Transaminase mutant, recombinant genetically engineered bacterium and application of recombinant genetically engineered bacterium in catalytic synthesis of (R)-1-Boc-3-aminopiperidine

Abstract

The invention belongs to the technical field of bioengineering, and relates to a transaminase mutant, a recombinant genetically engineered bacterium and application of the transaminase mutant in catalytic synthesis of (R)-1-Boc-3-aminopiperidine.The transaminase mutant is obtained by conducting single-point or combined mutation on the 131 site and / or the 197 site of an amino acid sequence shown in SEQ ID NO.2; the mutation sites comprise that the 131 phenylalanine is mutated into aspartic acid, threonine or tyrosine, and / or the 197 lysine is mutated into arginine or leucine. Experimental results show that compared with wild type transaminase, the catalytic activity, the thermal stability and the organic solvent tolerance of the obtained mutants, especially single-point mutants MyTA1-F131Y and MyTA1-K197R and a combined mutant MyTA1-F131Y-K197R, are all remarkably improved, and compared with the wild type transaminase, the catalytic activity, the thermal stability and the organic solvent tolerance of the obtained mutants are all remarkably improved. The mutant MyTA1-F131Y-K197R can be used for efficiently catalyzing asymmetric amination of N-Boc-3-piperidone to synthesize (R)-1-Boc-3-aminopiperidine, the conversion rate of the (R)-1-Boc-3-aminopiperidine after the (R)-1-Boc-3-aminopiperidine reacts for 24 hours under the condition that the substrate concentration is 100 g / L can reach 90% or above, and the mutant MyTA1-F131Y-K197R has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a transaminase mutant, a recombinant genetically engineered bacterium and its application in the catalytic synthesis of (R)-1-Boc-3-aminopiperidine. Background Technology

[0002] Chiral amines, as a class of chiral molecules with significant applications, are widely found in the structures of pharmaceuticals, pesticides, and fine chemicals due to the presence of one or more chiral centers in their structure. Transaminases are enzyme catalysts dependent on pyridoxal 5'-phosphate (PLP), capable of efficiently and selectively synthesizing chiral amine compounds via asymmetric transamination reactions. Compared to chemical catalysis methods, transaminase catalysis offers significant advantages such as milder conditions, environmental friendliness, and high stereoselectivity, meeting the requirements of green synthesis.

[0003] (R)-3-aminopiperidine is a key chiral intermediate in the synthesis of various dipeptidyl peptidase IV (DPP-IV) inhibitors (such as liraliptin, alogliptin, and trelagliptin), and there is significant market demand. Existing chemical synthesis and chiral resolution processes suffer from low efficiency, heavy pollution, and insufficient optical purity, while enzymatic synthesis, due to its high specificity, mild reaction, and green sustainability, is considered to have better application prospects. However, natural transaminases often face problems such as poor stability and easy inactivation in actual industrial environments, severely restricting their large-scale application.

[0004] Transaminases rely on the PLP cofactor for catalytic activity, achieving amino transfer through two half-reactions. Natural transaminases exhibit both (R)- and (S)-selective types. While protein engineering has made significant progress in enhancing transaminase catalytic activity, patent CN118755684A discloses a method for the efficient synthesis of (R)-N-Boc-3-aminopiperidine using a PLP-dependent transaminase, achieving a conversion rate >99% and optical purity >99% at a substrate concentration of 50 g / L for 20 hours. Patent CN120137930A discloses a transaminase mutant, its encoding gene, engineered bacteria, and its application in the synthesis of N-Boc-3-aminopiperidine; the mutant enzyme activity is 3.9 times that of the wild-type transaminase, with a conversion rate reaching 70.14%. However, existing mutants still fail to meet the requirements for industrial catalysis in terms of thermal stability, pH tolerance, organic solvent compatibility, and long-term operational stability.

