Application of R-transaminase and mutant thereof in asymmetric synthesis of chiral amine compounds

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to application of R-transaminase and a mutant thereof in asymmetric synthesis of chiral amine compounds. The amino acid sequence of the R-transaminase is as shown in SEQ ID NO. 1. The amino acid sequences of the R-transaminase mutants are respectively shown as SEQ ID NO: 2 (G66L), 3 (F127A), 4 (F127M), 5 (G66L / F127M), 6 (G66L / F127A), 7 (G66L / F127M / L160I) and 8 (G66L / H67N / F127M / L160I). The recombinant transaminase mutant obtained by the invention has the advantages of high catalytic activity, strict stereoselectivity, mild reaction conditions and the like for various cyclic ketone and N-heterocyclic ketone compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and specifically relates to application of an R-aminotransferase and a mutant thereof in asymmetric synthesis of chiral amine compounds. Background Art

[0002] Chiral N-heterocyclic amines are key intermediates for the preparation of various active pharmaceutical ingredients and have important application value in the pharmaceutical industry. For example, (R)-3-amino-N-Boc-pyrrole (1b), (R)-3-amino-N-Boc-piperidine (2b) and their N-Cbz and N-Bn substituted analogs are key chiral intermediates for the synthesis of the fifth-generation cephalosporin ceftobiprole, the quinolone antibacterial drug toloxacin, and the dipeptide kinase IV inhibitor drugs dulagliptin, alogliptin, linagliptin and trelagliptin.

[0003]

[0004] At present, there are many chemical and biological methods for synthesizing chiral N-heterocyclic amines, such as Hofmann rearrangement of chiral amide intermediates, kinetic resolution of racemic amines, and asymmetric synthesis of prochiral ketones or enamines (CN115557882A, CN107445887A, CN103865964A, Advanced Synthesis & Catalysis 2008, 350 (6), 807-812). Among them, transaminase-catalyzed asymmetric reductive amination of prochiral ketones has become an advantageous choice for synthesizing chiral N-heterocyclic amines due to its high atom economy and strict stereoselectivity.

[0005] Transaminase is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the transfer of amino groups from amine donors to prochiral ketones to generate corresponding chiral amines. However, wild-type transaminases are generally limited to narrow substrate spectra and inefficient conversion capabilities. So far, several transaminases have been able to be used to synthesize 1b and 2b, including Vfl-TA, Ade-TA, CbTA, RbTA, and commercial immobilized enzyme ATA-025-IMB (Advanced Synthesis & Catalysis 2008, 350 (6), 807-812, ACS Catalysis 2023, 13, 422-432, ACS Omega 2021, 6 (26), 17058-17070, Beilstein Journal of Organic Chemistry 2019, 15, 60-66), but they are faced with theoretical yield, low catalytic efficiency, and serious substrate inhibition. Therefore, developing more efficient enzyme catalysts to synthesize 1b and 2b to reduce reaction costs is of great significance for improving industrial applications. Summary of the invention

[0006] The present invention aims to solve the problems of low catalytic efficiency and severe substrate inhibition in the synthesis of N-heterocyclic amine compounds by existing transaminase, and to obtain excellent transaminase mutants for catalyzing the synthesis of chiral N-heterocyclic amine active drug intermediates through enzyme engineering strategies.

[0007] To achieve the above purpose, the present invention adopts the following technical solution:

[0008] The present invention provides R-aminotransferase, the amino acid sequence of which is shown as SEQ ID NO.1.

[0009] The present invention also provides an R-aminotransferase mutant, whose amino acid sequence is obtained by mutating one or more positions of the 66th, 67th, 127th, and 160th positions of the amino acid sequence of R-aminotransferase SEQ ID NO.1; the amino acid sequences of the mutants are respectively as shown in SEQ ID NO: 2 (G66L), 3 (F127A), 4 (F127M), 5 (G66L / F127M), 6 (G66L / F127A), 7 (G66L / F127M / L160I),

[0010] 8(G66L / H67N / F127M / L160I).

