sonicated to lyse the cells, centrifuged at 12000 rpm for 2 min, and the supernatant was the crude enzyme solution. The crude enzyme solution was freeze-dried to obtain a lyophilized crude enzyme powder. The crude enzyme solution was purified using a nickel affinity chromatography column to obtain a pure enzyme solution.

[0044] Example 3: Determination of enzyme activity and enzyme kinetic constant of leucine dehydrogenase mutant Y282L

[0045] The purified enzyme solution of the leucine dehydrogenase mutant Y282L obtained in Example 2 was added to the enzyme activity assay system, and the enzyme activity was measured. The assay system consisted of 10 mM 2-ketobutyrate, 0.5 mM NADH, and 0.9 mol / L NH4Cl-NH3•H2O buffer (pH 9.0). The enzyme activity was determined by detecting the change in absorbance of NADH at 340 nm during the reaction. Enzyme activity was defined as the amount of enzyme required to catalyze 1 mmol of NADH per minute, which was defined as 1 unit of enzyme activity (U).

[0046] The enzyme kinetic constant determination system consisted of 50 mM NH4Cl-NH3•H2O buffer (pH 7), 0.5 mM NADH, and 2-ketobutyric acid at concentrations ranging from 0.1 to 50 mM. The corresponding Km and kcat values ​​were obtained using the Michaelis-Menten kinetic equation.

[0047] Table 2. Enzyme activity and kinetic constants of leucine dehydrogenase and its mutant Y282L

[0048]

[0049] Example 4: Determination of optimal temperature, pH and stability of leucine dehydrogenase mutant Y282L

[0050] The purified enzyme solution obtained in Example 2 was placed at 20-60℃ and pH 5-9, and its optimal temperature and pH were determined according to the enzyme activity assay method described in Example 3. Under the optimal temperature and pH conditions, the purified enzyme solution was placed for 0-10 hours to determine its temperature and pH stability. The optimal temperature for the leucine dehydrogenase mutant Y282L was 20℃, and its enzyme activity reached more than 85% of the initial enzyme activity after 10 hours at 50℃. The optimal pH for the leucine dehydrogenase mutant Y282L was 7.0, and its enzyme activity reached more than 80% of the initial enzyme activity after 10 hours in a pH 8 buffer solution.

[0051] Example 5: Preparation of L-2-aminobutyric acid from whole cells using the leucine dehydrogenase mutant Y282L

[0052] The reaction system was prepared by sequentially adding 50 mM phosphate buffer (pH 7.0), L-threonine (final concentration 59 g / L), ammonium formate (final concentration 32 g / L), oxidized coenzyme I (NAD+) (final concentration 5 mM), whole-cell L-threonine deaminase (final concentration 70 g / L), whole-cell leucine dehydrogenase mutant Y282L (final concentration 70 g / L), and whole-cell formate dehydrogenase (final concentration 140 g / L). The reaction system was then stirred in a 5 L fermenter at 35°C for 24 h.

[0053] After the reaction, centrifuge at 12000 rpm for 5 min, take 600 μL of the supernatant sample or standard, add 300 μL of OPA derivatizing agent, mix well, let stand for 5 min, filter through a 0.22 μm microporous membrane, and then inject the sample. The liquid chromatography detection conditions were: mobile phase A: 8 g / L sodium acetate (+1% tetrahydrofuran), pH 7.2; mobile phase B: acetonitrile. Flow rate: 0.8 mL / min; column: ZORBAX SB-C18 column; column temperature: 40 ℃; detection wavelength: 338 nm.

[0054] As demonstrated by the above embodiments, this invention successfully provides a performance-improved leucine dehydrogenase mutant Y282L and its efficient preparation and application methods. The mutant can be successfully constructed, expressed, and prepared into various usable forms using mature genetic engineering techniques. Enzymatic characterization confirmed that the mutant exhibits significantly improved key catalytic performance and superior operational stability. Further catalytic application experiments showed that this mutant, particularly in whole-cell form, can effectively convert the substrate L-threonine into the target product L-2-aminobutyric acid (LABA), with a feasible catalytic process and a clearly defined product. In summary, the leucine dehydrogenase mutant Y282L possesses the potential to serve as a core biocatalyst for the industrial synthesis of L-2-aminobutyric acid.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A leucine dehydrogenase mutant, characterized in that, The leucine dehydrogenase mutant is derived from Bacillus cereus. Compared with the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the leucine dehydrogenase mutant includes the substitution of leucine at position 282 with tyrosine.

2. The leucine dehydrogenase mutant according to claim 1, characterized in that, The leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO.

4.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the leucine dehydrogenase mutant according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.

3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that, The recombinant vector is a bacterial plasmid, bacteriophage, yeast plasmid, or retroviral packaging plasmid.

7. A host cell, characterized in that, The host cell contains the nucleic acid molecule according to claim 3 or 4, or the recombinant vector according to claim 5 or 6.

8. The host cell according to claim 7, characterized in that, The host cell is a prokaryotic cell or a lower eukaryotic cell.

9. An enzyme system, characterized in that, The enzyme system comprises a leucine dehydrogenase mutant according to any one of claims 1 or 2.

10. A method for preparing L-2-aminobutyric acid, characterized in that, The method includes the step of catalyzing the production of L-threonine from L-2-aminobutyric acid in the presence of NAD or NADH using the leucine dehydrogenase mutant according to claim 1 or 2, or the enzyme system according to claim 9. The leucine dehydrogenase mutant is used in the form of crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme or whole cell. The catalytic reaction is carried out in a buffer solution with a pH of 6.0 to 7.5; The catalytic reaction is carried out at a temperature of 20°C to 50°C; The concentration of L-threonine in the method is from 0.1 mM to 500 mM.

Images