[0005] Therefore, there is an urgent need to carry out in-depth modifications to their stability in order to obtain a batch of engineered enzymes with excellent catalytic performance and suitable for harsh reaction environments, so as to promote the biomanufacturing process of chiral amine compounds. Summary of the Invention

[0006] The purpose of this invention is to provide a transaminase mutant, a recombinant genetically engineered bacterium, and their application in the catalytic synthesis of chiral amines. This invention establishes a catalytic synthesis process by constructing a transaminase mutant with further enhanced catalytic activity and stereoselectivity, laying the foundation for the industrial production of chiral amines, especially (R)-1-Boc-3-aminopiperidine, via transaminase synthesis, and meeting the needs of industrial synthesis of (R)-1-Boc-3-aminopiperidine.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a transaminase mutant, which is obtained by making a single mutation or multiple mutations at position 131 and / or position 197 of the amino acid sequence shown in SEQ ID NO.2.

[0009] Preferably, the transaminase mutant is one in which the amino acid sequence shown in SEQ ID NO.2 is mutated to at least one of the following:

[0010] (1) The phenylalanine at position 131 is mutated to any one of aspartic acid, threonine, or tyrosine;

[0011] (2) The lysine at position 197 is mutated to either arginine or leucine.

[0012] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of other amino acid sites in the above-mentioned transaminase mutants are also included within the scope of this invention.

[0013] Secondly, the present invention also provides a gene encoding the transaminase mutant. Due to the specificity of nucleotide sequences, any variant of the polynucleotide shown in this invention, provided it shares more than 90% homology with the aforementioned polynucleotide, falls within the scope of protection of this invention. The polynucleotide variant refers to a polynucleotide sequence with one or more nucleotide changes. This polynucleotide variant can be a live or non-live variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially alter the function of the encoded peptide protein.

[0014] Thirdly, the present invention also provides a recombinant vector comprising the coding gene of the transaminase mutant. The recombinant vector comprises a polynucleotide operatively linked to a control sequence suitable for directing expression in a host cell. Various vectors conventional in the art, such as various plasmids, phages, or viral vectors, linked to the novel transaminase mutant nucleotide sequence of the present invention, are all within the scope of protection of the present invention. Preferably, the recombinant vector uses plasmid pET-28a(+) as the expression vector, and the coding gene of the novel transaminase mutant is linked to the plasmid pET-28a(+).

[0015] Fourthly, the present invention also provides a recombinant genetically engineered bacterium containing the coding gene of the transaminase mutant or the recombinant vector.

[0016] Fifthly, the present invention also provides a method for preparing a transaminase mutant, comprising: introducing an exogenous transaminase mutant encoding gene into a host cell through genetic engineering technology to construct a genetically engineered bacterium and expressing it to obtain the transaminase mutant; wherein the host cell may be a bacterium, fungus, plant cell or animal cell, preferably Escherichia coli BL21(DE3) as the expression host.

[0017] The present invention also provides the application of the transaminase mutant in the enzymatic synthesis of (R)-1-Boc-3-aminopiperidine.

[0018] Preferably, the application includes using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the transaminase mutant encoding gene, immobilized cells of wet bacterial cells, enzymes extracted or immobilized enzymes after ultrasonic disruption of wet bacterial cells as catalysts, N-Boc-3-piperidinone as substrate, and phosphate buffer as reaction medium to form a reaction system, followed by separation and purification to obtain (R)-1-Boc-3-aminopiperidine.

[0019] Preferably, the application satisfies at least one of the following conditions:

[0020] (a) Methanol is used as a co-solvent for N-Boc-3-piperidinone, with a volume concentration of 10% to 30% in the reaction system;

[0021] (b) The pH of the reaction system is 8 to 9.5;

[0022] (c) The reaction temperature of the reaction system is 45℃;

[0023] (d) The concentration of pyridoxal phosphate in the reaction system is 2.5~4 mM;

[0024] (e) The amino donor is isopropylamine, and its concentration in the reaction system is 1.5~2.5 M;

[0025] (f) The catalyst is a wet bacterial cell, which is obtained by fermentation culture of recombinant genetically engineered bacteria containing the transaminase mutant encoding gene, and the concentration in the reaction system is 100~150 g / L;

[0026] (g) The concentration of N-Boc-3-piperidinone in the reaction system is 50 g / L or 100 g / L.

[0027] The nitrile hydrolase mutant described in this invention can be used in whole-cell engineered bacteria, as unpurified crude enzyme, or as partially or fully purified enzyme. Furthermore, the nitrile hydrolase mutant of this invention can be prepared into immobilized enzymes or immobilized cell-based biocatalysts using immobilization techniques known in the art.

