Use of an r-transaminase and mutants thereof in the asymmetric synthesis of chiral amine compounds

Abstract

The application 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 shown as 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 obtained recombinant transaminase mutant has the advantages of high catalytic activity, strict stereoselectivity, mild reaction conditions and the like for various cyclic ketones and N-heterocyclic ketone compounds.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to the application of an R-transaminase and its mutant in the asymmetric synthesis of chiral amine compounds. Background Technology

[0002] Chiral N-heterocyclic amines are key intermediates in the preparation of various active pharmaceutical ingredients and have important applications 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 in the synthesis of the fifth-generation cephalosporin cefepime, the quinolone antibacterial drug tofloxacin, and the dipeptide kinase IV inhibitors dogliptin, alogliptin, linagliptin, and trelagliptin.

[0003]

[0004] Currently, various chemical and biological methods are available for synthesizing chiral N-heterocyclic amines, such as Hoffmann 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 these, transaminase-catalyzed asymmetric reductive amination of prochiral ketones is a superior choice for synthesizing chiral N-heterocyclic amines due to its high atom economy and strict stereoselectivity.

[0005] Transaminases are pyridoxal phosphate (PLP)-dependent enzymes that catalyze the transfer of amino groups from amine donors to prochiral ketones to generate the corresponding chiral amines. However, wild-type transaminases are often limited by narrow substrate profiles and inefficient conversion capabilities. To date, several transaminases have been used to synthesize 1b and 2b, including Vfl-TA, Ade-TA, CbTA, RbTA, and the commercially available 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 all face problems such as low theoretical yield, low catalytic efficiency, and severe substrate inhibition. Therefore, developing more efficient enzyme catalysts for the synthesis of 1b and 2b to reduce reaction costs is of great significance for improving industrial applications. Summary of the Invention

[0006] The purpose of this invention is to solve the problems of low catalytic efficiency and severe substrate inhibition in the synthesis of N-heterocyclic amines by existing transaminases. Through enzyme engineering strategies, an excellent transaminase mutant is obtained to catalyze the synthesis of chiral N-heterocyclic amine active drug intermediates.

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

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

[0009] This invention also provides an R-transaminase mutant, the amino acid sequence of which is obtained by mutating one or more positions at positions 66, 67, 127, and 160 of the amino acid sequence of R-transaminase 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] As shown in 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] This invention also provides the application of the aforementioned R-transaminase and R-transaminase mutants in the asymmetric synthesis of chiral amine compounds.

[0028] Furthermore, in the above technical solution, the R-transaminase and R-transaminase mutant are used as biocatalysts in the preparation of chiral amines by catalyzing the transamination reaction of cyclic ketones or N-heterocyclic ketones.

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

[0030]

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

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

[0033]

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

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

[0036] In the above technical solution, 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 solution is phosphate buffer or Tris-HCl buffer.

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

[0038] SEQ ID NO.9:

[0039]

[0040] This invention modifies the R-transaminase MnTA using enzyme engineering technology, obtaining superior mutants capable of efficiently synthesizing active drug intermediates 1b and 2b. Furthermore, the optimal mutation significantly enhances the activity against various cyclic ketones or N-heterocyclic ketones. The superior mutants obtained in this invention enable efficient enzymatic synthesis of drug intermediates 1b and 2b. Compared with existing enzymatic synthesis processes, this method offers higher substrate loading, higher space-time yield, and can further reduce production costs, demonstrating its application value. Attached image description:

[0041] Figure 1 SDS-PAGE analysis of soluble protein expression.

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

[0043] Figure 3 Gas phase diagram for conversion detection of substrate 1a catalyzed by MnTA mutant; (A) 1a reference, (B) (R)-1b reference, (C) Detection of product (R)-1b synthesized from MnTA M5.

[0044] Figure 4 Gas phase diagram for conversion detection of substrate 2a catalyzed by MnTA mutant; (A) 2a reference, (B) (R)-2b reference, (C) Detection of product (R)-2b synthesized from MnTA M1.

[0045] Figure 5 Liquid chromatography plots showing the ee value of product (R)-1b generated from substrate 1a by MnTA mutant; (A) Racemic 1b reference, (B) (R)-1b reference, (C) Detection of product (R)-1b synthesized from MnTAM5.

[0046] Figure 6 Liquid chromatography plots showing the ee value of product (R)-2b generated from substrate 2a by MnTA mutant; (A) racemic 2b reference, (B) (R)-2b reference, (C) detection of product (R)-2b synthesized from MnTA M1.

[0047] Figure 7 Liquid chromatography plots showing the ee value of product (R)-3b generated from substrate 3a by MnTA mutant; (A) racemic 3b reference, (B) (R)-3b reference, (C) detection of product (R)-3b synthesized from MnTAM5.

[0048] Figure 8Liquid chromatography plots showing the ee value of product (R)-8b generated from substrate 8a by MnTA mutant; (A) racemic 8b reference, (B) (R)-8b reference, (C) detection of product (R)-8b synthesized from MnTA M1.

[0049] Figure 9 Liquid chromatography plots showing the ee value of product (R)-10b generated from substrate 10a catalyzed by MnTA mutant; (A) Racemic 10b reference, (B) (R)-10b reference, (C) Detection of product (R)-10b synthesized from MnTA M5. Detailed Implementation

[0050] The application of the R-transaminase and its mutants in the asymmetric synthesis of chiral amine compounds of the present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental procedures used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative and not limiting of the invention.

