Transaminase mutant and its application in synthesis of sitagliptin

Abstract

The application provides a transaminase, the amino acid sequence of which is shown as SEQ ID NO. 3, which can catalyze 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 in a methanol solution reaction system.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme catalysis, and particularly relates to a transaminase and application thereof in synthesis of sitagliptin. BACKGROUND

[0002] Sitagliptin (compound II) is a chemical name of 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, and its phosphate monohydrate is a best-selling dipeptidyl peptidase 4 (DPP-4) inhibitor drug, which can improve the blood glucose control of patients with type 2 diabetes by increasing the level of active intestinal incretin hormone, and has the advantages of good safety, low incidence of adverse reactions and the like.

[0003] At present, the transaminase biocatalyst is mainly used to synthesize sitagliptin in industry, which has the advantages of simple route, high optical purity of product and high substrate conversion rate.

[0004]

[0005] Phosphorylated pyridoxal (PLP) is added as a coenzyme in the reaction process, and the reversible transfer of amino group from the donor to the hydroxyl acceptor is catalyzed. Patent document CN102405281A discloses that on the basis of Arthrobacter sp. (R)-ω-transaminase, the 3D structure of the protein is simulated by computer-aided design, and strategies such as amino acid site combination mutation, site saturation mutation and random mutation of the whole gene sequence are used, and finally a new transaminase mutant is constructed, which can use a mixture of 50% water phase-50% DMSO organic phase as a reaction solvent, and can efficiently catalyze the synthesis of sitagliptin from (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-μ1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one (compound I). However, a large amount of expensive DMSO is used in the enzyme reaction system, which causes complex post-treatment and increases the production cost of the product. Compared with DMSO, alcohol such as methanol or ethanol is low in cost and easy to handle, so using methanol or ethanol to replace the cosolvent DMSO in the enzyme reaction system can significantly reduce the production cost of the product. The inventors disclose a Arthrobacter sp. R-type transaminase mutant ATA64 in patent document CN2021115167214, which can use 50% water phase-50% methanol organic phase as a reaction solvent to catalyze the synthesis of compound II from compound I. SUMMARY

[0006] In order to further improve the enzyme activity of the transaminase mutant ATA64 (SEQ ID NO: 1 herein) disclosed in CN2021115167214 in the methanol-containing reaction system, the inventors continue to modify the mutant enzyme ATA64, expecting to obtain a mutant enzyme that is resistant to a methanol environment and has higher catalytic efficiency, thereby further reducing the production cost of sitagliptin. On this basis, a large number of sites are screened, and a mutant capable of efficiently catalyzing the synthesis of sitagliptin from compound I in a methanol-containing reaction system is screened. Specifically, the present application comprises the following technical solutions:

[0007] A transaminase is a polypeptide selected from the following:

[0008] (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3;

[0009] (b) a polypeptide having more than 95%, preferably more than 96%, preferably more than 97%, preferably more than 98%, more preferably more than 99% homology with SEQ ID NO: 3, and improved enzyme activity in a methanol solution reaction system compared with SEQ ID NO: 3.

[0010]

[0011] Herein, the transaminase having an amino acid sequence of SEQ ID NO: 3 is named as ATA84, which is a mutant having aspartic acid at position 35 mutated to alanine (N35A), aspartic acid at position 120 mutated to glutamic acid (D120E), methionine at position 122 mutated to valine (M122V), phenylalanine at position 127 mutated to isoleucine (F127I), leucine at position 131 mutated to tyrosine (L131Y), aspartic acid at position 165 mutated to aspartic acid (N165D), alanine at position 169 mutated to leucine (A169L), and leucine at position 213 mutated to arginine (L213R) of SEQ ID NO: 1 (i.e. ATA64 disclosed in CN2021115167214).

[0012] The enzyme activity mentioned above refers to the enzyme activity when (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 converted into sitagliptin.

[0013] The present application also provides a gene encoding the above-mentioned transaminase.