[0028] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention, by using SEQ ID Mutations were made at positions 131 and / or 197 of the amino acid sequence shown in NO.2 to obtain mutants and strains with significantly improved catalytic activity, thermal stability, and organic solvent tolerance compared to wild-type transaminases. Among them, the single-point mutants MyTA1-F131Y, MyTA1-K197R, and the combined mutant MyTA1-F131Y-K197R all exhibited excellent catalytic efficiency and stability, and showed high activity and stereoselectivity in the catalytic synthesis of chiral amines such as (R)-1-Boc-3-aminopiperidine. They also had advantages such as high conversion rate, good optical selectivity, and strong adaptability to reaction conditions. The mutant MyTA1-F131Y-K197R can be used for efficient catalysis of the asymmetric amination synthesis of (R)-1-Boc-3-aminopiperidine from N-Boc-3-piperidinone. The conversion rate can reach more than 90% after 24 hours of reaction at a substrate concentration of 100 g / L, which is suitable for industrial biocatalytic processes. Attached Figure Description

[0029] Figure 1 The results are shown in Example 6, where the transaminase mutant catalyzes N-Boc-3-piperidone at different methanol concentrations.

[0030] Figure 2 The results are those of N-Boc-3-piperidone catalyzed by the transaminase mutant at a substrate concentration of 50 g / L in Example 7.

[0031] Figure 3 The results are those of N-Boc-3-piperidone catalyzed by the transaminase mutant at a substrate concentration of 100 g / L in Example 7. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0033] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0034] Example 1: Preparation of transaminase

[0035] 1. Construction of recombinant genetically engineered E. coli BL21(DE3) / pET28a(+)-MfTA1

[0036] Patent CN120818503A discloses a wild-type transaminase. The wild-type transaminase encoding gene MfTA1 (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) from Mycobacterium sp. is linked with the expression vector pET-28a to construct a heterologous expression vector pET28a(+)-MfTA1 containing the transaminase encoding gene. Then, the heterologous expression vector is transformed into the host bacterium E.coli BL21(DE3) competent cells to obtain the strain E.coli BL21(DE3) / pET28a(+)-MfTA1 containing the heterologous expression vector.

[0037] SEQ ID NO. 1:

[0038] SEQ ID NO. 2: MTIHTEFATSNLVSVEPGAIREDTPPGSVIQYSDYELDYSTEFAGGAAWIEGEYVPASEARISIFDTGFGRSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDPGMSKEELADITKRCVALSGLRESFVNLTVTRGYGKRKGEKDLSKLNHQVYIYAIPYLWIFPPEEQI FGTTAIVPRHVRRAGRNTVDPTIKNYQWGDLTAASFEAKDRGARTAILLDADNCVAEGPGFNVVIVKDGKLFSPSRNALPGITRKTVFEIADAIGIEATLCDVTSRELYDADELMAVTTAGGVTPIVSLDGEAFGDGRPGPITVAIRDRFWALMDEPSPLIEAIDYHHHHHH.

[0039] 2. Preparation of wet mycelium

[0040] After thawing the engineered transaminase strain *E. coli* BL21(DE3) / pET28a(+)-MfTA1 on ice, the bacterial culture was streaked onto LB agar plates containing a final concentration of 50 mg / L kanamycin and incubated at 37°C for 12 h. A single colony was picked and inoculated into a test tube containing 10 mL of LB liquid medium (final kanamycin concentration 50 mg / L), and cultured at 37°C with shaking at 180 rpm for 7–8 h. 2 mL of the bacterial culture from the test tube was then inoculated into a shake flask containing 100 mL of LB liquid medium (final kanamycin concentration 50 mg / L), and cultured at 37°C with shaking at 220 rpm for 2 h. 100 µL of 0.1 M IPTG was added, and the culture was incubated at 28°C with shaking at 220 rpm for 12–14 h. The cells were collected by centrifugation at 4°C and 8000 rpm for 10 min and stored at -20°C for later use.

