Transaminase mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to the technical field of enzyme engineering, and particularly relates to a transaminase mutant and application thereof. Specifically, the transaminase mutant is mutated on the basis of wild transaminase as shown in SEQ ID NO: 1 through a site-specific mutagenesis method (particularly, the 121st site is mutated into N from E, the 152nd site is mutated into A from Y, the 260th site is mutated into A from V and / or the 324th site is mutated into S from T), so that the amino acid sequence of the transaminase mutant is changed, and the change of the protein structure and function is realized; the transaminase mutant has the advantage that the enzyme activity is greatly improved, the enzyme activity is improved by multiple times compared with that of a transaminase parent, the stability is also greatly improved, and when the transaminase mutant is used for chiral amine production, the cost in chiral amine industrial production is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the field of enzyme engineering technology, specifically to a transaminase mutant and its applications. Background Technology

[0002] On June 22, 2021, savolitinib, a small-molecule MET inhibitor developed by Hutchison MediPharma, was approved in China. This marks the first approval of a selective MET inhibitor in China and the third MET inhibitor approved globally. Savolitinib is approved for locally advanced or metastatic non-small cell lung cancer with mesenchymal-epithelial transition factor (MET) exon 14 skipping. Savolitinib, or 3-[(1S)-1-imidazo[1,2-a]pyridin-6-ylethyl]-5-(1-methylpyrazol-4-yl)triazolo[4,5-b]pyrazine (1), is a small-molecule drug developed by Hutchison MediPharma Limited and AstraZeneca. It is a potent c-Met kinase inhibitor and is being tested in combination with osimertinib for the treatment of patients with non-small cell lung cancer and advanced or metastatic papillary renal cell carcinoma. The current chemical synthesis of severtinib involves five steps, the first of which involves transamination of the substrate ketone compound (2) to produce an enantioselective amine product compound (3) (WO2020053198A1). Engineered (S)-selective transaminases can be used for this conversion under industrial process conditions. While selectivity is suitable, improvements in enzyme activity are needed to accept higher substrate loadings, thereby optimizing industrial production.

[0003] Transaminases, catalyzing the stereoselective transfer of amino groups between amino donors and carbonyl substrates, are efficient biocatalytic tools for the synthesis of chiral amines. Transaminases (TAs) can be classified into two categories: α-transaminases (α-TAs) and ω-transaminases (ω-TAs), a classification that depends on the type of substrate being transformed. Unlike traditional α-transaminases (which act on α-amino groups), ω-TAs act on non-α-position (e.g., β, γ, or more distal) amino groups, transferring amino groups from amino donors (e.g., amino acids) to keto acid acceptors, generating the corresponding chiral amines and keto acids. As a star tool in the field of biocatalysis, ω-transaminases have demonstrated great potential in chiral amine synthesis and are expected to play a more central role in green pharmaceuticals and sustainable chemistry in the future.

[0004] While transaminase-catalyzed asymmetric transamination offers an economical and green synthetic approach for the synthesis of chiral amine compounds, transaminases face limitations and challenges in large-scale applications, including a relatively narrow substrate range, unfavorable thermodynamic equilibrium, and substrate / product inhibition. CN112930348B discloses a method for preparing severtinib using the commercially available enzyme ATA-436 from Codexis, which is very expensive, leading to high production costs. CN116209754A discloses engineered S-selective aminotransferase mutants with improved activity and substrate tolerance. The engineered transaminase was evolved to further improve activity and substrate tolerance in the asymmetric enantioselective transamination of the substrate ketone 1-imidazo[1,2a]pyridin-6-ylethylone to the product (1S)-1-imidazo[1,2a]pyridin-6-ylethylamine. However, obtaining this transaminase mutant requires dozens of mutations, involving a large screening workload and cumbersome procedures, and the enzyme gene source and sequence are not reported in detail.

[0005] Therefore, there is still a need for transaminases that are both highly selective and highly active, even with existing technologies. Summary of the Invention

[0006] To address the issues of low efficiency and high cost in the synthesis of cerivrinib, as well as the problems of low substrate feed rate, poor enzyme stability, and high cost in the biosynthesis process, one aspect of the present invention aims to provide a transaminase mutant with high catalytic activity, good thermal stability, large feed rate, and low cost, which can efficiently catalyze the synthesis of chiral amine compound intermediates of cerivrinib.