[0011] SEQ ID NO.1:

[0012] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFGHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESFVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAE QIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPPLIEAIEY

[0013] SEQ ID NO:2:

[0014] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFLHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESFVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0015] SEQ ID NO:3:

[0016] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFGHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESAVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0017] SEQ ID NO:4:

[0018] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFGHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESMVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0019] SEQ ID NO:5:

[0020] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFLHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESMVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0021] SEQ ID NO:6:

[0022] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFLHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESAVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYLWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0023] SEQ ID NO:7:

[0024] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFLHSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESMVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYIWAFPPAEQIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPLIEAIEY

[0025] SEQ ID NO:8:

[0026] MSTGTSNLVAVEPGAIREDTPPGSVIQYSDYELDHSSPFAGGVAWIEGEFLPAEDAKISIFDTGFLNSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDAGYTKDELADITKQCVSMSQLRESMVNLTVTRGYGKRRGEKDLSKLTHQVYIYAIPYIWAFPPAE QIFGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVCIVKDGKLASPSRNALPGITRKTVFEIADQMGIEATLRDVTSHELYDADELMAVTTAGGVTPINSLDGEAIGNGAPGPMTVAIRDRFWALMDEPGPPLIEAIEY

[0027] The present invention also provides the use of the aforementioned R-aminotransferase and R-aminotransferase mutant in asymmetric synthesis of chiral amine compounds.

[0028] In the above technical solution, further, the R-aminotransferase and the R-aminotransferase mutant are used as biocatalysts to catalyze the transamination reaction of cyclic ketones or N-heterocyclic ketone compounds to prepare chiral amines.

[0029] In the above technical solution, further, the cyclic ketone or N-heterocyclic ketone compound is selected from any one of the following:

[0030]

[0031] In the above technical solution, further, the R-aminotransferase and R-aminotransferase mutants are in the form of free cells, free enzymes, immobilized cells and immobilized enzymes as biocatalysts.

[0032] In the above technical scheme, further, the method for synthesizing chiral amine compounds is: in a buffer solution, in the presence of an amine donor and a cofactor, using the aforementioned R-transaminase or R-transaminase mutant as a catalyst, asymmetric amination of cyclic ketones or N-heterocyclic ketone compounds to generate chiral amines, the reaction formula:

[0033]

[0034] In the above technical scheme, further, the concentration of cyclic ketone or N-heterocyclic ketone compound in the reaction system is 0.1-500mM; the final concentration of the amine donor is 1-2000mM; the final concentration of the cofactor is 0.1-5mM; the pH value of the reaction system is 7.0-10.0, and the temperature of the reaction system is 20-45°C; the concentration of the cosolvent is 0.1-30% (v / v) of the reaction system; and the concentration of the buffer is 0.05-0.2M.

[0035] In the above technical solution, further, the concentration of the cyclic ketone or N-heterocyclic ketone compound in the reaction system is 0.1-300mM; the final concentration of the amine donor is 1-100mM;

[0036] In the above technical scheme, further, the cofactor is pyridoxal phosphate (PLP); the amine donor is alanine, phenylethylamine or isopropylamine, preferably isopropylamine; the cosolvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and acetone, preferably methanol or dimethyl sulfoxide; the buffer is phosphate buffer or Tris-HCl buffer.

[0037] The present invention also provides the gene of the aforementioned R-aminotransferase, whose nucleotide sequence is shown in SEQ ID NO.9.

[0038] SEQ ID NO.9:

[0039]

[0040] The present invention transforms R-aminotransferase MnTA through enzyme engineering technology, and obtains excellent mutants that can efficiently synthesize active drug intermediates 1b and 2b, and further, the optimal mutations also significantly improve the activity of various cyclic ketones or N-heterocyclic ketones. The excellent mutants obtained by the present invention can realize efficient enzymatic synthesis of drug intermediates 1b and 2b. Compared with the existing enzymatic synthesis process, this method has high substrate loading and high space-time yield, can further reduce production costs, and has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The soluble protein expression was analyzed by SDS-PAGE.

[0042] Figure 2 Reaction time-conversion curves of compounds 1a and 2a.

[0043] Figure 3 Gas phase diagram of the conversion rate detection of substrate 1a catalyzed by MnTA mutants; (A) 1a reference, (B) (R)-1b reference, (C) MnTA M5 synthesis (R)-1b product detection.

[0044] Figure 4 Gas phase diagram of the conversion rate detection of substrate 2a catalyzed by MnTA mutants; (A) 2a reference, (B) (R)-2b reference, (C) MnTA M1 synthesis (R)-2b product detection.