[0041] Example 2: Verification of the catalytic activity and chirality of transaminases

[0042] 1 mL reaction system: 0.1 g of the wet bacterial cells obtained in Example 1 was weighed and added to 500 µL of PB buffer (pH 9.5) containing 1 M isopropylamine and 1 mM PLP, and resuspended. 0.4 g of substrate (N-Boc-3-piperidinone) was weighed and dissolved in methanol to a final volume of 10 mL. 500 µL of this solution was added to the reaction mixture (N-Boc-3-piperidinone concentration in the reaction system was 20 g / L). The reaction temperature was controlled at 40 °C, and the reaction was carried out for 24 h. The conversion rate was detected by HPLC. After the reaction, the product was extracted with ethyl acetate, centrifuged and concentrated, and then reconstituted with anhydrous ethanol. The chirality was detected by HPLC. The results showed that the conversion rate after 24 h was 95%, and the ee value of the product was greater than 99%.

[0043] The quantitative analysis conditions for the product were as follows: C18 column (4.6 mm × 250 mm, 5.0 µm), mobile phase 26% acetonitrile: 72% 10 mM ammonium acetate (pH adjusted to 4.5 by acetic acid), flow rate 1 mL / min, UV absorption wavelength 210 nm, and injection volume 5 μL.

[0044] The chiral detection conditions were as follows: Daicel Chiralpak AD-H column (4.6 × 250 mm, 5.0 µm), n-hexane:0.1% ethylenediamine ethanol = 90%:10%, flow rate 1 mL / min, column temperature 30℃, detection wavelength 200 nm, and injection volume 5 μL.

[0045] Example 3: Construction of transaminase mutants

[0046] Using the heterologous expression vector pET28a(+)-MfTA1 prepared in Example 1 as a template, the site-directed mutagenesis primers described below (Table 1) were used, with the mutation sites marked underlined, and the mutations were introduced by PCR.

[0047] Table 1. Primer Table Primers Sequence 5'-3' F131Y-F <![CDATA[CTGGTCTGCGTGAATCT TAT GTTAACC<!-- 4 --> ]]> F131T-F <![CDATA[CTGGTCTGCGTGAATCT ACC GTTAACC]]> F131D-F <![CDATA[CTGGTCTGCGTGAATCT GAC GTTAACC]]> F131R AGATTCACGCAGACCAGACAGAGCAA K197R-F <![CDATA[GTTGACCCGACCATC CGA AACTACCAG]]> K197L-F <![CDATA[GTTGACCCGACCATC CGG AACTACCAG]]> K197-R GATGGTCGGGTCAACGGTGTTACGACC .

[0048] The PCR reaction procedure was as follows: 95℃ for 5 min, 98℃ for 30 s, 56℃ for 30 s, 72℃ for 3 min 40 s, repeated for 32 cycles; extension at 72℃ for 5 min. The PCR product was treated with DpnI at 37℃ for 2 h, transformed into E. coli BL21(DE3) recipient bacteria, plated on LB agar plates containing a final concentration of 50 mg / L kanamycin resistance, and incubated at 37℃ for 12 h. Single colonies were randomly selected for sequencing analysis, and wet cells of each transaminase mutant MyTA1-F131Y, MyTA1-F131T, MyTA1-F131D, MyTA1-K197R, and MyTA1-K197L were obtained according to the method in Example 1.

[0049] Furthermore, using plasmid DNA containing the MyTA1-F131Y and MyTA1-K197R genes as templates, mutations were introduced by PCR. The combined mutation primers are shown below, with the mutation sites underlined:

[0050] F131Y-K197R-F: 5'-GTTGACCCGACCATC CGA AACTACCAG -3';

[0051] F131Y-K197R-R: 5'- GATGGTCGGGTCAACGGTGTTACGACC -3'.

[0052] The PCR reaction procedure was as follows: 95℃ for 5 min, 98℃ for 30 s, 56℃ for 30 s, 72℃ for 3 min 30 s, repeated for 32 cycles; extension was performed at 72℃ for 5 min. The PCR product was inactivated by DpnI at 37℃ for 3 h and then transformed into E. coli BL21(DE3) recipient bacteria. The transformed bacteria were plated on LB agar plates containing a final concentration of 50 mg / L kanamycin and incubated at 37℃ for 12 h. Single colonies were randomly selected for sequencing analysis, and wet cells of the recombinant transaminase combination mutant MyTA1-F131Y-K197R (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4) were obtained according to the method in Example 1.

