Leucine dehydrogenase mutant with capability of catalytically synthesizing D-amino acid as well as preparation method and application of leucine dehydrogenase mutant

Abstract

The invention provides a leucine dehydrogenase mutant with the capability of catalytically synthesizing D-amino acid as well as a preparation method and application of the leucine dehydrogenase mutant, and belongs to the technical field of protease. The invention relates to a method for changing chiral selectivity of leucine dehydrogenase, which comprises the following steps of: replacing corresponding active center amino acid sites in the leucine dehydrogenase with active center amino acid sites of diaminopimelate dehydrogenase and active center amino acid sites of mutants of the diaminopimelate dehydrogenase; the amino acid sequence of the obtained leucine dehydrogenase mutant is as shown in SEQ ID NO: 4. The leucine dehydrogenase mutant can catalyze various D-amino acids (D-tertiary leucine, D-leucine and D-phenylglycine), shows excellent D-amino acid enzyme catalytic activity, and provides a powerful guarantee and a solid foundation for application of the leucine dehydrogenase mutant in the field of chiral amino acid synthesis.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteases, and in particular relates to a leucine dehydrogenase mutant capable of catalyzing the synthesis of D-amino acids, and a preparation method and application thereof. Background Art

[0002] As a class of amino acids with key biological activities, D-amino acids have broad application prospects in many fields, especially in medicine. For example, D-phenylglycine is an important precursor for semisynthetic antibiotics and actinomycin antibiotics; D-tert-leucine can be used in the synthesis of antiviral, anti-tumor, and anti-inflammatory drugs; and D-phenylalanine is an essential chiral synthetic raw material for the antidiabetic drug nateglinide. From the perspective of atom economy and mild reaction conditions, asymmetric reductive amination of keto acids using amino acid dehydrogenases is one of the most ideal pathways for synthesizing D-amino acids.

[0003] Leucine dehydrogenase, a member of the amino acid dehydrogenase family, is a NAD+-dependent enzyme that naturally catalyzes the reversible deamination of L-leucine and some other aliphatic branched-chain amino acids to produce the corresponding α-keto acids. It exhibits advantages such as high selectivity, high conversion rates, and mild reaction conditions. However, leucine dehydrogenase lacks catalytic activity towards D-amino acids, and D-amino acids can even inhibit its activity. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a leucine dehydrogenase mutant, which is prepared by replacing the active center of diaminopimelate dehydrogenase with the active center of leucine dehydrogenase, so that the mutant has the ability to catalyze the synthesis of D-amino acids.

[0005] The present invention provides a leucine dehydrogenase mutant, the amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO: 4.

[0006] The present invention provides a nucleic acid encoding the leucine dehydrogenase mutant.

[0007] The present invention provides a gene-derived product comprising the nucleic acid.

[0008] Preferably, it comprises at least one of the following: a gene expression cassette, a recombinant vector and a recombinant strain.

[0009] The present invention provides a method for preparing the leucine dehydrogenase mutant, comprising the following steps:

[0010] constructing a recombinant vector containing a gene sequence encoding the leucine dehydrogenase mutant;

[0011] introducing the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant into a host bacterium to obtain a recombinant strain;

[0012] The recombinant strain is induced to express and the recombinant protein is separated and purified, and the obtained recombinant protein is a leucine dehydrogenase mutant.

[0013] Preferably, the backbone vector in the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant includes pET32a; and the cloning site of the gene sequence encoding the leucine dehydrogenase mutant includes XhoⅠ and BamHI.

[0014] Preferably, the host bacteria of the recombinant strain includes a prokaryotic expression system.

[0015] The present invention provides the use of the leucine dehydrogenase mutant, the leucine dehydrogenase mutant prepared from the nucleic acid or the gene derivative product, or the leucine dehydrogenase mutant prepared by the preparation method in catalyzing the synthesis of D-amino acids.

[0016] Preferably, the D-amino acid comprises at least one of the following: D-tert-leucine, D-leucine and D-phenylglycine.