[0007] Specifically, the transaminase mutant of the present invention is based on the wild-type transaminase shown in SEQ ID NO: 1, and is mutated by site-directed mutagenesis (in particular, position 121 is mutated from E to N, position 152 from Y to A, position 260 from V to A, and position 324 from T to S), thereby changing its amino acid sequence and achieving changes in protein structure and function. Then, transaminase with the above-mentioned mutation sites is obtained by targeted screening. The transaminase mutant of the present invention has the advantage of significantly improved enzyme activity, which is many times higher than that of the original transaminase, and its stability is also greatly improved. When used in the production of chiral amines, it greatly reduces the cost of industrial production of chiral amines.

[0008] In this regard, the present invention includes, but is not limited to, the following technical solutions: In one aspect, the present invention provides a transaminase mutant obtained by mutating the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 as follows: (1) Y152A (tyrosine at position 152 is mutated to alanine); (2) V260A (valine at position 260 is mutated to alanine); (3) T324S (threonine at position 324 is mutated to serine); (4) E121N+Y152A+V260A (glutamic acid at position 121 is mutated to asparagine, tyrosine at position 152 is mutated to alanine, and valine at position 260 is mutated to alanine); or (5) E121N+Y152A+V260A+T324S (glutamic acid at position 121 is mutated to asparagine, tyrosine at position 152 is mutated to alanine, valine at position 260 is mutated to alanine and threonine at position 324 is mutated to serine).

[0009] In one aspect, the mutant of the present invention is obtained by mutating the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 as follows: (1) E121N+Y152A+V260A; or (2) E121N+Y152A+V260A+T324S.

[0010] In one aspect, the mutant of the present invention is obtained by mutating the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 as follows: (1) E121N+Y152A+V260A+T324S.

[0011] In the technical solution of the present invention concerning xylose reductase mutants, "+" represents the simultaneous presence of two or more mutation forms. For example, E121N+Y152A+V260A represents a transaminase mutant obtained by mutating the wild-type transaminase sequence shown in SEQ ID NO: 1 as follows: E121N, Y152A, and V260A; E121N+Y152A+V260A+T324S represents a transaminase mutant obtained by mutating the wild-type transaminase sequence shown in SEQ ID NO: 1 as follows: E121N, Y152A, V260A, and T324S.

[0012] In one aspect, the present invention also provides a polynucleotide characterized in that it encodes the transaminase mutant described in the present invention.

[0013] In one aspect, the polynucleotide of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0014] In one aspect, the present invention also provides a recombinant vector comprising the polynucleotides described herein.

[0015] In one aspect, the recombinant expression vector of the present invention is selected from pET-28a, pET-dute1, pRSF-dute1, and more preferably pET-28a.

[0016] In one aspect, the present invention also provides a host cell comprising the polynucleotides described herein or the recombinant vectors described herein.

[0017] In one aspect, the host cell described in this invention is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an *Escherichia coli* cell. It should be noted that the plant cell described in this invention is not intended to protect any specific plant, and this invention does not disclose any method for developing plant cells into a complete plant. The plant cell described in this invention is only used as an engineered cell for expressing the enzyme mutant of this invention.

[0018] In one aspect, the host cell of the present invention is specifically *Escherichia coli* MG1655, *Escherichia coli* BL21(DE3), or *Escherichia coli* BL21(DE3)pLysS. Preferably, the host cell is *Escherichia coli* BL21(DE3).

[0019] In another aspect, the present invention provides the application of the transaminase mutant or the host cell described herein in the catalytic preparation of chiral amines from carbonyl compounds.

[0020] In one aspect, the carbonyl compound of the present invention is 1 Imidazol[1,2] a]pyridine 6 Benzyl ketone, wherein the chiral amine is (1S). 1 Imidazol[1,2] a]pyridine 6 Methylamine.

[0021] In one aspect, the application of the present invention includes: in the presence of an amine source, causing 1 Imidazol[1,2] a]pyridine 6 The transaminase is contacted with methyl ethyl ketone, and preferably, the amine source is isopropylamine.

[0022] In another aspect, the present invention provides a method for preparing cevotinib, the method comprising the following steps: mixing the transaminase mutant described in the present invention or the host cell described in the present invention with 1 Imidazol[1,2] a]pyridine 6 (1S) is produced by mixing methyl ethyl ketone and an amine source under suitable conditions. 1 Imidazol[1,2] a]pyridine 6 Methylamine.

[0023] In one aspect, the transaminase mutant of the present invention also contains an enzyme cofactor in the process of catalyzing the reaction of carbonyl compounds to generate chiral amines.