[0053] SEQ ID NO. 3: ATGACCATCCACACCGAATTCGCTACCTCTAACCTGGTTTCTGTTGAACCGGGTGCTATCCGTGAAGACACCCCGCCGGGTTCTGTTATCCAGTACTCTGACTACGAACTGGACTACTCTACCGAATTCGCTGGTGGTGCTGCTTGGATCGAAGGTGAATACGTTCCGGCTTCTGAAGCTCGTATCTCTATCTTCGACACCGGTTTCGGTAGATCTGACCTGACCTACACCGTTGCTCACGTTTGGCACGGTAACATCTTCCGTCTGGGTGACCACCTGGACCGTCTGCTGGACGGTGCTCGTAAACTGCGTCTGGACCCGGGTATGTCTAAAGAAGAACTGGCTGACATCACCAAACGTTGCGTTGCTCTGTCTGGTCTGCGTGAATCT TAT GTTAACCTGACCGTTACCCGTGGTTACGGTAAACGTAAAGGTGAAAAAGACCTGTCTAAACTGAACCACCAGGTTTACATCTACGCTATCCCGTACCTGTGGATTTTCCCGCCGGAAGAACAGATCTTCGGTACCACCGCTATCGTTCCGCGTCACGTTCGTCGTGCTGGTCGTAACACCGTTGACCCGACCATC CGAAACTACCAGTGGGGTGACCTGACCGCTGCTTCTTTCGAAGCTAAAGACCGTGGTGCTCGTACCGCTATCCTGCTGGACGCTGACAACTGCGTTGCTGAAGGTCCGGGTTTCAACGTTGTTATCGTTAAAGACGGTAAACTGTTCTCTCCGTCTCGTAACGCTCTGCCGGGTATCACCCGTAAAACCGTTTTCGAAATCGCTGACGCTATCGGTATCGAAGCTACCCTGTGCGACGTTACCTCTCGTGAACTGTACGACGCTGACGAACTGATGGCTGTTACCACCGCTGGTGGTGTTACCCCGATCGTTTCTCTGGACGGTGAAGCTTTCGGTGACGGTCGTCCGGGTCCGATCACCGTTGCTATCCGTGACAGGTTCTGGGCTCTGATGGACGAACCGTCTCCGCTGATCGAAGCTATCGACTACCACCACCACCACCACCACTGA

[0054] SEQ ID NO. 4: MTIHTEFATSNLVSVEPGAIREDTPPGSVIQYSDYELDYSTEFAGGAAWIEGEYVPASEARISIFDTGFGRSDLTYTVAHVWHGNIFRLGDHLDRLLDGARKLRLDPGMSKEELADITKRCVALSGLRES Y VNLTVTRGYGKRKGEKDLSKLNHQVYIYAIPYLWIFPPEEQIFGTTAIVPRHVRRAGRNTVDPTI R NYQWGDLTAASFEAKDRGARTAILLDADNCVAEGPGFNVVIVKDGKLFSPSRNALPGITRKTVFEIADAIGIEATLCDVTSRELYDADELMAVTTAGGVTPIVSLDGEAFGDGRPGPITVAIRDRFWALMDEPSPLIEAIDYHHHHHH

[0055] Note: The underlined positions are the mutation sites.

[0056] Example 4: Screening of Transaminase Mutants

[0057] The transaminase obtained in Example 1 and the transaminase mutant obtained in Example 3 were analyzed by enzyme activity determination. A 1 mL reaction system was constructed according to the method shown in Example 2, the reaction temperature was controlled at 45℃, and the reaction was carried out for 4 h. The conversion rate was detected by HPLC. The relative enzyme activity was calculated as (mutant conversion rate / wild-type conversion rate) * 100%.

[0058] The results are shown in Table 2. Site-directed mutagenesis further improved the catalytic activity of transaminases. The optimal single mutant was MyTA1-F131Y, which showed a 5.12% increase compared to the wild type. The optimal mutant obtained by combining different mutation sites was MyTA1-F131Y-K197R, which showed an 11.78% increase compared to the wild type.