[0017] The present invention provides a method for changing the chiral selectivity of leucine dehydrogenase, comprising the following steps:

[0018] The active center amino acid sites R35, R71, G228, N308 of diaminopimelate dehydrogenase StDAPDH and the active center amino acid sites L151, G155, I170, M196, N245 of the diaminopimelate dehydrogenase mutant CgDAPDH are used to replace the corresponding active center amino acid sites in leucine dehydrogenase PfLeuDH to obtain the leucine dehydrogenase mutant.

[0019] The present invention provides a leucine dehydrogenase mutant, the amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO: 4. The present invention replaces the corresponding amino acid site of the leucine dehydrogenase active center with a specific amino acid site in the active center of diaminopimelate dehydrogenase StDAPDH and the active center of the mutant CgDAPDH of diaminopimelate dehydrogenase, thereby obtaining a leucine dehydrogenase mutant with the ability to catalyze the synthesis of D-amino acids. The present invention utilizes recombinantly expressed leucine dehydrogenase mutants to perform high pressure liquid chromatography (HPLC) detection on the product of the enzyme-catalyzed reaction, and the results show that the chiral selectivity of the leucine dehydrogenase mutant changes, and it can catalyze a variety of D-amino acids (D-tert-leucine, D-leucine and D-phenylglycine). The leucine dehydrogenase provided by the present invention exhibits excellent D-amino acid enzyme catalytic activity, providing a strong guarantee and a solid foundation for its application in the field of chiral amino acid synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the sequence alignment result of meso-diaminopimelate (a type of amino acid dehydrogenase) and leucine dehydrogenase (from Planifilum fimeticola) from different sources using MEGA11 and GeneDoc software;

[0021] Figure 2 The figure shows the SDS-PAGE analysis results of the expression of leucine dehydrogenase PfLeuDH and its mutant LeuDH-PL9 proteins, where M: Marker; 1: pET-28(a); 2: pET-32(a); 3: PfLeuDH crude enzyme solution; 4: PfLeuDH pure enzyme solution; 5: LeuDH-PL9 crude enzyme solution; 6: LeuDH-PL9 pure enzyme solution (the molecular weights of the target proteins are as follows: PfLeuDH: 40.30 kDa; LeuDH-PL9: 59.1 kDa);

[0022] Figure 3 Schematic diagram comparing the yield of D-amino acids catalyzed by the original leucine dehydrogenase WT (PfLeuDH) and the leucine dehydrogenase mutant LeuDH-PL9. DETAILED DESCRIPTION

[0023] The present invention provides a method for changing the chiral selectivity of leucine dehydrogenase, comprising the following steps:

[0024] The active center amino acid sites R35, R71, G228, N308 of diaminopimelate dehydrogenase StDAPDH and the active center amino acid sites L151, G155, I170, M196, N245 of the diaminopimelate dehydrogenase mutant CgDAPDH were used to replace the corresponding active center amino acid sites in leucine dehydrogenase PfLeuDH to obtain a leucine dehydrogenase mutant.