[0024] In one aspect, the enzyme cofactor described in this invention is pyridoxal phosphate.

[0025] In another aspect, the present invention provides a method for producing the transaminase mutant described herein, the method comprising: (a) The host cells of the present invention are cultured under conditions suitable for the expression of the transaminase mutant; and (b) The transaminase mutant was recovered.

[0026] The beneficial effects of the present invention include at least the following: 1. The present invention provides a transaminase mutant having the amino acid sequence shown in SEQ ID NO: 3, which exhibits high catalytic activity for sterically hindered substrates; the 24-hour conversion rate of the transaminase mutant is 80%; while the wild-type enzyme has less than 1% activity for such substrates; furthermore, this enzyme mutant has a half-life of more than 60 hours at 55°C, while the wild-type enzyme has a half-life of only 24 hours under the same conditions.

[0027] 2. The transaminase mutant provided by this invention exhibits high catalytic activity and good heat resistance under alkaline conditions, and can be applied to the biosynthesis of chiral amines. Compared with chemical synthesis methods, the transamination reaction it catalyzes is simple and mild, with high reaction selectivity and low preparation cost, and has good application prospects.

[0028] 3. The transaminase mutant of the present invention exhibits significantly improved catalytic activity for ketone substrates compared to the wild type, enabling the synthesis of sterically hindered chiral amine compounds using transaminase catalysis without the need for heavy metal catalysts, achieving green chemical synthesis with lower usage costs. Furthermore, using the transaminase with enhanced activity for the transamination reaction readily yields highly active and selective chiral amine compounds. Attached Figure Description

[0029] Figure 1The polyacrylamide gel electrophoresis results are shown for the wild-type transaminase (amino acid sequence SEQ ID NO: 1) and mutant (amino acid sequence SEQ ID NO: 3) produced on a small scale in shake flasks in Example 3. From left to right, the lanes represent: Marker, blank control, wild-type whole cells, wild-type whole cell lysate supernatant, wild-type whole cell lysate, mutant whole cells, mutant whole cell lysate supernatant, and mutant whole cell lysate.

[0030] Figure 2 1 Imidazol[1,2] a]pyridine 6 (1S) methyl ethyl ketone is produced by a transaminase mutant-catalyzed reaction. 1 Imidazol[1,2] a]pyridine 6 A schematic diagram of the reaction of methylethylamine.

[0031] Figure 3 The HPLC chromatograms of the reaction in Example 6 are shown. The retention time of 4.128 min is for compound (3), and the retention time of 6.576 min is for compound (2). Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments. These embodiments are illustrative of the invention, and the invention is not limited to them. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0033] Example 1: Establishment of Wild-type Transaminase Genetically Engineered Bacteria According to NCBI records Alphaproteobacteria bacterium The wild-type gene of aminotransferase class III-fold pyridoxal phosphate-dependent enzyme (GenBank: MBM3604520.1) was sequence-optimized and the whole gene fragment (nucleotide sequence shown in SEQ ID NO.2) was artificially synthesized. The gene was then inserted into the pET-28a plasmid by Azenta using XbaI and XhoI restriction enzymes. The ligated vector was then transformed into Escherichia coli BL21(DE3) to establish a wild-type transaminase genetically engineered bacterium. After kanamycin resistance screening, sequencing was performed to verify the results.

[0034] Example 2: Obtaining the transaminase mutant and the target gene The three-dimensional structure of SEQ ID NO: 1 was obtained using an online protein structure prediction tool, and the three-dimensional structure of the transaminase with the highest structural similarity (4E3Q, homology 84.77%) was obtained through PDB for structural comparison. Then, the binding simulation of the substrate and the three-dimensional structure of the transaminase protein in formula (2) was performed using AutoDock. Finally, the three-dimensional structural features and the interaction between amino acid residues were analyzed using Pymol, and amino acids that may be related to substrate binding were selected as mutant amino acids.

