Transaminase mutant and application thereof in sitagliptin synthesis

Abstract

The invention provides transaminase, the amino acid sequence of the transaminase is shown as SEQ ID NO.3, and the transaminase can catalyze (2Z)-4-oxo-4-[3-(trifluoromethyl)-5, 6-dihydro [1, 2, 4] triazolo [4, 3-a] pyrazine-7-(8H)-yl]-1-(2, 4, 5-trifluorophenyl) butan-2-one to be converted into sitagliptin in a methanol solution reaction system.

Description

A transaminase mutant and its application in the synthesis of sitagliptin Technical Field

[0001] The present invention belongs to the technical field of enzyme catalysis, and in particular relates to a transaminase and an application thereof in the synthesis of sitagliptin. Background Art

[0002] The chemical name of sitagliptin (Compound II) is 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-trifluoromethyl-1,2,4-triazolo[4,3-a]pyrazine. Its phosphate monohydrate is a best-selling dipeptidyl peptidase 4 (DPP-4) inhibitor drug that can improve blood sugar control in patients with type 2 diabetes by increasing the level of active incretin hormone. It has the advantages of good safety and low incidence of adverse reactions.

[0003] Currently, the industry mainly uses transaminase biocatalysts to synthesize sitagliptin, which has the advantages of a simple route, high product optical purity, and high substrate conversion rate.

[0004]

[0005] During the reaction, pyridoxal phosphate (PLP) is added as a coenzyme to catalyze the reversible transfer of the amino group from the donor to the hydroxyl acceptor. Patent document CN102405281A discloses that based on the (R)-ω-transaminase from Arthrobacter sp., computer-aided design was used to simulate the protein's 3D structure, combined with strategies such as amino acid site combination mutagenesis, site-directed saturation mutagenesis, and random mutagenesis of the entire gene sequence. Ultimately, a novel transaminase mutant was constructed that can efficiently catalyze the synthesis of sitagliptin from (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one (Compound I) using a mixed system of 50% aqueous phase and 50% DMSO organic phase as the reaction solvent. However, the large amount of expensive DMSO used in the enzyme reaction system complicates post-processing and increases product production costs. Compared to DMSO, alcohols such as methanol or ethanol are inexpensive and easy to post-process. Therefore, using methanol or ethanol to replace the cosolvent DMSO in the enzyme reaction system can significantly reduce product production costs. In patent document CN2021115167214, the inventors disclosed an Arthrobacter sp.-derived R-type aminotransferase mutant ATA64, which can catalyze the synthesis of compound I into compound II in a 50% aqueous phase-50% methanol organic phase as the reaction solvent.

[0006] Summary of the Invention

[0007] In order to further improve the enzyme activity of the transaminase mutant ATA64 (SEQ ID NO: 1 herein) disclosed in CN2021115167214 in a methanol-containing reaction system, the inventors continued to transform the mutant enzyme ATA64, hoping to obtain a mutant enzyme with higher catalytic efficiency and tolerance to methanol environment, and further reduce the production cost of sitagliptin. On this basis, many sites were screened to screen out a mutant that can efficiently catalyze compound I to synthesize sitagliptin in a methanol-containing reaction system. Specifically, the present invention includes the following technical solutions:

[0008] A transaminase, which is a polypeptide selected from the group consisting of:

[0009] (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3;

[0010] (b) A polypeptide that has 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, and more preferably 99% or more homology to SEQ ID NO: 3, and has increased enzyme activity in a methanol solution reaction system compared to SEQ ID NO: 3.

[0011]

[0012] Herein, the transaminase with the amino acid sequence of SEQ ID NO: 3 is named ATA84, which is a mutant in which the asparagine at position 35 in SEQ ID NO: 1 (i.e., ATA64 disclosed in CN2021115167214) is mutated to alanine (N35A), the aspartic acid at position 120 is mutated to glutamate (D120E), the methionine at position 122 is mutated to valine (M122V), the phenylalanine at position 127 is mutated to isoleucine (F127I), the leucine at position 131 is mutated to tyrosine (L131Y), the asparagine at position 165 is mutated to aspartic acid (N165D), the alanine at position 169 is mutated to leucine (A169L), and the leucine at position 213 is mutated to arginine (L213R).