[0025] In the present invention, the leucine dehydrogenase PfLeuDH is derived from thermophilic bacteria (Planifilumfimeticola), and the amino acid sequence is as shown in SEQ ID NO: 1 (MKWFDYMERYDYEQLLLCQDKNSG LKAIIAIHDTTLGPALGG V RMWNYASEEEAIIDALRLARGMTYKAAAAGLNLGGGK T VIIGDPRKDKNEAMFRALGRFIQGLNGRYITAEDVGTTEEDMDIIHQETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKIAFGSDSL E GRT V AVQGVGSVAYHLCKHLHEEGARLIVTDIVRENVERAVRDFGAESVDPDKIYDVECDIFSPCALGAIINDETLPRLKCRVVAGSANNQLKEERHGDRLEELGIIY V PDYVINAGGLINVADELLGYNRERAM KKVETIYDNVLKVFEIAKRDGIPSYKAADRMAEERIQSMARSRSTFLQNERHVLNMV). The diaminopimelate dehydrogenase StDAPDH is derived from Symbiobacterium thermophilum, and its amino acid sequence is shown in SEQ ID NO: 2 (MDKLRVAVVGYGNVGRYALEAVQAAPDMELVGVVRRKVLAATPPELTGVRVVTDISQLEGVQGALLCVPTRSVPEYAEAMLRRGIHTVDSYDIHGDLADLRRRLDPVAREHGAAAVISAGWDPGTDSIIRALLEFMAPKGITYTNFGPGMSMGHSVAVKAIPGVRDALSMTIPAGMGVHKRAVYVELEPGADFAEVERAIKTDPYFVRDETRVTQVESVSALMDVGHGVVMERKGVSGATHNQLFRFEMRINNPALTAQVMVAALRAAARQKPGCYTMIEIPVIDYLPGDREAWIRKLV). The mutant of diaminopimelate dehydrogenase, CgDAPDH, is derived from a mutant of Corynebacterium glutamicum, and its amino acid sequence is shown in SEQ ID NO: 3 (MTNIRVAIVGYGNLGRSVEKLIAKQPDMDLVGIFSRRATLDTKTPVFDVAD VDKHADDVDVLFLCMGSATDIPEQAPKFAQFACTVDTYDNHRDIPRHRQVMNEAATAAGNVALVSTGWDPGMFSINRVYAAAVLAEHQQHTFWGPGLSQLHSDGLRRIPGVQKAVQQYILPSEDALEKARRGEAGLTGKQTHKRMCFVVADAADHERIENDIRTMPDYFVGAYEVEVNFIDEATFDSEHTGMPNGGHVITTGDTGGFNHTVEYILKLDRNPDFTASQIAFGRAAHRMKQQGQSGAFTVLEVAPYLLSPENLDDLIARDV).

[0026] The present invention provides a leucine dehydrogenase mutant, wherein the amino acid sequence of the leucine dehydrogenase mutant is as shown in SEQ ID NO: 4 (MKWFDYMERYDYEQLLLCQDKNSGLKAIIAIHDTTL GPALGG RRMWNYASEEEAIIDALRLARGMTYKAAAAGLNLGGGK R VIIGDPRKDKNEAMFRALGRFIQGLNGRYITAEDVGTTEEDMDIIHQETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKIAFGSDSL L GRT G AVQGVGSVAYHLCK I LHEEGARLIVTDIVRENVERAVRDFGAESVDPDKIYMVECDIFSPCALGAIINDETLPRLKCRVVAGSANNQLKEERHGDRLEELGIIY N PGYVINAGGLINVADELLGYNRERAM N KVETIYDNVLKVFEIAKRDGIPSYKAADRMAEERIQSMARSRSTFLQNERHVLNMV).

[0027] The present invention provides a nucleic acid encoding the leucine dehydrogenase mutant.

[0028] In the present invention, the nucleic acid preferably includes a codon-optimized nucleic acid. In this embodiment, in order to improve the recombinant expression level of the leucine dehydrogenase mutant, the nucleic acid is optimized according to the codon preference of the host bacteria to obtain a codon-optimized nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0029] The present invention provides a gene-derived product comprising the nucleic acid.

[0030] In the present invention, the gene-derived product preferably comprises at least one of the following: a gene expression cassette, a recombinant vector, and a recombinant strain. The gene expression cassette is an independently expressible DNA fragment that contains all or part of the elements required for gene expression, for example, including, in addition to the target nucleic acid, genetic elements such as a promoter and enhancer.

[0031] In the present invention, the recombinant vector is a recombinant plasmid comprising the nucleic acid. The present invention does not impose any particular limitation on the type of the backbone vector of the recombinant plasmid, and any plasmid well known in the art can be used, such as a prokaryotic expression vector and / or a eukaryotic expression vector, selected according to the expression system.

[0032] In the present invention, the recombinant strain is an engineered strain containing the nucleic acid. The present invention does not impose any particular restrictions on the type of host bacteria for the engineered strain; any host bacteria known in the art may be used, such as a prokaryotic expression system and / or a eukaryotic expression system. The prokaryotic expression system includes Escherichia coli (E. coli) or Bacillus. The eukaryotic expression system preferably includes yeast.

[0033] The present invention provides a method for preparing the leucine dehydrogenase mutant, comprising the following steps:

[0034] constructing a recombinant vector containing a gene sequence encoding the leucine dehydrogenase mutant;

[0035] introducing the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant into a host bacterium to obtain a recombinant strain;

[0036] The recombinant strain is induced to express and the recombinant protein is separated and purified, and the obtained recombinant protein is a leucine dehydrogenase mutant.