[0045] Figure 5 Liquid phase diagram of ee value detection of substrate 1a to product (R)-1b catalyzed by MnTA mutant; (A) racemic 1b reference, (B) (R)-1b reference, (C) MnTAM5 synthesis (R)-1b product detection.

[0046] Figure 6 Liquid phase diagram of ee value detection of substrate 2a to product (R)-2b catalyzed by MnTA mutant; (A) racemic 2b reference, (B) (R)-2b reference, (C) MnTA M1 synthesis (R)-2b product detection.

[0047] Figure 7 Liquid phase diagram of ee value detection of substrate 3a to product (R)-3b catalyzed by MnTA mutant; (A) racemic 3b reference, (B) (R)-3b reference, (C) MnTAM5 synthesis of (R)-3b product detection.

[0048] Figure 8 Liquid phase diagram of ee value detection of substrate 8a to product (R)-8b catalyzed by MnTA mutant; (A) racemic 8b reference, (B) (R)-8b reference, (C) MnTA M1 synthesis (R)-8b product detection.

[0049] Fig. 9 Liquid phase diagram of ee value detection of substrate 10a to product (R)-10b catalyzed by MnTA mutant; (A) racemic 10b reference, (B) (R)-10b reference, (C) MnTA M5 synthesis (R)-10b product detection. DETAILED DESCRIPTION

[0050] The application of the R-aminotransferase and its mutants in the asymmetric synthesis of chiral amine compounds is further explained below in conjunction with the accompanying drawings and specific examples. Unless otherwise specified, the experimental schemes used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the present invention.

[0051] Example 1 Obtaining the transaminase MnTA gene

[0052] The gene sequence encoding MnTA (WP_030134134.1) was obtained from the NCBI database (https: / / www.ncb.nlm.nih.gov / genome / ). Primers at both ends were designed to obtain the gene fragment from the Mycobacterium neoaurum genome by PCR. The designed primers are shown in Table 1. The gene obtained by PCR was digested and connected with the vector plasmid pET-28a(+) to obtain the recombinant plasmid.

[0053] Example 2: Obtaining the transaminase MnTA mutant gene

[0054] The mutant genes were obtained by overlapping extension PCR technique. The mutant genes were obtained by overlapping extension PCR method using the recombinant pET-28a(+) plasmid containing the wild-type MnTA gene obtained above as a template. The primers for each mutant are shown in Table 1.

[0055] Table 1 Primer list involved

[0056]

[0057]

[0058] The PCR amplification system is shown in Table 2:

[0059] Table 2 PCR amplification system

[0060]

[0061]

[0062] Amplification program: 94°C: 10 min, (94°C: 30 s, 48°C: 30 s, 72°C: 90 s) 35 cycles, 72°C, 10 min.

[0063] Two fragments can be obtained through one round of PCR. After verifying the correctness of the band size by agarose gel electrophoresis, the two fragments are recovered with a common DNA product gel recovery kit as templates for the second round of PCR. The second round of PCR uses the two-end primers MnTA-Nde IF and MnTA-Xho IR to amplify the mutant gene according to the system described in Table 2. After the second round of PCR is completed, the product is recovered by a DNA purification kit to obtain a DNA product containing the mutation site.

[0064] Example 3: Construction of recombinant engineering plasmid

[0065] The purified DNA products and the vector pET-28a(+) were double-digested with NdeⅠ and XhoⅠ (Table 3), respectively, and incubated in a 37℃ incubator for 4-6h. The digested products were recovered by a DNA product gel recovery kit to obtain mutant DNA fragments and vector fragments with the same sticky ends. The digested target gene was connected to the vector plasmid using T4 DNA ligase to construct recombinant engineering plasmids of different mutants, and the recombinant engineering plasmids were transformed into competent Escherichia coli Rosetta2 (DE3), coated on LB agar plates containing Kana resistance, and cultured at 37℃ overnight.

[0066] Table 3 Enzyme digestion system

[0067]

[0068] Example 4: Transaminase protein expression

[0069] Different positive transformants verified on LB agar plates were inoculated into 4 mL of LB liquid medium containing Kana resistance, and cultured at 37°C, 200 rpm for 6-8 h to obtain seed solutions of different mutants. 1 mL of seed solutions of different mutants were inoculated into 100 mL of LB liquid medium containing Kana resistance, and cultured at 37°C, 200 rpm until the culture solution OD reached 0. 600 When the concentration reaches 0.8-1.0, add IPTG solution with a final concentration of 0.1 mM, and lower the temperature to 20-25°C to induce expression for 15-20 hours. Centrifuge at 3000 r / min for 10 minutes to collect the cells, wash with physiological saline, and centrifuge again to collect the cells to obtain the whole-cell biocatalyst.