[0035] Based on the above analysis results, at least one site in the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 was mutated: position 21 from F to A, position 58 from L to V, position 121 from E to N, position 152 from Y to A, position 167 from Y to F, position 230 from A to G, position 260 from V to A, and position 324 from T to S. Based on the docking results, the hydrophobic amino acids at a distance of 5 Å from the substrate binding region were analyzed. Preferably, at least one site in the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 was mutated: E121N, Y152A, V260A, T324S. Based on Pymol analysis, the final designed combination of mutation sites was: position 121 from E to N, position 152 from Y to A, position 260 from V to A, and position 324 from T to S. In this process, the transaminase gene mutation was performed using gene synthesis. The synthesized fragment was combined with the pET28a plasmid vector, and the gene was inserted into the pET-28a plasmid using XbaI and XhoI restriction enzymes. The ligated vector was then transformed into *E. coli* BL21(DE3) to create a transaminase gene mutant. *E. coli* BL21(DE3) was used as the host, and the pET28a plasmid as the vector to express the mutated transaminase. Specifically, the encoding nucleotide sequence of the transaminase mutant containing mutation sites E121N, Y152A, V260A, and / or T324S was based on SEQ ID NO: 2, with codons GAA at positions 361-363 changed to AAC, codons TAT ​​at positions 454-456 changed to GCG, codons GTG at positions 778-780 changed to GCG, and / or codons ACC at positions 970-972 changed to AGC. For example, the encoding nucleotide sequence of the transaminase mutant (containing the E121N+Y152A+V260A+T324S mutation) shown in SEQ ID NO: 3 is shown in SEQ ID NO. 4.

[0036] The following table shows the activity of mutant strains screened using the enzyme activity detection method described in Example 5. The detection results are shown in Table 1 below: Table 1 Enzyme Activity

[0037] Note: In the table above, + represents a conversion rate greater than or equal to 0% and less than 20%, ++ represents a conversion rate greater than or equal to 20% and less than 40%, +++ represents a conversion rate greater than or equal to 40% and less than 80%, and ++++ represents a conversion rate greater than or equal to 80%.

[0038] Example 3: Small-scale production of transaminase mutant protein in shake flasks Recombinant cells of the *E. coli* transaminase mutant SEQ ID NO: 3 were cultured overnight in LB liquid medium (100 μg / ml kanamycin) at 37°C and 220 rpm. The culture was then transferred at a 1:100 ratio to 50 mL of fresh LB medium (250 mL shake flask) and grown at 37°C. When the optical density at 600 nm (OD600) reached approximately 0.6, isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown at 25°C for 16 hours. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was discarded. The cell pellet was resuspended in pre-chilled 100 mM Tris-HCl buffer (pH 8.5) at 200 g / L, sonicated, and then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C. The crude enzyme solution was subjected to polyacrylamide gel electrophoresis; the results are shown below. Figure 1 .

[0039] Example 4: High-density fermentation preparation of transaminase mutant protein The recombinant mutant strain SEQ ID NO: 3 obtained in Example 2 was inoculated into 3 mL of liquid LB medium and cultured overnight at 37°C and 220 rpm with shaking. Then, approximately 1% of this strain was inoculated into 400 mL of liquid LB medium and cultured until the OD600 reached 4. This seed culture was then transferred to 2 L of fermentation medium for high-density fermentation. The initial temperature was 37°C, the stirring speed was 300 rpm, the aeration rate was 1.5 vvm / L / min, and the pH was 6.8. The stirring speed was then continuously increased, up to a maximum of 1000 rpm. The fermentation process consisted of two stages. In the first stage, after inoculation, the culture was carried out for approximately 4 hours until the carbon source in the medium was completely consumed. Feedback was then initiated according to DO (dissolved oxygen) levels. After feeding, the temperature was lowered to 25°C, and dissolved oxygen was maintained above 30%. Eight hours after feeding, isopropyl thiogalactoside (IPTG) was added for induction. After 12 hours of induction, the culture was transferred to the fermentation tank. The bacterial cells obtained by centrifuging at 8000 rpm for 10 min and discarding the supernatant were suspended in 100 mM Tris-HCl buffer (pH 8.5). The suspended cells were then broken up using a high-pressure homogenizer to obtain a crude enzyme solution of high-density fermented transaminase.

[0040] Example 5: Detection of product (1S)-1-imidazo[1,2-a]pyridin-6-ylethylamine by HPLC-UV analysis Instrument: Waters e2695 Chromatographic column: HP-C18, 4.6*250 mm, 5 μm Column temperature: 40℃ Mobile phase A: 30 mM, pH 6.8, KH₂PO₄ : ACN = 9:1 (V / V) Mobile phase B: ACN Flow rate: 1 mL / min Detection wavelength: 280 nm gradient:

[0041] Example 6: Preparation of (1S)-1-imidazo[1,2-a]pyridin-6-ylethylamine The substrate used in this embodiment is Figure 2 The compound shown in formula (2) is (1) Imidazol[1,2] a]pyridine 6 (1S) methyl ethyl ketone: This compound is reacted by the transaminase catalytic reaction of the present invention to form the compound shown in formula (3). 1 Imidazol[1,2] a]pyridine 6 (methylethylamine).