[0037] The present invention has no particular limitation on the method for constructing a recombinant vector containing the gene sequence encoding the leucine dehydrogenase mutant, and both homologous recombination methods and gene synthesis methods well known in the art can be used.

[0038] In an embodiment of the present invention, the backbone vector in the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant preferably includes pET32a or pET28a; the cloning site of the gene sequence encoding the leucine dehydrogenase mutant preferably includes XhoⅠ and BamHI.

[0039] In the present invention, the method for introducing the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant into the host bacteria preferably includes a freeze-thaw method. The present invention has no particular limitation on the preparation method of the freeze-thaw method, and the freeze-thaw method well known in the art can be used.

[0040] In the embodiment of the present invention, the host bacteria of the recombinant strain preferably includes a prokaryotic expression system, specifically a competent Escherichia coli BL21.

[0041] The method of inducing expression of the recombinant strain in the present invention is preferably to determine the OD of the recombinant strain. 600When the pH value is 0.6 to 0.8, TIPT inducer is added to 0.3 mM and then induced culture is carried out. The temperature of the induction culture is preferably 15 to 19°C, and can be 17°C. The rotation speed of the induction culture is preferably 180 to 220 rpm, and can be 200 rpm. The time of the induction culture is 10 to 14 h, and can be 12 h. The method for separating and purifying the recombinant protein is preferably to separate the bacteria from the induced bacterial liquid, crush it, collect the contents and perform nickel column chromatography separation and purification. The present invention has no special restrictions on the nickel column chromatography separation and purification method, and the nickel column chromatography separation and purification method well known in the art can be used.

[0042] The present invention provides the use of the leucine dehydrogenase mutant, the leucine dehydrogenase mutant prepared from the nucleic acid or the gene derivative product, or the leucine dehydrogenase mutant prepared by the preparation method in catalyzing the synthesis of D-amino acids.

[0043] In the present invention, the D-amino acid preferably comprises at least one of the following: D-tert-leucine, D-leucine, and D-phenylglycine. The substrate for catalytic synthesis of the D-amino acid preferably comprises a ketoacid. The ketoacid is selected based on the type of D-amino acid being synthesized. Trimethylpyruvate is used as a substrate to catalytically produce D-tert-leucine, 4-methyl-2-oxopentanoic acid is used to catalytically produce D-leucine, and phenylglyoxylic acid is used to catalytically produce D-phenylglycine.

[0044] In the present invention, the system for catalytic synthesis of D-amino acids preferably also includes glucose dehydrogenase. The temperature for catalytic synthesis of D-amino acids is preferably 38 to 42° C., and may be 40° C. The pH value of the system for catalytic synthesis of D-amino acids is preferably 9.2 to 9.7, and may be 9.5.

[0045] In the present invention, the system for catalytic synthesis of D-amino acids comprises the following components according to 1 mL: 100 μL of leucine dehydrogenase mutant enzyme solution, 100 μL of glucose dehydrogenase enzyme solution, 10 mM keto acid, 10 mM NAD + 1.5mM, 24mM glucose, and ammonium chloride to adjust the pH value of the system to 9.2-9.7. The concentration of the glucose dehydrogenase solution is preferably 1-10mg / L, 3-8mg / L, or 5mg / L.

[0046] In the present invention, the D-amino acid content in the catalytic product was detected by high performance liquid chromatography. The results showed that leucine dehydrogenase (LeuDH-PL9) was able to produce D-amino acids, among which D-phenylglycine had the highest yield, followed by D-leucine, and D-tert-leucine was relatively low.

