Transaminase and use thereof in preparing sitagliptin or its intermediates

Abstract

The present invention provides a transaminase and a use for preparing sitagliptin or an intermediate thereof. The amino acid sequence of the transaminase comprises one, two or three amino acid residue differences at residue positions compared to the amino acid sequence shown in SEQ ID NO:1. The present invention also provides a nucleic acid encoding the transaminase, a recombinant expression vector including the nucleic acid, and a transformant comprising the nucleic acid or the recombinant expression vector, and their applications. The present invention also provides an enzyme preparation comprising the transaminase or a combination of the transaminase. The present invention also provides a method for preparing (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, the method using the transaminase, with high conversion rate, good stability, high stereoselectivity, thereby reducing production costs, and being conducive to the progressive effect of industrialized production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a transaminase, use of the transaminase in preparing sitagliptin or an intermediate thereof, and a method for preparing sitagliptin or its intermediate (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone. Background Art

[0002] Diabetes is a metabolic disease caused by altered insulin secretion, leading to insulin deficiency and weakened action, or decreased insulin activity, or a combination of both. It is characterized by hyperglycemia and disturbances in protein, sugar, and fat metabolism. Diabetes and its complications rank third in the list of health hazards after cardiovascular disease and cancer, making them a significant threat to human health. Of the four types of diabetes, type 2 diabetes accounts for over 90% and is most common in people over 30 years old. It is primarily caused by insulin insensitivity.

[0003] Sitagliptin phosphate is the first dipeptidyl peptidase IV (DPP-4) inhibitor approved by the FDA in 2006 for the treatment of type 2 diabetes. Sitagliptin phosphate has a significant hypoglycemic effect when used alone or in combination with metformin or pioglitazone. It is safe, well-tolerated, and has few adverse reactions.

[0004] WO2019011236A1 reports that 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (referred to as morpholinedione in the present invention) can be catalyzed by transaminase to produce (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, and (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone can be further used to produce Boc-(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid (referred to as sitagliptin-Boc butyric acid). Sitagliptin-Boc butyric acid is then reacted to produce sitagliptin phosphate, but the enzyme activity in the reaction needs to be further improved.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of the prior art, such as low conversion rate, when using a transaminase to catalyze a morpholinedione substrate to produce a sitagliptin intermediate or sitagliptin. A transaminase and its use for preparing a sitagliptin intermediate or sitagliptin are provided. When the transaminase of the present invention is used to catalyze a morpholinedione substrate to produce a sitagliptin intermediate or sitagliptin, the conversion rate is high and the stability is good, thereby reducing production costs and facilitating industrial production.

[0007] To solve the above technical problems, the present invention provides a transaminase in a first aspect, wherein the amino acid sequence of the transaminase comprises amino acid residue differences at one, two or three residue positions selected from the group consisting of:

[0008] Amino acid residue 150 is A, L, Q, V, W, T, E, or C;

[0009] Amino acid residue 152 is A, H, I, K, L, M, N, Q, S, T, V, or Y;

[0010] The amino acid residue at position 155 is A, K, N or R.

[0011] In other words, the transaminase has S150A / L / Q / V / W / T / E / C, C152A / H / I / K / L / M / N / Q / S / T / V / Y and / or Q155A / K / N / R changes compared to the amino acid sequence shown in SEQ ID NO: 1. In the present invention, the changes do not necessarily require mutations based on SEQ ID NO: 1. As long as the transaminase ultimately has amino acid differences of S150A / L / Q / V / W / T / E / C, C152A / H / I / K / L / M / N / Q / S / T / V / Y and / or Q155A / K / N / Q / R compared to the amino acid sequence shown in SEQ ID NO: 1, it also falls within the scope of protection of the present invention.

[0012] In some embodiments, the amino acid sequence of the transaminase may have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, or 1-20 amino acid residue differences at positions other than the aforementioned specific positions; these residue differences include substitutions with conservative amino acid residues. Generally, these substitutions do not affect the enzymatic activity of the transaminase.