[0042] At room temperature, 24 g of wet cells (solid after high-speed centrifugation of fermentation broth) containing the above-mentioned transaminase mutants were added to a 250 mL glass reactor along with 23 mL of 6 M isopropylamine, 100 mg of pyridoxal phosphate, and 72 mL of 100 mM sodium borate buffer (pH 8.5). The mixture was stirred at 100 rpm for 20 min to thoroughly mix all solids, and the pH was adjusted to 8.5 by adding 10% sodium hydroxide solution. The temperature was then raised to 55 °C using a water bath. 3 g of substrate was then dissolved in 5 mL of DMSO and slowly added to the stirred mixture at 100 rpm in the 250 mL reactor. The pH was monitored and maintained at 8.5 during the reaction, and samples of the mixture were taken for HPLC analysis. Figure 3 After 24 h and 48 h, the conversion rates of the substrate (compound (2)) under the mutant catalytic conditions were 80% and 95.50%, respectively. The conversion rate was calculated as follows: Conversion rate (%) = (initial substrate concentration - final substrate concentration) / initial substrate concentration × 100%.

[0043] After the reaction was complete, the reaction system was adjusted to pH=12 with sodium hydroxide, 20% diatomaceous earth was added, and the mixture was stirred for 1 hour before filtration. The filtrate was extracted with 50 mL nBuOH and concentrated under vacuum. The mixture was cooled and filtered, and washed with nBuOH to obtain the target product compound (3). The purity was >98%, the de value was >99%, and the yield was about 90% as determined by HPLC.

[0044] 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.

[0045] sequence list SEQ ID NO: 1 MTMTTPQSWEARADEYSLYGFTDLPSVQKRGAVVLTHGEGPYVVDVNGNRYLDANSGLWNMVAGFDHKGLAEAAKAQYDRFPGYHAFFGRMSDQTVMLSEKLVEVSPFSRGKV FYTNSGSEANDTMVKMLWFLHASEGNPQKRKILTRWNAYHGVTAVSASMTGKPYNEVFGLPLPGFIHLTCPHYWRYGEEGETETQFTQRLARELDDTIQREGADTIAGFFAEPV QGAGGVIPPSEGYFQAIMPVLKKHGIPMIADEVITGFGRTGNTWGSQTYDFVPDAIISSKNLTAGLFPMGAVILGPELADRVQAAVERIEEFPHGFTASGHPVGCAIALKAIDV VMNEGLADNVRRLAPRMEDGLRQLAQNPNIGEYRGVGFMWALEAVRDKATKTPFEGHLSVSERIANTCTDMGLICRPLGQSIVLCPPFILTEAQMDEMFEKLDKALGKVFAELA SEQ ID NO: 2 SEQ ID NO: 3 MTMTTPQSWEARADEYSLYGFTDLPSVQKRGAVVLTHGEGPYVVDVNGNRYLDANSGLWNMVAGFDHKGLAEAAKAQYDRFPGYHAFFGRMSDQTVMLSEKLVEVSPFSRGKVFYTNSGSNANDTMVKMLWFLHASEGNPQKRKILTRWNAAHGVTAVSASMTGKPYNEVFGLPLPGFIHLTCPHYWRYGEEGETETQFTQRLARELDDTIQREGADTIAGFFAEPVQGAGGVIPPSEGYFQAIMPVLKKHGIPMIADEAITGFGRTGNTWGSQTYDFVPDAIISSKNLTAGLFPMGAVILGPELADRVQAAVERIEEFPHGFSASGHPVGCAIALKAIDVVMNEGLADNVRRLAPRMEDGLRQLAQNPNIGEYRGVGFMWALEAVRDKATKTPFEGHLSVSERIANTCTDMGLICRPLGQSIVLCPPFILTEAQMDEMFEKLDKALGKVFAELA SEQ ID NO: 4, the underlined part is the codon encoding the mutated amino acid residue ATGACCATGACCACCCCGCAGAGCTGGGAAGCGCGCGCGGATGAATATAGCCTGTATGGCTTTACGGATCTGCCGAGCGTGCAGAAACGCGGCGCGGTGGTGCTGACCCATGGCGAAGGCCCGTATGTGGTGGATGTGAACGGCAACCGCTATCTGGATGCGAACAGCGGCCTGTGGAACATGGTGGCGGGCTTTGATCATAAAGGCCTGGCGGAAGCGGCGAAAGCGCAGTATGATCGCTTTCCGGGCTATCATGCGTTTTTTGGCCGCATGAGCGATCAGACCGTGATGCTGAGCGAAAAACTGGTGGAAGTGAGCCCGTTTAGCCGCGGCAAAGTGTTTTATACCAACAGCGGCAGC AACGCGAACGATACCATGGTGAAAATGCCTGTGGTTTCTGCATGCGAGCGAAGGCAACCCGCAGAAACGCAAAATTCTGACCCGCTGGAACGCG GCG CATGGCGTGACCGCGGTGAGCGCGAGCATGACCGGCAAACCGTATAACGAAGTGTTTGGCCTGCCGCTGCCGGGCTTTATTCATCTGACCTGCCCGCATTATTGGCGCTATGGCGAAGAAGGCGAAACCGAAACGCAGTTTACGCAGCGCCTGGCGCGCG AACTGGATGATACCATTCAGCGCGAAGGCGCGGATACCATTGCGGGCTTTTTTGCGGAACCGGTGCAAGGCGCGGGCGGCGTGATTCCGCCGAGCGAAGGCTATTTTCAAGCGATTATGCCGGTGCTGAAAAAACATGGCATTCCGATGATTGCGGATGAA GCG ATTACCGGCTTTGGCCGCACCGGCAACACCTGGGGCAGTCAGACCTATGATTTTGTGCCGGATGCGATTATTAGCAGCAAAAACCTGACCGCGGGCCTGTTTCCGATGGGCGCGGTGATTCTGGGCCCGGAACTGGCGGATCGCGTGCAAGCGGCGGTGGAACGCATTGAAGAATTTCCGCACGGCTTT AGCGCGAGCGGCCATCCGGTGGGCTGCGCGATTGCGCTGAAAGCGATTGATGTGGTGATGAACGAAGGTTTAGCGGATAACGTGCGCCGCCTGGCGCCGCGCATGGAAGATGGCCTGCGTCAGCTGGCGCAGAACCCGAACATTGGCGAATATCGCGGCGTGGGCTTTATGTGGGCGCTGGAAGCGGTGCGCGATAAAGCGACCAAAACCCCGTTTGAAGGCCATCTGAGCGTGAGCGAACGCATTGCGAACACCTGCACCGATATGGGCCTGATTTGTCGCCCGCTGGGTCAGAGCATTGTGCTGTGCCCGCCGTTTATTCTGACCGAAGCGCAGATGGATGAAATGTTTGAAAAACTGGATAAAGCGCTGGGCAAGGTGTTTGCCGAGCTGGCG。