[0070] Example 5: Gas chromatography method for measuring conversion rate

[0071] SCION 456-GC system, detection conditions: injection temperature 240℃, split ratio 20:1, FID detector temperature 240℃. Initial temperature 60℃, rising to 240℃ at a rate of 20℃ / min.

[0072] Table 4 Gas phase analysis conditions and retention times of substrates 1a-12a and their products 1b-12b

[0073]

[0074] Note: (0.25mm×30m,0.25μm film thickness; Restek); (0.32mm×30m,0.25μm film thickness; Restek).

[0075] Example 6: Liquid chromatography method for measuring product ee value

[0076] JASCO LC-1500 system, Daicel column, sample Boc derivatization, detection conditions: 210 nm, n-hexane:isopropanol=90:10; flow rate 0.7 mL / min.

[0077] Table 5 Chiral analysis conditions and retention time of products

[0078]

[0079] Example 7: Activity screening of transaminase MnTA mutants

[0080] Reaction:

[0081]

[0082] 1a reaction system: 80 mg / mL of wet cells of different mutants as described in Example 4, substrate 1a (50 mM), isopropylamine hydrochloride (250 mM), PLP (1 mM), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M).

[0083] 2a reaction system: 50 mg / mL of wet cells of different mutants as described in Example 4, substrate 2a (100 mM), isopropylamine hydrochloride (500 mM), PLP (1 mM), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M).

[0084] The reaction temperature was 25°C, and the wild type was used as a control. After 5 hours of reaction, the reaction conversion rate was measured by gas chromatography, and the results are shown in Table 6:

[0085] Table 6 Determination of the conversion rate of substrates 1a and 2a catalyzed by MnTA mutants

[0086]

[0087]

[0088] Example 8: Transaminase MnTA mutant catalyzes cyclic ketones and N-heterocyclic ketones

[0089] Reaction system: 80 mg / mL wet cells of different mutants, substrate (50 mM), isopropylamine hydrochloride (250 mM), PLP (1 mM), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M) as described in Example 4. (a indicates 150 mM substrate, 500 mM isopropylamine hydrochloride, 50 mg / mL wet cell catalyst; b indicates 5 mM substrate).

[0090] The reaction temperature was 25°C. After 5 hours of reaction, the reaction conversion rate was measured by gas chromatography. The results are shown in Table 7:

[0091] Table 7 Determination of the conversion rate of cyclic ketones and N-heterocyclic ketones catalyzed by transaminase MnTA mutants

[0092]

[0093] Note: M1 represents the MnTA F127M mutant, and M5 represents the MnTA G66L / H67N / F127M / L160I mutant.

[0094] Example 9. Synthesis of (R)-3-amino N-tert-butyloxycarbonyl-pyrrole (R-1b) catalyzed by transaminase MnTA mutant

[0095] The reaction system was sequentially added with substrate 1a (final concentration of 150 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (700 mM), mutant M5 wet cell catalyst (200 g / L), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). The reaction was carried out on a constant temperature shaker, the temperature was set at 25° C., the speed was 200 rpm, and the reaction time was 5 hours. After the reaction was completed, the reaction conversion rate was greater than 95%, and the product ee value was greater than 99%.

[0096] Example 10. Synthesis of (R)-3-amino N-tert-butyloxycarbonyl-piperidine (R-2b) catalyzed by transaminase MnTA mutant

[0097] The reaction system was sequentially added with substrate 2a (final concentration of 250 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (700 mM), mutant M1 wet cell catalyst (200 g / L), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). The reaction was carried out on a constant temperature shaker, the temperature was set at 25° C., the speed was 200 rpm, and the reaction time was 5 hours. After the reaction was completed, the reaction conversion rate was greater than 99%, and the product ee value was greater than 99%.