[0013] The above enzyme activity refers to the enzyme activity when catalyzing the conversion of (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one into sitagliptin.

[0014] The present invention also provides a gene encoding the above transaminase.

[0015] For example, the gene encoding the transaminase SEQ ID NO: 3 can be a polynucleotide represented by the nucleotide sequence SEQ ID NO: 4, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, preferably 97% or more, preferably 98% or more, and more preferably 99% or more homology to SEQ ID NO: 4.

[0016] The present invention also provides a plasmid comprising the above-mentioned encoding gene. For example, the above-mentioned plasmid can be a pET vector such as pET22b, pET24a, pET28a, or other commonly used vectors such as pSH plasmid.

[0017] Another aspect of the present invention provides a microorganism for expressing the above transaminase, such as SEQ ID NO: 3, wherein the above encoding gene, such as SEQ ID NO: 4, is integrated into the genome of the microorganism, or the microorganism is transformed with the above plasmid.

[0018] The above-mentioned plasmid can be transformed into competent cells by conventional chemical transformation or electroporation. The above-mentioned gene editing technology is, for example, selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0019] Preferably, the microorganism is a microorganism with a fast growth rate and suitable for expressing exogenous recombinant proteins, for example, selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida magnolii, Pichia pastoris, and Saccharomyces cerevisiae. Preferably, the microorganism is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0020] Obviously, the above-mentioned transaminase or the above-mentioned microorganism can be used to produce sitagliptin. For example, in a reaction system containing an organic solvent, preferably an alcohol such as methanol or ethanol as a cosolvent, (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one is used as a reaction substrate, and the above-mentioned transaminase or the above-mentioned microorganism is used to catalyze the transamination reaction to obtain sitagliptin.

[0021] An organic solvent is added to the above reaction system as a co-solvent for the substrate, and the organic solvent includes but is not limited to methanol, ethanol, propanol, isopropanol, DMSO, or a mixture of two or more thereof.

[0022] When the cosolvent is methanol, the concentration of methanol in the reaction system is 10%-60%, preferably 50%.

[0023] In one embodiment, the reaction system contains pyridoxal phosphate as a coenzyme.

[0024] Preferably, the reaction system may further comprise o-phenylenediamine dihydrochloride and / or isopropylamine as an amino group donor.

[0025] Furthermore, the reaction temperature can be 35-50° C., preferably 38-49° C., preferably 39-48° C., preferably 40-46° C., preferably about 45° C. The reaction pH can be 7.0-9.5, preferably pH 7.5-9.2, preferably pH 7.8-9.0, preferably pH 8.0-8.8, preferably pH 8.3-8.6, for example, about pH 8.5.

[0026] Furthermore, the methanol concentration in the reaction system is 40%-60%, preferably 50%. The concentration of substrate I is 20-200 g / L, preferably 150 g / L.

[0027] The present invention uses the solvent methanol as the environmental screening pressure, combines genetic engineering mutation technology, and continues to perform multiple rounds of mutation on the transaminase mutant ATA64 (SEQ ID NO: 1 herein) reported in patent document CN2021115167214. After screening, a mutant SEQ ID NO: 3 that is tolerant to methanol environment and has higher enzyme activity is obtained. The conversion rate of the catalytic reaction of compound I is high, and the stereoselectivity of product II is high, thereby improving the economic efficiency of the enzymatic synthesis process route of sitagliptin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is an HPLC spectrum of the synthesis of sitagliptin (Compound II) catalyzed by the mutant strain EcATA84.

[0029] FIG2 is an HPLC spectrum of sitagliptin synthesized by the starting strain EcATA64 under the same reaction conditions. DETAILED DESCRIPTION

[0030] The transaminase of the present invention is a mutant obtained by further mutation of ATA64 (i.e., SEQ ID NO: 1) disclosed in patent document CN2021115167214, which has significantly improved tolerance to alcohols (especially methanol) and increased enzyme activity. It can be applied to the sitagliptin enzyme-catalyzed reaction system in which alcohols such as methanol / ethanol are used as cosolvents for substrate I.