[0014] For example, the gene encoding the transaminase of SEQ ID NO: 3 can be a polynucleotide as set forth in SEQ ID NO: 4, or a polynucleotide having more than 90%, preferably more than 92%, preferably more than 95%, preferably more than 97%, preferably more than 98%, more preferably more than 99% homology with SEQ ID NO: 4.

[0015] The present application 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.

[0016] Another aspect of the present application provides a microorganism for expressing the above-mentioned transaminase such as SEQ ID NO: 3, which has integrated into its genome the above-mentioned encoding gene such as SEQ ID NO: 4, or which has been transformed with the above-mentioned plasmid.

[0017] The above-mentioned plasmid can be transformed into a cell competent by conventional chemical transformation or electroporation method. The above-mentioned gene editing technology is selected from the group consisting of homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), and the like.

[0018] Preferably, the above-mentioned microorganism is a microorganism with fast proliferation speed and suitable for expressing exogenous recombinant proteins, for example, selected from the group consisting of Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida mihlanga, Pichia pastoris, Saccharomyces cerevisiae. Preferably, the microorganism is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0019] 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, using (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 as the reaction substrate, the above-mentioned transaminase or the above-mentioned microorganism is used to catalyze the aminotransfer reaction to obtain sitagliptin.

[0020] An organic solvent is added as a cosolvent for the substrate in the above-mentioned reaction system, which includes but is not limited to methanol, ethanol, propanol, isopropanol, DMSO, or a mixture of two or more thereof.

[0021] When the co-solvent is methanol, the concentration of methanol in the reaction system is 10%-60%, preferably 50%.

[0022] In an embodiment, pyridoxal phosphate is included in the reaction system as a coenzyme.

[0023] Preferably, o-phenylenediamine dihydrochloride and / or isopropylamine can also be included in the above reaction system as an amino donor.

[0024] Further, the reaction temperature can be 35-50℃, preferably 38-49℃, preferably 39-48℃, preferably 40-46℃, preferably about 45℃. The reaction pH value 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.

[0025] Further, the concentration of methanol in the reaction system is 40%-60%, preferably 50%. The concentration of substrate I is 20-200g / L, preferably 150g / L.

[0026] The present application uses solvent methanol as an environmental screening pressure, combined with genetic engineering mutation technology, after further mutation of the transaminase mutant ATA64 (SEQ ID NO: 1 herein) reported in patent document CN2021115167214, a mutant SEQ ID NO: 3 with higher tolerance to methanol environment and higher enzyme activity is screened, which has high conversion rate in catalyzing the reaction of compound I, high stereoselectivity of product II, and improves the economy of the enzyme synthesis process of sitagliptin. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the HPLC spectrum of the mutant strain EcATA84 catalyzing the synthesis of sitagliptin (compound II).

[0028] Figure 2 is the HPLC spectrum of the starting strain EcATA64 catalyzing the synthesis of sitagliptin under the same reaction conditions. DETAILED DESCRIPTION

[0029] The transaminase of the present application is a mutant obtained by further mutation of ATA64 (i.e. SEQ ID NO: 1) disclosed in patent document CN2021115167214, which has significantly improved alcohol (especially methanol) tolerance and improved enzyme activity, and can be applied to the sitagliptin enzyme catalytic reaction system using methanol / ethanol and other alcohols as co-solvents.

[0030] In this technical solution, ATA64 with amino acid sequence SEQ ID NO: 1 is used as the initial enzyme for mutation.

[0031]

[0032] In the present text, the terms "starting enzyme", "initial enzyme", "starting enzyme" mean the same and refer to transaminase ATA64 having the amino acid sequence of SEQ ID NO: 1. Sometimes for the sake of convenience, the starting enzyme and its mutants such as SEQ ID NO: 3 and the like can be collectively referred to as "transaminase" in the present text.

[0033] In the present text, the terms "(enzyme activity) increase" or "increase" mean an increase of at least 100% compared to a reference level, for example 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.