[0013] In some embodiments, the amino acid sequence of the transaminase comprises amino acid residue differences at residue positions selected from the following two compared to the amino acid sequence shown in SEQ ID NO: 1: amino acid residue 150 is A, L, Q, V or W; amino acid residue 152 is L, Q, T, A or S; and amino acid residue 155 is A or R.

[0014] In some embodiments, the amino acid sequence of the transaminase comprises amino acid residue differences at residue positions selected from the following three compared to the amino acid sequence shown in SEQ ID NO: 1: amino acid residue 150 is A, V, W, T, E, or C; amino acid residue 152 is H, I, K, L, M, N, Q, S, T, V, or Y; and amino acid residue 155 is A, K, N, or R.

[0015] In some preferred embodiments, the amino acid sequence of the transaminase comprises amino acid residue differences at residue positions selected from the following two compared to the amino acid sequence shown in SEQ ID NO: 1: the amino acid residue at position 150 is A, L, V or W; the amino acid residue at position 152 is L, T, A or S.

[0016] In some preferred embodiments, the amino acid sequence of the transaminase comprises an amino acid residue difference at a residue position selected from the following two compared to the amino acid sequence shown in SEQ ID NO: 1: the amino acid residue at position 150 is Q or V; the amino acid residue at position 155 is A or R.

[0017] In some preferred embodiments, the amino acid sequence of the transaminase comprises an amino acid residue difference at a residue position selected from the following two compared to the amino acid sequence shown in SEQ ID NO: 1: the amino acid residue at position 152 is L or Q; the amino acid residue at position 155 is R.

[0018] In some more preferred embodiments, the amino acid sequence of the transaminase comprises an amino acid residue difference selected from the following groups compared to the amino acid sequence shown in SEQ ID NO: 1, as shown in the following table:

[0019]

[0020]

[0021] In some further more preferred embodiments, the nucleotide sequence encoding the transaminase comprises a nucleotide difference selected from the following group compared to the nucleotide sequence shown in SEQ ID NO: 2, as shown in the following table:

[0022]

[0023]

[0024]

[0025] In the above table, 152CTG indicates that the codon encoding the 152nd amino acid residue is replaced by TGC to CTG, 155CGT indicates that the codon encoding the 155th amino acid residue is replaced by CAA to CGT, and 150ACC indicates that the codon encoding the 150th amino acid residue is replaced by AGT to ACC. The replacement of codons for other amino acid residues in this table is represented in a similar manner.

[0026] To solve the above technical problems, the second aspect of the present invention provides an isolated nucleic acid encoding the transaminase as described in the first aspect of the present invention.

[0027] To solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the second aspect of the present invention.

[0028] Preferably, the backbone of the recombinant expression vector is plasmid pET21a.

[0029] To solve the above technical problems, the fourth aspect of the present invention provides a transformant, which is a host into which the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention is introduced.

[0030] Preferably, the host is Escherichia coli; for example, Escherichia coli BL21.

[0031] To solve the above technical problems, the fifth aspect of the present invention provides a method for preparing the transaminase as described in the first aspect of the present invention, which comprises culturing the transformant as described in the fourth aspect of the present invention under conditions suitable for expressing the transaminase.

[0032] In a preferred embodiment, the method specifically includes: (1) inoculating the transformant containing the transaminase into a culture medium containing antibiotics, such as LB culture medium, and shaking culture to obtain a seed solution; (2) transferring the seed solution in (1) to a culture medium containing antibiotics, such as LB culture medium, and shaking culture; (3) adding IPTG to the culture medium in (2) for induction overnight, and collecting the bacteria after centrifugation; (4) washing and resuspending the bacteria collected in (3), crushing and centrifuging to obtain a crude enzyme solution containing the transaminase. The antibiotic can be preferably 50 μg / mL kanamycin. The shaking culture conditions in (1) can be preferably 37°C and 12h. The inoculation amount of the seed solution in (2) can be preferably 2% by volume. The shaking culture conditions can be preferably 37°C and until OD600 is 0.75-0.85, for example, 0.8. The final concentration of IPTG in (3) can preferably be 0.05-5 mM. The temperature for overnight induction can preferably be 15-30° C., for example, 18° C. The centrifugation conditions can preferably be 4000-12000 rpm, for example, 10000 rpm, for 5-30 min, for example, 10 min.