[0098] Example 11. Synthesis of (R)-3-amino N-benzyloxycarbonyl-pyrrole (R-3b) catalyzed by transaminase MnTA mutant

[0099] The reaction system was sequentially added with substrate 3a (final concentration of 50 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (250 mM), mutant M5 wet cell catalyst (100 g / L), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). The reaction was carried out on a constant temperature shaker, the temperature was set at 25° C., the speed was 200 rpm, and the reaction time was 5 hours. After the reaction was completed, the reaction conversion rate was greater than 99%, and the product ee value was greater than 99%.

[0100] Example 12. Synthesis of (R)-3-amino N-benzyloxycarbonyl-piperidine (R-8b) catalyzed by transaminase MnTA mutant

[0101] The reaction system was sequentially added with substrate 8a (final concentration of 50 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (250 mM), mutant M1 wet cell catalyst (100 g / L), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). The reaction was carried out on a constant temperature shaker, the temperature was set at 25° C., the speed was 200 rpm, and the reaction time was 5 hours. After the reaction was completed, the reaction conversion rate was greater than 99%, and the product ee value was greater than 99%.

[0102] Example 13. Synthesis of (R)-3-amino N-benzyloxy-piperidine (R-10b) catalyzed by transaminase MnTA mutant

[0103] The reaction system was sequentially added with substrate 10a (final concentration of 50 mM), cofactor PLP (1 mM), isopropylamine hydrochloride (250 mM), mutant M5 wet cell catalyst (100 g / L), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). The reaction was carried out on a constant temperature shaker, the temperature was set at 25° C., the speed was 200 rpm, and the reaction time was 5 hours. After the reaction was completed, the reaction conversion rate was greater than 99%, and the product ee value was greater than 99%.

Claims

1. An R-aminotransferase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. An R-aminotransferase mutant, characterized in that The amino acid sequence is obtained by mutating one or more of the 66th, 67th, 127th and 160th positions of the amino acid sequence of R-aminotransferase SEQ ID NO.1; the amino acid sequences of the mutants are SEQ ID NO: 2 (G66L), 3 (F127A), 4 (F127M), 5 (G66L / F127M), 6 (G66L / F127A), 7 (G66L / F127M / L160I), 8(G66L / H67N / F127M / L160I).

3. Use of the R-aminotransferase according to claim 1 or the R-aminotransferase mutant according to claim 2 in asymmetric synthesis of chiral amine compounds.

4. The use according to claim 3, characterized in that: The R-aminotransferase and the R-aminotransferase mutant are used as biocatalysts in catalyzing the transamination reaction of cyclic ketones or N-heterocyclic ketone compounds to prepare chiral amines.

5. The use according to claim 3, characterized in that: The cyclic ketone or N-heterocyclic ketone compound is selected from any one of the following:

6. The use according to claim 3, characterized in that: The R-aminotransferase and R-aminotransferase mutants are used as biocatalysts in the form of free cells, free enzymes, immobilized cells and immobilized enzymes.

7. The use according to claim 3, characterized in that: The method for synthesizing chiral amine compounds is: in a buffer solution, in the presence of an amine donor and a cofactor, using the R-aminotransferase of claim 1 or the R-aminotransferase mutant of claim 2 as a catalyst, asymmetric amination of cyclic ketones or N-heterocyclic ketone compounds to generate chiral amines, the reaction formula is:

8. The use according to claim 7, characterized in that: The concentration of the cyclic ketone or N-heterocyclic ketone compound in the reaction system is 0.1-500mM; the final concentration of the amine donor is 1-2000mM; the final concentration of the cofactor is 0.1-5mM; the pH value of the reaction system is 7.0-10.0, and the temperature of the reaction system is 20-45°C; the concentration of the cosolvent is 0.1-30% (v / v) of the reaction system; the concentration of the buffer is 0.05-0.2M; preferably, the concentration of the cyclic ketone or N-heterocyclic ketone compound in the reaction system is 0.1-300mM; and the final concentration of the amine donor is 1-100mM.

9. The use according to claim 7, characterized in that: The cofactor is pyridoxal phosphate (PLP); the amine donor is alanine, phenylethylamine or isopropylamine, preferably isopropylamine; the cosolvent is selected from one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and acetone, preferably methanol or dimethyl sulfoxide; and the buffer is a phosphate buffer or a Tris-HCl buffer.

10. A gene encoding the R-aminotransferase according to claim 1, wherein the nucleotide sequence thereof is shown as SEQ ID NO.9.

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