[0031] In this technical solution, ATA64 with the amino acid sequence SEQ ID NO: 1 is used as the initial enzyme for mutation.

[0032]

[0033] As used herein, the terms "starting enzyme," "initial enzyme," and "starting enzyme" have the same meaning and refer to the transaminase ATA64 having the amino acid sequence of SEQ ID NO: 1. For convenience, the starting enzyme and its mutants, such as SEQ ID NO: 3, may be collectively referred to as "transaminase" herein.

[0034] As used herein, the term "(enzyme activity) increase" or "increase" refers to an increase of at least 100% compared to a reference level, for example, an increase of at least about 1-fold, at least about 2-fold, or at least about 3-fold, or at least about 5-fold, or at least about 10-fold, or at least about 20-fold compared to a reference level.

[0035] The "mutation" includes, but is not limited to, replacement, deletion, insertion, chemical modification of amino acid residues, preferably a forward mutation, i.e., a mutation that improves enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution. "Conservative" amino acid substitutions or mutations refer to the interchangeability of residues with similar side chains, and therefore generally include substitutions of amino acids in the same or similar amino acid definition classes with amino acids in the polypeptide. However, as used herein, if a conservative mutation can instead be aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restricted residue to restricted residue substitution, then a conservative mutation does not include substitutions of hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue. As is well known in the art, common conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions include:

[0036]

[0037] "Non-conservative substitutions" refer to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions can use amino acids between, rather than within, the defined groups listed above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the region of the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk.

[0038] "Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, while retaining enzymatic activity and / or retaining the improved properties of the engineered aldolase. Deletions can be directed to the interior and / or ends of the polypeptide. In various embodiments, deletions can comprise continuous segments or can be discontinuous.

[0039] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered aldolase enzymes include the insertion of one or more amino acids into naturally occurring aldolase enzymes and the insertion of one or more amino acids into other improved aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl or amino termini. As used herein, insertion includes fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids from a naturally occurring polypeptide.

[0040] The mutant enzyme SEQ ID NO: 3, which was screened from a random mutation point library constructed by error-prone PCR under methanol pressure, is a mutant of the initial enzyme SEQ ID NO: 1 in which individual amino acids are replaced. The specific changes are asparagine at position 35 is mutated to alanine (N35A), aspartic acid at position 120 is mutated to glutamic acid (D120E), methionine at position 122 is mutated to valine (M122V), phenylalanine at position 127 is mutated to isoleucine (F127I), leucine at position 131 is mutated to tyrosine (L131Y), asparagine at position 165 is mutated to aspartic acid (N165D), alanine at position 169 is mutated to leucine (A169L), and leucine at position 213 is mutated to arginine (L213R).

[0041] The transaminase mutant of the present invention, SEQ ID NO: 3, has 330 amino acids and a well-defined structure. Therefore, those skilled in the art can easily obtain its encoding genes, expression cassettes and plasmids containing these genes, and transformants containing these plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering methods well known to those skilled in the art.

[0042] In order to express transaminase optimally in the most commonly used intestinal bacteria of genetic engineering, the expression genes of these enzymes can be codon optimized. Codon optimization is a kind of technology that can be used for maximizing protein expression in organism by increasing the translation efficiency of gene of interest. Different organisms usually illustrate the special preference of one of some codons for encoding identical amino acids due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as intestinal bacteria, the optimization codon reflects the composition of its respective genome tRNA library. Therefore, in fast-growing microorganisms, amino acid whose low frequency codon can be used for identical amino acid but high frequency codon replacement. Therefore, the expression of optimized dna sequence dna is improved in fast-growing microorganisms.

[0043] For example, to express a transaminase in E. coli, the gene encoding the codon-optimized starting transaminase SEQ ID NO: 1 can be SEQ ID NO: 2; and the gene encoding the transaminase mutant SEQ ID NO: 3 can be SEQ ID NO: 4.

[0044] The transformant host can be any microorganism suitable for expressing transaminase, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0045] When used as a biocatalyst for the production of sitagliptin, the transaminase of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, and enzymes immobilized on carriers; the bacterial cells include living cells, dead cells, and immobilized cells.