Claims

1. A transaminase mutant, characterized in that, The mutant was obtained by mutating the amino acid sequence of the wild-type transaminase shown in SEQ ID NO: 1 as follows: (1) Y152A; (2) V260A; (3) T324S; (4) E121N+Y152A+V260A; or (5) E121N+Y152A+V260A+T324S.

2. A polynucleotide, characterized in that, The transaminase mutant according to claim 1 is encoded.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the polynucleotide according to claim 2 or the recombinant vector according to claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an Escherichia coli cell.

6. The application of the transaminase mutant according to claim 1 or the host cell according to claim 4 or 5 in the catalytic preparation of chiral amines from carbonyl compounds.

7. The application according to claim 6, characterized in that, The carbonyl compound is 1 Imidazol[1,2] a]pyridine 6 Benzyl ketone, wherein the chiral amine is (1S). 1 Imidazol[1,2] a]pyridine 6 Methylamine.

8. The application according to claim 7, characterized in that, The application methods include: in the presence of an amine source, making 1 Imidazol[1,2] a]pyridine 6 The transaminase is contacted with methyl ethyl ketone, and preferably, the amine source is isopropylamine.

9. A method for preparing severtinib, characterized in that, The method includes the following steps: mixing the transaminase mutant according to claim 1 or the host cell according to claim 4 or 5 with 1 Imidazol[1,2] a]pyridine 6 (1S) is produced by mixing methyl ethyl ketone and an amine source under suitable conditions. 1 Imidazol[1,2] a]pyridine 6 Methylamine.

10. A method for producing a transaminase mutant according to claim 1, characterized in that, The method includes: (a) The host cells of claim 4 or 5 are cultured under conditions suitable for the expression of the transaminase mutant; and (b) The transaminase mutant was recovered.

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