[0033] After the transformant expresses the transaminase, it can be extracted by conventional technical means in the art, for example, a crude enzyme solution can be prepared, and the crude enzyme solution can be subjected to conventional concentration and replacement after preparation, or the crude enzyme solution can be further subjected to one or more purification steps such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography and molecular sieve chromatography to purify the transaminase. In a preferred embodiment, the following steps can be adopted: (1) the transformant containing the transaminase is inoculated into a culture medium containing antibiotics, such as LB culture medium, and shaken to obtain a seed solution; (2) the seed solution in (1) is transferred to a culture medium containing antibiotics, such as LB culture medium, and shaken to obtain a seed solution; (3) IPTG is added to the culture medium in (2) for induction overnight, and the cells are collected after centrifugation; (4) the cells collected in (3) are washed and resuspended, crushed and then centrifuged to obtain a crude enzyme solution containing the transaminase. In a preferred embodiment, the cells collected after centrifugation and the buffer solution are homogenized in a ratio (e.g., 1:7) to obtain a crude enzyme solution.

[0034] In order to solve the above technical problems, the sixth aspect of the present invention provides an enzyme preparation, which comprises the transaminase as described in the first aspect of the present invention.

[0035] Preferably, the enzyme preparation generally includes a cofactor for the transaminase, such as pyridoxal phosphate. In the present invention, the enzyme preparation generally can be an enzyme preparation obtained from a culture of a transformant host cell containing the transaminase or its culture fluid, or a product obtained by processing the same; wherein the product refers to an extract obtained from the transformant host cell, an isolated product obtained by isolating or purifying the transaminase in the extract, or an immobilized product obtained by immobilizing the transformant cell, its extract, or an isolated product of the extract.

[0036] In order to solve the above technical problems, the seventh aspect of the present invention provides an enzyme combination, which comprises two or more of the transaminases described in the first aspect of the present invention.

[0037] To solve the above technical problems, the eighth aspect of the present invention provides a method for preparing (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, which includes the step of catalyzing the substrate 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (abbreviated as morpholinedione) to obtain (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone in the presence of an amino donor in a reaction solvent using the transaminase described in the first aspect of the present invention, the enzyme preparation described in the sixth aspect of the present invention, or the enzyme combination described in the seventh aspect of the present invention.

[0038] Preferably, the reaction solvent is isopropanol and water.

[0039] Preferably, the amino donor is isopropylamine hydrochloride.

[0040] Preferably, the molar ratio of the amino donor to the substrate is 1:1 to 10:1.

[0041] Preferably, the concentration of the substrate is 5-100 g / L, for example 10 g / L.

[0042] Preferably, the mass ratio of the transaminase to the substrate is 1:1 to 6:1, for example 3:1; wherein the transaminase exists in the form of bacteria or protein.

[0043] Preferably, the prepared reaction system also includes a cofactor of transaminase such as pyridoxal phosphate, and the concentration thereof is preferably 0.5-5 mM, such as 1 mM.

[0044] Preferably, the reaction temperature is 30-60°C, such as 45°C.

[0045] Preferably, the rotation speed during the reaction is 100-300 rpm, such as 200 rpm.

[0046] In a preferred embodiment, the transaminase is in the form of a crude enzyme solution. The preparation of the crude enzyme solution may include the following steps:

[0047] The engineered bacteria containing the transaminase gene are cultured in a liquid culture medium such as LB at 37°C until the OD600 reaches 0.6-0.8, IPTG is added at a final concentration of 0.5 mM, and the culture is induced at 20-30°C, such as 18°C, for 16-24 hours. The culture solution is centrifuged at 8000-14000 rpm, such as 10000 rpm, for 5-30 minutes, such as 10 minutes, to collect the bacteria;

[0048] The transaminase-expressing bacteria are homogenized with phosphate buffer at a ratio of 1:7 M / V (g / mL) and centrifuged; the phosphate buffer is, for example, 50 mM phosphate buffer, pH 6.0.

[0049] To solve the above technical problems, the ninth aspect of the present invention provides a method for preparing sitagliptin or sitagliptin phosphate, which comprises the step of preparing (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone according to the preparation method described in the eighth aspect of the present invention.