[0046] When microorganisms such as Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli are no longer fermentative and proliferating, but instead used for enzymatic reactions, they become natural immobilized enzymes. These enzymes can be used as enzyme preparations for catalytic reactions without the need for fragmentation or even extraction and purification. Because both the reaction substrates and products are small molecules that easily cross the cell membrane, the biological barrier of the bacteria, fragmentation is unnecessary, which is economically advantageous.

[0047] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0048] Example

[0049] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0050] Materials and methods

[0051] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0052] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were performed primarily with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined by simple experiments when necessary.

[0053] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0054] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (Add 20 g / L agar powder to LB solid medium.)

[0055] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, pH 7.0-7.5. (TB solid medium should also contain 20g / L agar powder.)

[0056] 20X electroporation masterbatch: 80 g / L glycine, 2% Tween 80.

[0057] HPLC detection conditions: chromatographic column C18 (4.6×250 mm, 5 μm); mobile phase A:mobile phase B=7:3 (mobile phase A: 10 mM K2HPO4, 0.1% triethylamine, pH 3.5; mobile phase B: acetonitrile); flow rate 1.0 mL / min; detection wavelength 210 nm.

[0058] Chiral detection chromatographic conditions: chromatographic column CHIRALPAK AD-H (4.6×250 mm, 5 μm); mobile phase: n-hexane:ethanol:triethylamine (40:60:0.1); flow rate: 0.7 mL / min; detection wavelength: 268 nm.

[0059] It should be noted that, for the sake of convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0060] Example 1: Construction of Initial Transaminase Gene Recombinant Escherichia coli

[0061] 1.1 Referring to the method of Example 1 in patent document CN2021115167214, the amino acid sequence of the initial enzyme ATA64, SEQ ID NO: 1, was used as the basis. Codon optimization was performed according to the codon preference of Escherichia coli. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize its encoding gene, SEQ ID NO: 2, and cloned into the NcoI and BamHI sites of plasmid pET28a to obtain plasmid pET-ATA64.

[0062] 1.2 The recombinant plasmid pET-ATA64 was transformed into the expression host Escherichia coli BL21 (DE3) by electroporation to obtain recombinant Escherichia coli EcATA64 expressing the initial transaminase.

[0063] Example 2: Construction of random mutation point library and high-throughput screening from round 1 to round 2

[0064] 2.1 Construction of random mutation point library by error-prone PCR

[0065] A random mutant library was constructed using error-prone PCR technology using plasmid pET-ATA64 as a template.

[0066] The following primer pair ATA-5 / ATA-3 was designed:

[0067] Forward primer ATA-5: 5'-CTTTAAGAAGGAGATATACCATG-3',

[0068] Reverse primer ATA-3: 5′-GAGCTCGAATTCGGATCCTTA-3′.

[0069] Using plasmid pET-ATA64 as a template, PCR amplification was performed to obtain a transaminase mutant DNA sequence of approximately 1.0 kb.

[0070] The 50 μL error-prone PCR reaction system included: 10 ng of plasmid (pET-ATA64) template, 50 pmol of a pair of primers ATA-5 and ATA-3, 1× Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme (Takara).

[0071] PCR reaction conditions were as follows: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min / kbp, 30 cycles; 72°C for 10 min.

[0072] PCR products were electrophoresed and recovered using the Axygen DNA Gel Recovery Kit AP-GX-50. Using plasmid pET-ATA64 as a template and the approximately 1.0 kb recovered product (randomly mutated fragment) as a large primer, KOD-plus DNA polymerase was used for MegaPrimer PCR: 94°C for 5 minutes, 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 2 minutes / kb, for 25 cycles, and 68°C for 10 minutes. The plasmid template was digested with DpnI restriction endonuclease (Thermo Fisher Scientific) and electroporated into E. coli BL21 (DE3), yielding over 10 4 Random mutation library of 10 clones.