[0051] Example 1: Obtaining the MnTA transaminase gene

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

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

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

[0055] Table 1 lists the primers 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℃: 10min, (94℃: 30s, 48℃: 30s, 72℃: 90s) 35 cycles, 72℃, 10min.

[0063] Two fragments were obtained from the first round of PCR. After verifying the correct band size by agarose gel electrophoresis, the two fragments were recovered using a standard DNA product gel extraction kit and used as templates for the second round of PCR. The second round of PCR amplified the mutant gene using the end primers MnTA-Nde IF and MnTA-Xho IR according to the system described in Table 2. After the second round of PCR, the products were recovered using a DNA purification kit to obtain the DNA product containing the mutation site.

[0064] Example 3: Construction of recombinant engineered plasmids

[0065] The purified DNA products and the vector pET-28a(+) were double-digested with NdeI and XhoI, respectively (Table 3). After incubation at 37°C for 4-6 hours, the digested products were recovered using a DNA product gel extraction kit to obtain mutant DNA fragments and vector fragments with the same sticky ends. The digested target gene was ligated to the vector plasmid using T4 DNA ligase to construct recombinant engineered plasmids of different mutants. These recombinant engineered plasmids were then transformed into competent cells of *E. coli* Rosetta2(DE3) and plated on LB agar plates containing Kana resistance, and incubated overnight at 37°C.

[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. After incubation at 37°C and 200 rpm for 6-8 h, seed cultures of different mutants were obtained. 1 mL of each seed culture was then inoculated into 100 mL of LB liquid medium containing Kana resistance and incubated at 37°C and 200 rpm until the culture medium reached OD. 600 When the concentration reaches 0.8-1.0, add IPTG solution to a final concentration of 0.1 mM and lower the temperature to 20-25℃ to induce expression for 15-20 h. Centrifuge at 3000 r / min for 10 min to collect the bacterial cells. After washing with physiological saline, centrifuge again to collect the bacterial cells and obtain the whole-cell biocatalyst.

[0070] Example 5: Gas Chromatography Method for Determining Conversion Rate

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

[0072] Table 4. Gas phase analysis conditions and retention times for 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 Determining the ee Value of Products

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

[0077] Table 5. Chiral analysis conditions and retention times of the products

[0078]

[0079] Example 7: Screening for the activity of transaminase MnTA mutants

[0080] Reaction formula:

[0081]

[0082] 1a reaction system: 80 mg / mL of different mutant wet cells 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 different mutant wet cells 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℃, with the wild type used as a control. After 5 hours of reaction, the conversion rate was determined by gas chromatography, and the results are shown in Table 6.

[0085] Table 6. Conversion rates of substrates 1a and 2a catalyzed by the MnTA mutant.

[0086]

[0087]

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

[0089] Reaction system: 80 mg / mL of different mutant wet cells as described in Example 4, substrate (50 mM), isopropylamine hydrochloride (250 mM), PLP (1 mM), methanol (5% v / v), and pH 9.0 Tris-HCl buffer (0.1 M). (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℃, and after 5 hours of reaction, the reaction conversion rate was determined by gas chromatography. The results are shown in Table 7.

[0091] Table 7. Determination of conversion rates of cyclic ketones and N-heterocyclic ketones catalyzed by MnTA transaminase 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-butoxycarbonyl-pyrrole (R-1b) catalyzed by transaminase MnTA mutant

[0095] The following components were added sequentially to the reaction system: substrate 1a (final concentration 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 in a constant temperature shaker at 25°C and 200 rpm for 5 hours. After the reaction, the conversion rate was greater than 95%, and the ee value of the product was greater than 99%.

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

[0097] The following components were added sequentially to the reaction system: substrate 2a (final concentration 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 in a constant temperature shaker at 25°C and 200 rpm for 5 hours. After the reaction, the conversion rate was greater than 99%, and the product ee value was greater than 99%.

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

[0099] The following components were added sequentially to the reaction system: substrate 3a (final concentration 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 in a constant temperature shaker at 25°C and 200 rpm for 5 hours. After the reaction, the conversion rate was greater than 99%, and the product ee value was greater than 99%.

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

[0101] The following components were added sequentially to the reaction system: substrate 8a (final concentration 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 in a constant temperature shaker at 25°C and 200 rpm for 5 hours. After the reaction, the conversion rate was greater than 99%, and the ee value of the product was greater than 99%.

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

[0103] The following components were added sequentially to the reaction system: substrate 10a (final concentration 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 in a constant temperature shaker at 25°C and 200 rpm for 5 hours. After the reaction, the conversion rate was greater than 99%, and the product ee value was greater than 99%.

Claims

1. An R-transaminase mutant, characterized in that, The R-transaminase mutant was obtained by mutating G66L, H67N, F127M, and L160I based on the amino acid sequence SEQ ID NO.

1. The amino acid sequence of the R-transaminase mutant is shown in SEQ ID NO:

8.

2. The application of the R-transaminase mutant according to claim 1 as a biocatalyst in the preparation of chiral amines, characterized in that, The R-transaminase mutant catalyzes the transamination reaction of N-heterocyclic ketones to prepare chiral amines, wherein the N-heterocyclic ketones are shown below: ； The chiral amine is (R)-3-aminoN-tert-butoxycarbonyl-pyrrole (R-1b).

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