[0034] The term "mutation" includes, but is not limited to, substitution, deletion, insertion, chemical modification of an amino acid residue, preferably a mutation which is a positive mutation, i.e. an increase in enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution or a combination of non-conservative and conservative substitutions. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues having similar side chains and thus generally includes the substitution of amino acids within a given class definition for one another in a polypeptide. However, as used herein, a conservative mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitutions if the conservative mutation can alternatively be a substitution of aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or a restriction residue to a restriction residue. It is well known in the art that common instances of conservative substitutions include: interchanges among the aromatic amino acids F, W, Y; interchanges among the hydrophobic amino acids L, I, V; interchanges among the polar amino acids Q, N; interchanges among the basic amino acids K, R, H; interchanges among the acidic amino acids D, E; interchanges among the hydroxyl amino acids S, T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions are, for example:

[0035]

[0036] A "non-conservative substitution" refers to a substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. A non-conservative substitution can use amino acids from groups listed above, but outside of the groups, rather than within. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the region of the substitution (e.g., a proline in place of glycine), (b) the charge or hydrophobicity, or (c) the side chain bulk.

[0037] A "deletion" refers to a modification made to a polypeptide by removing one or more amino acids from a reference polypeptide. A deletion can include 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 enzyme activity and / or retaining the improved properties of the engineered aldolase. Deletions can be to the interior and / or the ends of the polypeptide. In various embodiments, deletions can comprise contiguous segments or can be non-contiguous.

[0038] An "insertion" refers to a modification made to a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered aldolases include one or more amino acids inserted into a naturally occurring aldolase and one or more amino acids inserted into other improved aldolase polypeptides. The insertion can be to the interior of the polypeptide, or to the carboxy- or amino-terminus. As used herein, insertions include fusion proteins as known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more amino acids in the naturally occurring polypeptide.

[0039] The mutant enzyme SEQ ID NO: 3 screened from the random mutation point library constructed by error-prone PCR method by methanol environment pressurization is a mutant with individual amino acid replacement of the initial enzyme SEQ ID NO: 1, and the specific changes are that the asparagine at position 35 is mutated to alanine (N35A), the aspartic acid at position 120 is mutated to glutamic acid (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).

[0040] The amino acid number of the transaminase mutant SEQ ID NO: 3 of the present application is 330, and the structure is clear, so that the coding gene, the expression cassette and plasmid containing the gene, and the transformant containing the plasmid can be easily obtained by those skilled in the art. These genes, expression cassettes, plasmids, and transformants can be obtained by gene engineering construction methods well known to those skilled in the art.

[0041] In order to express the transaminases optimally in E. coli, which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon-optimized. Codon-optimization is a technique that can be used to maximize protein expression in an organism by increasing the efficiency of translation of the gene of interest. Different organisms generally show a particular bias for one of the codons that encodes the same amino acid due to mutational bias and natural selection. For example, in fast-growing microorganisms such as E. coli, the optimized codons reflect the composition of their respective genomic tRNA pool. Thus, in fast-growing microorganisms, the low-frequency codons for an amino acid can be replaced with a codon for the same amino acid but of high frequency. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0042] For example, in order to express the transaminases in E. coli, the coding gene of the initial transaminase SEQ ID NO: 1 codon-optimized can be SEQ ID NO: 2; the coding gene of the transaminase mutant SEQ ID NO: 3 can be SEQ ID NO: 4.

[0043] The transformant host described above can be any microorganism suitable for expressing the transaminase, including bacteria and fungi. The preferred microorganism is Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or E. coli, preferably E. coli, more preferably E. coli BL21 (DE3).

[0044] When used as a biocatalyst for the production of sitagliptin, the transaminase of the present application can be in the form of an enzyme or in the form of a bacterial cell. The form of the enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier-immobilized enzyme, etc.; the form of the bacterial cell includes viable bacterial cell, dead bacterial cell, immobilized bacterial cell, etc.

[0045] When the microorganism such as Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or E. coli is no longer proliferated by fermentation, but is used for enzyme catalysis, it is a natural immobilized enzyme itself, and does not need to be broken down, even extracted and purified, and can be used as an enzyme preparation for catalysis. Since the reaction substrate and the reaction product are both small molecular compounds, they can easily pass through the biological barrier of the bacterial cell membrane, so the bacterial cell does not need to be broken down, which is economically advantageous.