[0073] 2.2 High-throughput screening of mutant libraries

[0074] Pick a single colony from the LB plate (containing Kan) of the candidate strain and inoculate it into a 96-well plate (each well contains 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 hours, remove 60 μL of the bacterial solution from each well and transfer it to a 96-well deep-well plate (each well contains 240 μL of liquid TB-Kan-0.2 mM IPTG) and incubate at 25°C and 400 rpm for 12-16 hours. Collect the cells by centrifugation at 4°C and 4000 rpm for 10 minutes, and remove the supernatant culture medium. Subsequently, wash the cells with pre-chilled saline, collect the cells by centrifugation at 4°C and 4000 rpm for 10 minutes, and remove the supernatant. Add 200 μL of enzyme reaction solution (100 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 20 g / L Compound I, 55% methanol) to each well and adjust the pH to 8.5 for reaction. Resuspend the cells and incubate at 45°C and 250 rpm for 1-5 h. Detect the value at a wavelength of 475 nm; a larger value indicates higher enzyme activity.

[0075] 2.3 Select strains with significantly improved activity for nucleic acid sequencing to determine the amino acid mutation sites. At the same time, HPLC is used to detect the ee values ​​of the reaction products of these strains. The strains with product ee values ​​greater than 99.95% and the highest improvement in enzyme activity are selected as the starting strains for the next round of random mutant library construction. Suzhou Jinweizhi Biotechnology Co., Ltd. is commissioned to perform genome sequencing and comparison on the strains with the highest enzyme activity to determine the changes in their amino acid sequences. Repeat the random mutation library establishment and high-throughput screening in the reaction system with compound I as the substrate and methanol as the solubilizer. The screening results are shown in Table 1.

[0076] Table 1. Results of high-throughput screening of the first and second rounds of random mutation libraries

[0077]

[0078] Note: "+" represents that the percentage of viability is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the percentage of viability is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the percentage of viability is greater than 100% and less than or equal to 200% relative to the respective starting strain; "++++" represents that the percentage of viability is greater than 200% relative to the respective starting strain.

[0079] After comparison, the strain EcATA73 with high enzyme activity was selected for the next round of mutagenesis.

[0080] Example 3: Construction of random mutation point library and high-throughput screening from rounds 3 to 5

[0081] 3.1 Construction of random mutation point library by error-prone PCR

[0082] The selected bacterial plasmids were used as templates to construct a random mutation library. The construction method was similar to the method for constructing a transaminase random mutation library in Example 2.

[0083] 3.2 High-throughput screening of mutant libraries

[0084] Single colonies were picked from LB plates (containing Kan) of candidate strains and inoculated into 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 hours, 60 μL of the bacterial culture was removed from each well and transferred to a 96-well deep-well plate (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG) and incubated at 25°C and 400 rpm for 12-16 hours. The cells were harvested by centrifugation at 4°C and 4000 rpm for 10 minutes, and the supernatant was removed. Subsequently, the cells were washed with pre-chilled saline and harvested by centrifugation at 4°C and 4000 rpm for 10 minutes, and the supernatant was removed. Resuspend the cells in 100 μL of a 50% water-50% methanol solution per well and incubate at 45°C and 800 rpm for 1 hour. Subsequently, add enzyme reaction solution to each well to a final volume of 200 μL. The final volume contains 70 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 30 g / L Compound I, and 55% methanol. The reaction is performed at a pH of 8.5. Resuspend the cells and incubate at 45°C and 250 rpm for 1–5 hours. The activity is measured at a wavelength of 475 nm. A higher value indicates higher enzyme activity.

[0085] 3. Strains with significantly improved activity were selected for nucleic acid sequencing to identify amino acid mutation sites. The ee values ​​of the reaction products of these strains were also measured by HPLC. Strains with product ee values ​​greater than 99.95% and the highest enzyme activity were selected as the starting strains for the next round of random mutant library construction. Random mutant library construction and high-throughput screening using Compound I as a substrate and methanol as a solubilizer were repeated. The screening results are shown in Table 2.

[0086] Table 2. Results of high-throughput screening of random mutation libraries from rounds 3 to 5

[0087]

[0088]

[0089] Note: "+" represents that the percentage of viability is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the percentage of viability is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the percentage of viability is greater than 100% and less than or equal to 200% relative to the respective starting strain; "++++" represents that the percentage of viability is greater than 200% relative to the respective starting strain.