[0050] To solve the above technical problems, the tenth aspect of the present invention provides a transaminase as described in the first aspect of the present invention, the enzyme preparation as described in the sixth aspect of the present invention, or the enzyme combination as described in the seventh aspect of the present invention for use in the preparation of (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, sitagliptin, or sitagliptin phosphate.

[0051] Preferably, the sitagliptin phosphate is sitagliptin phosphate monohydrate.

[0052] In the present invention, the 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione (also referred to as morpholinedione in the present invention) has the following specific structural formula:

[0053]

[0054] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive progress of the present invention is that when the transaminase of the present invention is used to catalyze the ketoamide substrate to produce the sitagliptin intermediate, the conversion rate is high, the stability is good, and the stereoselectivity is high, thereby reducing the production cost and facilitating industrial production. DETAILED DESCRIPTION

[0057] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0058] The experimental methods in the present invention are conventional methods unless otherwise specified. For details on gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

[0059] Unless otherwise specified, the abbreviated symbols for amino acids in the present invention are conventional in the art. The amino acids corresponding to the specific abbreviated symbols are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] The codons corresponding to the amino acids are also conventional in the art, and the specific correspondence between amino acids and codons is shown in Table 2.

[0064] Table 2

[0065]

[0066] Pet21a was purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher; BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0067] Conversion rate UPLC method: Chromatographic column: ZORBAX Eclipse plus C18 (RRHD, 1.8 μm, 50×2.1 mm); mobile phase A: 0.1% TFA aqueous solution; mobile phase B: 0.1% TFA methanol solution; gradient elution: 60% A+40% B (0.01 min), 30% A+70% B (1.5 min), 100% B (1.6 min), 100% B (2.0 min), 60% A+40% B (2.1 min), 60% A+40% B (3.5 min); column temperature: 35°C; flow rate: 0.5 mL / min; injection volume: 1 μL.

[0068] Retention time of morpholinodione reference substance: 1.408min;

[0069] (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)-1-butanone reference substance retention time: 0.696 min;

[0070] Among them, the morpholinedione substrate raw material and the reference substance (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone were synthesized by our company, and the synthesis method was referenced to WO2019011236A1; the 3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone racemate was synthesized by our company in the laboratory, and was obtained by amination and catalytic hydrogenation of morpholinedione. Determination of the configuration of the reference substance (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone: (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone can be further reacted to produce (3R)-N-tert-butyloxycarbonyl-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid (reference WO2019011236A1). Using the (3R)-N-tert-butyloxycarbonyl-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid standard (purchased from Anhui Haikang Pharmaceutical Co., Ltd.) as a reference, it can be determined to be the R configuration.

[0071] HPLC method for configuration determination:

[0072] Chromatographic column: Daicel Chiralpak AD-H (4.6 mm*250 mm, 5 μm); mobile phase: n-hexane:isopropanol = 90:10; detection wavelength: 210 nm; flow rate: 1.0 ml / min; injection volume: 10 μl; column temperature: 25°C; run time: 40 min.

[0073] The chiral HPLC method for detecting the ee value of the product is as follows:

[0074] Chromatographic column: Daicel Chiralpak AD-H column 4.6 mm × 250 mm, 5 μm; mobile phase: n-hexane:isopropanol:diethylamine = 40:60:0.1; detector: UV 268 nm; column temperature: 25°C; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0075] Retention time of 3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)-1-butanone racemate reference: 10.290 min and 28.087 min;

[0076] (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)-1-butanone reference substance retention time: 28.093 min.

[0077] Example 1 Preparation of Transaminase Mutant Enzyme Solution

[0078] According to the transaminase genes in Table 3, the full genes were synthesized using SEQ ID NOs: 2, 4, and 6, with restriction sites NdeI and HindIII, and ligated to the pET21a vector to obtain recombinant plasmids containing the transaminase genes. The gene synthesis company was Suzhou Jinweizhi Biotechnology Co., Ltd. (Building C3, BioNano Technology Park, 218 Xinghu Street, Suzhou Industrial Park). The recombinant plasmids were transformed into host Escherichia coli BL21 competent cells to obtain engineered strains containing the transaminase genes Enz.1 to Enz.3.