[0046] The present application is further described in conjunction with the following specific examples. It should be understood that the following examples are intended to illustrate the present application and are not intended to limit the scope of the present application.

[0047] Embodiments

[0048] The amount of addition, content and concentration of various substances are referred to in this paper, wherein the percentage content refers to the mass percentage content unless otherwise specified.

[0049] Materials and methods

[0050] The total gene synthesis, primer synthesis and sequencing in the examples were completed by Suzhou JUNWENZHI Biotechnology Co., Ltd.

[0051] The molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., which are mainly carried out according to the Molecular Cloning Laboratory Guide (3rd Edition), J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translation, Science Press, Beijing, 2002. If necessary, the specific experimental conditions can be determined by simple tests.

[0052] PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple test.

[0053] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium plus 20 g / L agar powder.)

[0054] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4.3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20 g / L agar powder.)

[0055] 20X electrotransformation mother liquor: 80 g / L glycine, 2% Tween 80.

[0056] HPLC detection conditions: column C18 (4.6 x 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.

[0057] Chiral detection chromatographic conditions: column CHIRALPAK AD-H (4.6 x 250 mm, 5 μm); mobile phase is n-hexane: ethanol: triethylamine (40:60:0.1); flow rate is 0.7 mL / min, detection wavelength is 268 nm.

[0058] It should be noted that for the convenience of description, in the examples, the strain number, plasmid number, enzyme number, and enzyme coding gene number can share a number, which is easily understood by those skilled in the art, i.e., the same number can refer to different biological forms in different environments.

[0059] Example 1: Construction of recombinant Escherichia coli of initial transaminase gene

[0060] 1.1 According to the method of Example 1 in the patent document CN2021115167214, based on the amino acid sequence SEQ ID NO: 1 of the initial enzyme ATA64, the codon optimization was carried out according to the codon preference of Escherichia coli, and the coding gene SEQ ID NO: 2 thereof was synthesized by Suzhou Jinyuzhi Biological Technology Co., Ltd., and was cloned into the NcoI, BamHI site of plasmid pET28a to obtain plasmid pET-ATA64.

[0061] 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.

[0062] Example 2: Establishment of random mutation point library and high-throughput screening from the first round to the second round

[0063] 2.1 Construction of random mutation point library by error-prone PCR

[0064] The plasmid pET-ATA64 was used as the template to construct the random mutant library by error-prone PCR technology.

[0065] The following primer pair ATA-5 / ATA-3 was designed:

[0066] Forward primer ATA-5: 5'-CTTTAAGAAGGAGATATACCATG-3',

[0067] Reverse primer ATA-3: 5'-GAGCTCGAATTCGGATCCTTA-3'.

[0068] The plasmid pET-ATA64 was used as the template for PCR amplification to obtain a transaminase mutant DNA sequence of about 1.0 kb.

[0069] 50 μL of the error-prone PCR reaction system comprises: 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, 2.5 units of Taq enzyme (Takara).

[0070] The PCR reaction conditions are: 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.

[0071] The PCR product is electrophoresed and gel recovered (Axygen DNA Gel Recovery Kit AP-GX-50). The plasmid pET-ATA64 is used as a template, the recovered product (randomly mutated fragment) of about 1.0 kb is used as a large primer, KOD-plus DNA polymerase is used for MegaPrimer PCR: 94 °C for 5 min; 98 °C for 10 s, 60 °C for 30 s, 68 °C for 2 min / kb, 25 cycles; 68 °C for 10 min. The plasmid template is digested by DpnI restriction endonuclease (Thermo), and E. coli BL21 (DE3) is electroporated to obtain a random mutation library of more than 10 4 clones.