[0090] After multiple rounds of mutation and screening, a relatively ideal mutant enzyme ATA84 was obtained, whose amino acid sequence is SEQ ID NO: 3.

[0091] Example 4: Fermentation culture of starting strain EcATA64 and mutant strain EcATA84

[0092] The starting strain EcATA64 and the mutant strain EcATA84 were cultured in fermenters. A single colony was picked from the LB plate (containing Kan) of the strain and inoculated into 5 mL of liquid LB medium containing Kan. The culture was incubated at 37°C and 220 rpm overnight. The next day, the inoculum was transferred to a shake flask containing 100 mL of liquid TB medium at a volume concentration of 5% v / v and cultured at 37°C and 220 rpm until the OD 600nm After reaching 6, transfer the seed solution to a 5L fermenter. After inoculation, culture at 400-800 rpm / min and 37°C, controlling the dissolved oxygen within the range of 20-30%. 600nm After reaching 20, IPTG was added to induce transaminase expression at a final concentration of 0.2 mM. Culture was continued at 28-30°C for 16-24 hours. The cells were collected by centrifugation and used to catalyze the synthesis of sitagliptin in a 1 L reaction system. Ammonia was used to control the pH of the entire fermentation process to 6.8-7.2.

[0093] Example 5: Application of transaminase mutants in the synthesis of sitagliptin

[0094] In a 1L reaction system, whole cells of strains EcATA64 and EcATA84 were used to catalyze the asymmetric synthesis of sitagliptin (Compound II) from the sitagliptin precursor ketone (Compound I). The overall reaction system consisted of: 100mM triethanolamine, 1M isopropylamine, 0.5g / L pyridoxal phosphate, 150g / L Compound I, 50g / L cells (wet weight), and 50% methanol. 150g of Compound I was dissolved in methanol and added to the reaction system at a flow rate of 1mL / min. The pH of the reaction system was controlled at approximately 8.5 using hydrochloric acid or isopropylamine.

[0095] HPLC analysis revealed that after 20 hours of reaction, the mutant strain EcATA84 achieved a molar yield of over 95% (as shown in Figure 1), with an ee value exceeding 99.95%. Under the same reaction conditions, the starting strain EcATA64 whole cells catalyzed the asymmetric synthesis of sitagliptin from the sitagliptin precursor ketone, achieving a molar yield of only approximately 77% (as shown in Figure 2), with an ee value exceeding 99.95%. This experiment demonstrates that the transaminase mutant ATA84 exhibits excellent tolerance to formazan and significantly higher enzyme activity than the starting enzyme ATA64, demonstrating promising application prospects and paving the way for the industrialization of enzymatic production of sitagliptin.

Claims

1. A transaminase, which is a polypeptide selected from the following: (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3; (b) A polypeptide having more than 95% homology with SEQ ID NO: 3 and having an increased enzyme activity in a methanol solution reaction system compared to SEQ ID NO:

3.

2. The transaminase according to claim 1, wherein The enzyme activity refers to the enzyme activity when catalyzing the conversion of (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one into sitagliptin.

3. A gene encoding the transaminase according to claim 1 or 2.

4. The gene according to claim 3, wherein The gene encoding the transaminase of SEQ ID NO: 3 is a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4, or a polynucleotide having 90% or more homology with SEQ ID NO:

4.

5. A plasmid comprising the gene according to claim 4.

6. A microorganism for expressing the transaminase according to claim 1, characterized in that: A microorganism having the gene according to claim 4 integrated into its genome, or having been transformed with the plasmid according to claim 5.

7. The microorganism according to claim 6, wherein The microorganism is Escherichia coli.

8. Use of the transaminase according to claim 1 or the microorganism according to claim 6 in producing sitagliptin.

9. The use according to claim 7, characterized in that In a reaction system containing an organic solvent, preferably an alcohol as a cosolvent, (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one is used as a reaction substrate, and a transaminase as described in claim 1 or a microorganism as described in claim 6 is used to catalyze a transamination reaction to obtain sitagliptin.

10. The use according to claim 8, characterized in that The reaction system contains pyridoxal phosphate as a coenzyme.

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