[0079] Similarly, the genes of transaminases Enz.4 to Enz.36 obtained by site-directed mutagenesis of the Enz.1 gene in Table 4 were ligated with the restriction sites NdeI and HindIII, and the vector pET21a was used to obtain recombinant plasmids containing the genes of transaminases Enz.4 to Enz.36, respectively. Each recombinant plasmid was transformed into the host Escherichia coli BL21 competent cells to obtain the engineered strains Enz.4 to Enz.36 containing the transaminase genes in Table 4. 152CTG in the table indicates that the codon encoding the 152nd amino acid residue is replaced by TGC to CTG, 155CGT indicates that the codon encoding the 155th amino acid residue is replaced by CAA to CGT, and 150ACC indicates that the codon encoding the 150th amino acid residue is replaced by AGT to ACC. The replacement of codons for other amino acid residues in this table is indicated in a similar manner.

[0080] After activation by streaking, the engineered bacteria containing the transaminase gene were inoculated into 5 ml of LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C with shaking for 12 hours. A 2% (v / v) inoculum was then transferred to 150 ml of fresh LB liquid medium also containing 50 μg / ml kanamycin and shaken at 37°C until the OD600 reached approximately 0.8. IPTG was then added to a final concentration of 0.5 mM and induced at 18°C ​​for 16 hours. Following incubation, the culture was centrifuged at 10,000 rpm for 10 minutes, the supernatant discarded, and the cells harvested and stored in a -80°C freezer until ready for use.

[0081] Table 3

[0082]

[0083] Table 4

[0084]

[0085]

[0086] Example 2 Various transaminase mutants are used to catalyze the reaction of morpholinodione

[0087] The bacterial cells obtained in Example 1 were homogenized with 50 mM phosphate buffer (pH 7.0) at a ratio of 1:7 (M / V), and the supernatant was collected to obtain a crude transaminase enzyme solution.

[0088] In a 5 mL reaction system (Table 5), 2.5 mL of isopropanol, 50 mg of morpholinodione, 0.1 mL of a 12.5 mg / mL PLP (pyridoxal phosphate) aqueous solution, 0.2 mL of 4 M isopropylamine hydrochloride, 0.5 mL of 1 M triethanolamine, and 1.1 mL of crude enzyme solution were added. The reaction was shaken at 45 ° C and 200 rpm. After 18 hours of reaction, a sample was taken for enzyme inactivation and centrifugation. The supernatant was diluted 20 times with methanol and the conversion rate and ee value were detected.

[0089] The method for determining conversion rate is described in the UPLC method section above. Testing revealed that the retention times of each substrate and product were consistent with those of their respective references. The test results are shown in Tables 6-1, 6-2, 6-3, and 6-4 below. The method for determining ee value is described in the chiral HPLC method described above. Testing revealed that the ee values ​​of the products catalyzed by each mutant enzyme exceeded 99%.

[0090] Table 5 Reaction system

[0091]

[0092] Table 6-1

[0093]

[0094]

[0095] Table 6-2 Conversion rate of the first batch of enzyme mutants

[0096]

[0097] Table 6-3 Conversion rate of the second batch of enzyme mutants

[0098]

[0099]

[0100] Table 6-4 Conversion rate of the third batch of enzyme mutants

[0101]