[0072] 2.2 High-throughput screening of the mutant library

[0073] A single colony is picked from an LB plate (containing Kan) of an alternative strain, inoculated into a 96-well plate (each well containing 110 μL of liquid LB-Kan medium), incubated at 37 °C, 400 rpm for 5 h, and then 60 μL of the bacterial solution is taken from each well into a 96-well deep well plate (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG), incubated at 25 °C, 400 rpm for 12-16 h. The bacterial cells are collected by centrifugation at 4 °C, 4000 rpm for 10 min, and the supernatant is removed. Then, the bacterial cells are washed with pre-cooled physiological saline, collected by centrifugation at 4 °C, 4000 rpm for 10 min, and the supernatant is removed. 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, pH 8.5) is added to each well. The bacterial cells are resuspended and incubated at 45 °C, 250 rpm for 1-5 h, and the value at 475 nm is detected. The greater the value, the higher the enzyme activity.

[0074] 2.3 Select the strain with significantly improved activity for nucleic acid sequencing to determine the amino acid mutation site, and HPLC to detect the ee value of the reaction product of these strains, select the strain with product ee value greater than 99.95% and the highest enzyme activity improvement as the starting strain for the next round of random mutant library construction. Commission Suzhou Jinyuizhi Biological Technology Co., Ltd. to perform genome sequencing comparison on the strain with the highest enzyme activity to determine the amino acid sequence change. Repeat the establishment of random mutation library and high-throughput screening in the reaction system with compound I as the substrate and methanol as the cosolvent. The screening results are shown in Table 1.

[0075] Table 1, high-throughput screening results of the first and second rounds of random mutation library

[0076]

[0077] Note: "+" represents that the activity percentage is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the activity percentage is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the activity percentage is greater than 100% and less than or equal to 200% relative to the respective starting strain; "++++" represents that the activity percentage is greater than 200% relative to the respective starting strain.

[0078] After comparison, the strain EcATA73 with high enzyme activity was selected for the next round of mutation.

[0079] Example 3: Establishment of random mutation point library and high-throughput screening in the third to fifth rounds

[0080] 3.1 Construction of random mutation point library by error-prone PCR

[0081] The plasmid of the selected strain was used as the template for the construction of the random mutation library, and the construction method referred to the method for constructing the transaminase random mutation point library in Example 2.

[0082] 3.2 High-throughput screening of the mutant library

[0083] Single colony was picked from LB plate (containing Kan) of alternative strain, inoculated into 96-well plate (containing 110 μL of liquid LB-Kan medium in each well), incubated at 37°C, 400 rpm for 5 h, then 60 μL of bacterial solution was taken from each well into 96-well deep well plate (containing 240 μL of liquid TB-Kan-0.2 mM IPTG in each well), incubated at 25°C, 400 rpm for 12-16 h. The bacterial cells were collected by centrifugation at 4°C, 4000 rpm for 10 min, and the supernatant was removed. Then the bacterial cells were washed with pre-cooled normal saline, collected by centrifugation at 4°C, 4000 rpm for 10 min, and the supernatant was removed. 100 μL of 50% water-50% methanol solution was added to each well to resuspend the cells, and the cells were incubated at 45°C, 800 rpm for 1 h. Then enzyme reaction solution was added to each well to make up to 200 μL, and the final reaction solution contained 70 mM triethanolamine, 70 mM o-phenylenediamine dihydrochloride, 0.5 g / L pyridoxal phosphate, 30 g / L compound I, 55% methanol, and the pH value was 8.5. The resuspended bacterial cells were incubated at 45°C, 250 rpm for 1-5 h, and the value at 475 nm was detected. The greater the value, the higher the enzyme activity.

[0084] 3. The strains with significantly improved activity were selected for nucleic acid sequencing to determine the amino acid mutation site, and HPLC was used to detect the ee value of the reaction product of these strains. The strain with the ee value of the product greater than 99.95% and the highest enzyme activity was selected as the starting strain for the next round of random mutant library construction, and the random mutant library construction and high-throughput screening of the reaction system with compound I as the substrate and methanol as the cosolvent were repeated. The screening results are shown in Table 2.

[0085] Table 2. High-throughput screening results of the third to fifth rounds of random mutant library

[0086]

[0087]

[0088] Note: "+" represents that the activity percentage is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the activity percentage is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the activity percentage is greater than 100% and less than or equal to 200% relative to the respective starting strain; "++++" represents that the activity percentage is greater than 200% relative to the respective starting strain.