[0047] The following describes in detail a leucine dehydrogenase mutant capable of catalyzing the synthesis of D-amino acids, a preparation method thereof, and applications thereof, in conjunction with the examples provided by the present invention. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Cloning, expression and purification of leucine dehydrogenase mutants

[0050] Step 1: Perform full gene synthesis on the gene sequences encoding the two leucine dehydrogenases in the amino acid sequences (SEQ ID NO: 1, SEQ ID NO: 4) to obtain the PfLeuDH and LeuDH-PL9 gene sequence fragments, and further insert the PfLeuDH and LeuDH-PL9 gene fragments into plasmids (pET28a) and plasmids (pET32a) by double enzyme digestion (Xho I / Bam HI), respectively, to obtain recombinant plasmids pET28a-PfLeuDH and pET32a-LeuDH-PL9;

[0051] Step 2: Transform the above-constructed recombinant plasmids into host E. coli BL21 (DE3) and E. coli BL21 (DE3) pLysS competent cells respectively, using the heat shock method. The specific operation process is as follows:

[0052] (1) Take the competent cells out of the -80°C freezer and quickly place them in an ice box to wait for them to thaw;

[0053] (2) Carefully add the constructed recombinant plasmid to the competent cells and mix gently. Place on ice and let stand for 30 minutes.

[0054] (3) Heat shock the cells in a water bath preheated to 42°C for 45 seconds. After 45 seconds, place the cells back on ice and allow to stand for 2 minutes (do not shake the cells during this process). Add 700 μL of antibiotic-free sterile LB liquid medium to the cells in a clean bench and mix thoroughly.

[0055] (4) Culture under shaking conditions at 37°C and 200 rpm for 1 hour, draw an appropriate volume and evenly spread it on LB solid medium containing the corresponding antibiotic until the liquid is fully absorbed, invert and place it in a 37°C incubator for overnight culture to obtain the recombinant strain.

[0056] Step 3: Prokaryotic expression and purification

[0057] The above recombinant strain was further expanded and induced to express. The specific operation process is as follows: After successfully obtaining the recombinant strain, an appropriate amount of bacterial liquid was measured and added to 200 mL of LB liquid medium to expand the culture. The culture was continued until the bacterial liquid OD 600After reaching 0.6-0.8, IPTG inducer (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.3 mM and cultured at 17°C and 200 rpm for 12 hours. The induced cells were collected by centrifugation and ultrasonically disrupted to obtain the corresponding crude enzyme solution.

[0058] The process of further purification to obtain pure enzyme solution, given that the leucine dehydrogenase plasmid constructed in this experiment carries a 6×His tag, is purified using a nickel column Ni-NTA His Bind Resin purification column and corresponding supporting reagents. The specific steps are as follows:

[0059] A. Set up the purification apparatus and then equilibrate the column with a volume of loading buffer 10 times the volume of the column.

[0060] B. Filter the crude enzyme solution through a 0.45 μm filter membrane and load the filtered solution onto the equilibrated purification column. Any residual enzyme solution on the column wall should be rinsed with approximately 10 column volumes of loading buffer to ensure cleanliness and accurate loading.

[0061] C. Use 10 to 20 column volumes of wash buffer to wash away the impurities. If the amount of impurities is too high, increase the volume of wash buffer to more fully remove the impurities and improve the purity of the target protein.

[0062] D. After washing, use 4 to 10 column volumes of elution buffer to elute the target protein. It should be noted that the collection process can only begin after one column volume of liquid has flowed out, so as to ensure that the target protein collected is relatively pure and meets the requirements;

[0063] E. After collection, wash the purification column with 5 column volumes of 8M urea, then approximately 20 column volumes of distilled water. Finally, add approximately one column volume of 20% ethanol to seal the upper and lower ends of the purification column. Store in a refrigerator at 4°C.

[0064] F. Since the pure enzyme solution obtained through the above steps contains a high concentration of imidazole, it is necessary to use Merck Millipore ultrafiltration tubes (cut-off capacity 10kDa) for desalination. The specific steps are as follows:

[0065] (S1) Adding pure enzyme solution containing high concentration of imidazole to the inner tube of the ultrafiltration tube, centrifuging at 8000 rpm at 4°C for 30 minutes, and discarding the liquid in the outer tube after centrifugation;

[0066] (S2) Add an appropriate amount of 20 mM PBS buffer to the inner cannula, centrifuge at 8000 rpm for 30 minutes at 4°C, and discard the liquid in the outer cannula;

[0067] (S3) Repeat the corresponding operation twice according to the operation flow of the above step (S2);

[0068] (S4) Add an appropriate amount of 20 mM PBS buffer to the inner cannula again, and then use a rubber-tipped dropper to collect all the liquid in the inner cannula. The collected liquid is the pure enzyme solution after desalination, and it is stored at 4°C for subsequent use;

[0069] (S5) Clean the ultrafiltration tube with 0.2 M sodium hydroxide solution and deionized water. After cleaning, add deionized water to the outer tube and add 0.2 M sodium hydroxide solution to the inner tube, and store at 4°C.