[0059] Table 2. Comparison of relative enzyme activities enzymes Relative enzyme activity (%) Wild type (MyTA1) 100.00 MyTA1-F131Y 108.09 MyTA1-F131T 106.45 MyTA1-F131D 102.7 MyTA1-K197R 105.12 MyTA1-K197L 104.56 MyTA1-F131Y-K197R 111.78 .

[0060] Example 5: Screening for transaminase mutants with high thermal stability

[0061] 1 mL reaction system: Weigh 0.1 g of the wet bacterial cells obtained in Example 1, add 500 µL of PB buffer containing 1 M isopropylamine, 1 mM PLP, pH=9.5, and resuspend. Weigh 0.4 g of substrate (N-Boc-3-piperidinone) and dissolve it in methanol to a final volume of 10 mL. Take 500 µL of this solution and add it to the reaction mixture (the concentration of N-Boc-3-piperidinone in the reaction system is 20 g / L). Incubate at 70 °C for 2 h, then react at 45 °C for 1 h. Take a sample and perform HPLC to detect the conversion rate.

[0062] As shown in Table 3, the results of the residual enzyme activity determination show that the site-directed mutagenesis technology in Example 1 improved the stability of transaminase. The optimal mutant with single mutant was MyTA1-F131Y, which was 4.6 times higher than the wild type. The optimal mutant obtained after combining mutations at different mutation sites was MyTA1-F131Y-K197R, which was 5.0 times higher than the wild type.

[0063] Table 3. Comparison of Residual Enzyme Activities enzymes Remaining enzyme activity (%) Wild type (MyTA1) 13.52 MyTA1-F131Y 65.30 MyTA1-F131T 25.93 MyTA1-F131D 29.70 MyTA1-K197R 54.28 MyTA1-K197L 33.56 MyTA1-F131Y-K197R 68.10 .

[0064] Example 6: Catalysis of N-Boc-3-piperidinone with different concentrations of methanol

[0065] 0.1 g of the wet bacterial cell MyTA1-F131Y-K197R prepared in Example 3 was used to construct a 1 mL reaction system according to the method shown in Example 2. The substrate (N-Boc-3-piperidinone) was dissolved in methanol of different concentrations as a co-solvent. After reacting at 45°C and 800 rpm for 1 h, 200 µL of 1 M hydrochloric acid was added to terminate the reaction. The conversion rate was then measured by HPLC. The results are shown in Table 4. The highest catalytic efficiency (77.91%) was achieved with 10% methanol as the co-solvent. Furthermore, high conversion rates were observed in concentrations ranging from 10% to 30%. Considering substrate solubility, subsequent experiments used 25% methanol to dissolve the substrate.

[0066] Table 4. Catalytic effect at different methanol concentrations Methanol concentration (%) Conversion rate (%) 50 32.29 40 60.59 30 69.67 25 70.11 20 74.00 10 77.91 .

[0067] Example 7: Optimal transaminase mutant catalyzes 50 g / L N-Boc-3-piperidinone

[0068] 1 g of the wet bacterial cell MyTA1-F131Y-K197R prepared in Example 3 was used to construct a 10 mL reaction system. Different masses of substrate (N-Boc-3-piperidinone) were dissolved in 25% methanol. The pH of the PB buffer was 9, the PLP concentration was 2.5 mM, and the isopropylamine concentration was 2 M. The reaction was carried out at 45 °C and 800 rpm for 48 h. Samples were taken periodically, and 200 µL of 1 M hydrochloric acid was added to terminate the reaction. The conversion rate was then analyzed by HPLC. The results are as follows: Figure 2 As shown.

[0069] The results showed that under the reaction conditions of 25% methanol, 2.5 mM PLP, 2 M isopropylamine, pH=9, and 45℃, the conversion rate reached 99% after 24 h of reaction with 50 g / L substrate; and the conversion rate was >90% after 24 h of reaction with 100 g / L substrate.

[0070] Example 8: Optimal transaminase mutant catalyzes 100 g / L N-Boc-3-piperidinone

[0071] 1 g of the wet bacterial cell MyTA1-F131Y-K197R prepared in Example 3 was used to construct a 10 mL reaction system. Different masses of substrate (N-Boc-3-piperidinone) were dissolved in 25% methanol. The pH of the PB buffer was 9, the PLP concentration was 2.5 mM, and the isopropylamine concentration was 2 M. The reaction was carried out at 45 °C and 800 rpm for 48 h. Samples were taken periodically, and 200 µL of 1 M hydrochloric acid was added to terminate the reaction. The conversion rate was then analyzed by HPLC. The results are as follows: Figure 3 As shown.