[0089] After the above-mentioned multiple rounds of mutation and screening, a relatively ideal mutant enzyme ATA84 was obtained, and the amino acid sequence thereof was SEQ ID NO: 3.

[0090] Example 4: Fermentation culture of the starting strain EcATA64 and the mutant strain EcATA84

[0091] Fermentation tank cultivation was carried out for the starting strain EcATA64 and the mutant strain EcATA84 respectively. Single colony was picked from LB plate (containing Kan) of the strains and inoculated into 5 mL liquid LB medium containing Kan, and incubated at 37°C, 220 rpm overnight. The next day, the inoculation volume was 5% v / v, and the inoculum was transferred into a flask containing 100 mL liquid TB medium, and incubated at 37°C, 220 rpm until OD 600nm 6 was reached, and then transferred into a 5 L fermentation tank as seed liquid. After inoculation, the cultivation was carried out at 37°C, 400-800 rpm / min, and the dissolved oxygen was controlled within 20-30%. When the OD 600nm 20 was reached, IPTG was added to induce the expression of transaminase, and the final concentration of IPTG was 0.2 mM. The cultivation was continued at 28-30°C for 16-24 h, and the bacterial cells were collected by centrifugation for use in 1 L reaction system for catalyzing the synthesis of sitagliptin. Ammonia was used to control the pH value of the whole fermentation process to be 6.8-7.2.

[0092] Example 5: Application of the transaminase mutant for the synthesis of sitagliptin

[0093] In a 1 L reaction system, the whole cells of the strains EcATA64 and EcATA84 were respectively used to catalyze the asymmetric synthesis of sitagliptin (compound II) from the sitagliptin precursor ketone (compound I). The total reaction system included: 100 mM triethanolamine, 1 M isopropylamine, 0.5 g / L pyridoxal phosphate, 150 g / L compound I, 50 g / L cells (wet weight), 50% methanol. 150 g of compound I was dissolved in methanol, and added to the reaction system at a flow rate of 1 mL / min, and hydrochloric acid or isopropylamine was used to control the pH value of the reaction system to be about 8.5.

[0094] The HPLC detection results showed that after 20 h of reaction, the molar yield of the catalytic product involved in the mutant strain EcATA84 was more than 95% (as shown in Figure 1 ), and the e.e. value of the product was greater than 99.95%. Under the same reaction conditions, the whole cells of the starting strain EcATA64 were used to catalyze the asymmetric synthesis of sitagliptin from the sitagliptin precursor ketone, and the molar yield of the product was only about 77% (as shown in Figure 2 ), and the e.e. value of the product was greater than 99.95%. This experiment proved that the transaminase mutant ATA84 had good methanol solvent tolerance, and the enzyme activity of ATA84 was significantly higher than that of the initial enzyme ATA64, which had good application prospect, and laid a foundation for the industrialization of enzyme catalytic production of sitagliptin.

Claims

1. A transaminase, which is a polypeptide with the amino acid sequence shown in SEQ ID NO:

3.

2. The gene encoding the transaminase as described in claim 1.

3. The gene as described in claim 2, characterized in that, The gene encoding the transaminase SEQ ID NO: 3 is a polynucleotide as shown in the nucleotide sequence SEQ ID NO:

4.

4. A plasmid, characterized in that, It contains the gene as described in claim 3.

5. A microorganism for expressing the transaminase as described in claim 1, characterized in that, The genome integrates the gene as described in claim 3, or is transformed with the plasmid as described in claim 4.

6. The microorganism as described in claim 5, characterized in that, The microorganism in question is Escherichia coli.

7. Use of the transaminase as described in claim 1 or the microorganism as described in claim 5 in the production of sitagliptin.

8. The use as described in claim 7, characterized in that, In a reaction system containing an organic solvent as a co-solvent, sitagliptin is obtained by using (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)but-2-one as the reaction substrate and employing the transaminase as described in claim 1 or the microbial-catalyzed aminotransfer reaction as described in claim 5.

9. The use as described in claim 8, characterized in that, The organic solvent is an alcohol.

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

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