[0070] The molecular weights of the target proteins were verified by SDS-PAGE as follows: PfLeuDH: 40.30 kDa; LeuDH-PL9: 59.1 kDa. Figure 2 As shown, the enzyme bands prepared and purified by the technical solution of the present application are clear and of high purity.

[0071] Example 2

[0072] catalytic reaction

[0073] D-tert-leucine, D-leucine and D-phenylglycine were produced at 40°C and pH 9.5 using the catalytic system shown in Table 1.

[0074] Table 1 Dual enzyme coupled catalytic system

[0075]

[0076] Note: Glucose dehydrogenase was purchased from Sigma, CAS#: 9028-53-9.

[0077] A chiral analytical column, Chiralpak MA(+) (4.6 mm × 50 mm, 3 μm), an Agilent 1200 high-performance chromatographic column, was used. The analysis method was as follows: a 2 mM copper sulfate solution containing 5% by volume acetonitrile was used as the mobile phase; the detection wavelength was the maximum absorption wavelength corresponding to the target product; the flow rate was 0.5 mL / min, and the column temperature was 30°C.

[0078] Table 2 Standard curve of target product

[0079] product Reaction time / min Standard curve (y: peak area; x: concentration in mM) D-tert-Leucine 7.3 y=3370.1099x+2.9104 D-phenylglycine 10.2 y=2994.4172x-2.2888 D-Leucine 8.6 y=2781.2874x+1.3520

[0080] The yield of target product is Figure 3 As shown, the highest yield of the following three products is taken as 100%. It can be seen that the leucine dehydrogenase (LeuDH-PL9) in the present application can be used to prepare D-amino acids, among which D-phenylglycine has the highest yield.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A leucine dehydrogenase mutant, characterized in that: The amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO:

4.

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

3. A gene-derived product comprising the nucleic acid of claim 2.

4. The gene-derived product according to claim 3, characterized in that: The invention comprises at least one of the following gene expression cassette, recombinant vector and recombinant strain.

5. A method for preparing the leucine dehydrogenase mutant according to claim 1, characterized in that: The following steps are involved: Constructing a recombinant vector containing the gene sequence encoding the leucine dehydrogenase mutant according to claim 1; introducing the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant into a host bacterium to obtain a recombinant strain; The recombinant strain is induced to express and the recombinant protein is separated and purified, and the obtained recombinant protein is a leucine dehydrogenase mutant.

6. The preparation method according to claim 5, characterized in that: The backbone vector in the recombinant vector encoding the gene sequence of the leucine dehydrogenase mutant includes pET32a; the cloning site of the gene sequence encoding the leucine dehydrogenase mutant includes XhoⅠ and BamHI.

7. The preparation method according to claim 5, characterized in that: The host bacteria of the recombinant strain include a prokaryotic expression system.

8. Use of the leucine dehydrogenase mutant according to claim 1, a leucine dehydrogenase mutant prepared from the nucleic acid according to claim 2 or the gene derivative according to claim 3 or 4, or a leucine dehydrogenase mutant prepared by the preparation method according to any one of claims 5 to 7 in catalyzing the synthesis of D-amino acids.

9. The application according to claim 8, characterized in that: The D-amino acid includes at least one of the following: D-tert-leucine, D-leucine and D-phenylglycine.

10. A method for changing the chiral selectivity of leucine dehydrogenase, characterized in that: The following steps are involved: The active center amino acid sites R35, R71, G228, N308 of diaminopimelate dehydrogenase StDAPDH and the active center amino acid sites L151, G155, I170, M196, N245 of the diaminopimelate dehydrogenase mutant CgDAPDH are used to replace the corresponding active center amino acid sites in leucine dehydrogenase PfLeuDH to obtain the leucine dehydrogenase mutant according to claim 1.

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