[0072] The results showed that under the reaction conditions of 25% methanol, 2.5 mM PLP, 2 M isopropylamine, pH=9, and 45℃, the conversion rate reached 99% after 24 h of reaction with 100 g / L substrate; the conversion rate was >90% after 24 h of reaction with 100 g / L substrate.

[0073] The transaminase mutant obtained in this invention significantly enhances catalytic activity and structural stability through precise amino acid site modification (such as single / multi-point mutations at positions 131 and 197). Conformation optimization of the mutant allows it to maintain high activity even under high temperature or extreme pH conditions, significantly broadening its industrial application scope. By constructing a recombinant vector containing the mutant's encoding gene and transforming it into host bacteria, efficient expression of the enzyme protein was achieved. The recombinant genetically engineered bacteria can produce the mutant on a large scale, significantly reducing production costs. The design of the recombinant vector ensures stable gene delivery and high-level expression, providing reliable technical support for industrial enzyme production. This mutant exhibits excellent stereoselectivity in catalyzing the synthesis of (R)-1-Boc-3-aminopiperidine from N-Boc-3-piperidinone, significantly improving product purity and effectively reducing subsequent purification steps. In summary, this transaminase mutant, through genetic engineering optimization, achieves efficient and highly selective chiral amine synthesis. Its application not only improves reaction efficiency and product quality but also simplifies the production process and reduces environmental impact, making it of significant value in the pharmaceutical and chemical industries.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A transaminase mutant, characterized in that, The transaminase mutant was obtained by making a single or multiple mutations at position 131 and / or position 197 of the amino acid sequence shown in SEQ ID NO.

2.

2. The transaminase mutant according to claim 1, characterized in that, The transaminase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO. 2 to at least one of the following: (1) The phenylalanine at position 131 is mutated to any one of aspartic acid, threonine, or tyrosine; (2) The lysine at position 197 is mutated to either arginine or leucine.

3. The transaminase mutant according to claim 2, characterized in that, The transaminase mutant was obtained by mutating phenylalanine at position 131 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine and lysine at position 197 to arginine.

4. A gene encoding a transaminase mutant according to any one of claims 1 to 3.

5. A recombinant vector containing the encoding gene of claim 4.

6. A recombinant genetically engineered bacterium containing the encoding gene of claim 4.

7. A transaminase mutant according to any one of claims 1 to 3 in the catalytic synthesis ( R Application of 1-Boc-3-aminopiperidine.

8. The application according to claim 7, characterized in that, The method of application includes: fermenting and culturing recombinant genetically engineered bacteria containing the transaminase mutant encoding gene, centrifuging to obtain wet bacterial cells, immobilized wet bacterial cells, and using enzymes extracted or immobilized after ultrasonic disruption of wet bacterial cells as catalysts, and under the conditions of adding an amino donor and pyridoxal phosphate, using N-Boc-3-piperidone as a substrate and phosphate buffer as a reaction medium to form a reaction system, followed by separation and purification to obtain ( R )-1-Boc-3-aminopiperidine.

9. The application as described in claim 8, characterized in that, The application meets at least one of the following conditions: (a) Methanol is used as a co-solvent for N-Boc-3-piperidinone, with a volume concentration of 10% to 30% in the reaction system; (b) The pH of the reaction system is 8 to 9.5; (c) The reaction temperature of the reaction system is 45℃; (d) The concentration of pyridoxal phosphate in the reaction system is 2.5~4 mM; (e) The amino donor is isopropylamine, and its concentration in the reaction system is 1.5~2.5 M; (f) The catalyst is a wet bacterial cell, which is obtained by fermentation culture of recombinant genetically engineered bacteria containing the transaminase mutant encoding gene, and the concentration in the reaction system is 100~150 g / L; (g) The concentration of N-Boc-3-piperidinone in the reaction system is 50 g / L or 100 g / L.

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