[0102] As can be seen from Tables 6-2 to 6-4 above, the conversion rates of most of the above enzyme mutants are more than 10% higher than that of Enz.1. SEQUENCE LISTING <110> Yikelai Biotechnology (Shanghai) Co., Ltd. <120> Transaminase and use thereof in preparing sitagliptin or its intermediates <130> P21015155C <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Enz.1 <400> 1 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr 115 120 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Thr Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Arg Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 2 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Enz.1 nucleotide sequence <400> 2 atggcattct cagcagacac gccggaaatt gtttacaccc acgatacggg cctggactac 60 attacctaca gcgactacga actggacccg gcaaacccgc tggctggcgg tgcagcatgg 120 attgagggtg cgtttgtgcc gccgagtgaa gcccgtattc caatctttga tcagggtttc 180 tatacgtctg acgcaaccta caccacgttt catgtttgga acggtaatgc tttccgtctg 240 ggcgaccaca ttgaacgcct gttcagcaat gcagaatcta ttcgcctgat cccgccgctg 300 acgcaagatg aagtcaaaga aatcgcgctg gaactggtgg ccaagaccga actgcgtgaa 360 gcccaggtca ccgtgacgat tacccgcggc tatagctcta cgccgtttga acgtgatatc 420 accaaacatc gcccgcaggt gtatatgagt gcgtgcccgt accaatggat tgttccgttc 480 gatcgtatcc gcgacggtgt gcacctgatg attgcacaga gcgtccgtcg caccccgcgt 540 agttccattg atccgcaggt gaagaacttt caatggggcg acctgattcg tgcaatccaa 600 gaaacccatg atcgcggttt cgaactgccg ctgctgctgg attgtgacaa cctgctggct 660 gaaggtacgg gctttaatgt ggttgtcatc aaagatggtg tggttcgtag cccgggtcgt 720 cgcgctctgc cgggtattac gcgcaagacc gttctggaaa tcgcggaatc tctgggccac 780 gaagcgattc tggccgatat cacgccggca gaactgtacg atgctgacga agttctgggt 840 tgctcaaccg gcggtggcgt ctggccgttc gtttcggtcg atggtaattc aatttcggac 900 ggtgtgccgg gtccggttac ccagagcatt atccgtcgtt actgggaact gaatgtggaa 960 ccgtcgtcgc tgctgacccc ggtgcaatac 990 <210> 3 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Enz.2 <400> 3 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 4 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Enz.2 nucleotide sequence <400> 4 atggcgttct cagcggacac ccctgaaatc gtttacaccc acgacaccgg tctggactat 60 atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120 atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggtttt 180 [[ID=,28]]tatacttctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240 ggggaccaca tcgaacgtct gttctctaat gcggaatcta ttcgtttgat cccgccgctg 300 acccaggacg aagttaaaga gatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360 gcgatggtta ccgttacgat cacccgtggt tactcttcta ccccattcga gcgtgacatc 420 accaaacatc gtccgcaggt ttacatgagc gcttgcccgt accagtggat cgtaccgttt 480 gaccgcatcc gtgacggtgt tcacctgatg gttgctcagt cagttcgtcg tacaccgcgt 540 agctctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgcaattcag 600 gaaacccacg accgtggttt cgagttgccg ctgctgctgg actgcgacaa cctgctggct 660 gaaggtccgg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720 gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780 gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840 tgctcaaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900 ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960 ccttcttctc tgctgacccc ggtacagtac 990 <210> 5 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Enz.3 Amino Acid Sequence <400> 5 Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr 1 5 10 15 Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn 20 25 30 Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp 50 55 60 Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu 85 90 95 Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu 100 105 110 Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr 115 120 125 Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg 165 170 175 Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp 180 185 190 Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Pro Gly 210 215 220 Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg 225 230 235 240 Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu 245 250 255 Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu 260 265 270 Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr 325 330 <210> 6 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Enz.3 Nucleotide Sequence <400> 6 atggcgttct cagcggacac ccctgaaatc gtttacaccc acgacaccgg tctggactat 60 atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120 attggaggtg ctttcgttcc gccgtcggaa gctcgtatcc cgatcttcga ccagggtttt 180 tatacttctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240 ggggaccaca tcgaacgtct gttctctaat gcggaatcta ttcgtttgat cccgccgctg 300 acccaggacg aagttaaaga gatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360 gcgatggtta ccgttacgat cacccgtggt tactcttcta ccccattcga gcgtgacatc 420 accaaacatc gtccgcaggt ttacatgttc gctagcccgt acctgcagat cgtaccgttt 480 gaccgcatcc gggacggtgt tcacctgatg gttgctcagt cagttcgtcg tacaccgcgt 540 agctctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgcaattcag 600 gaaacccacg atcgtggttt cgagttgccg ctgctgctgg acggggacaa cctgctggct 660 gaaggtccgg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720 gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780 gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840 tgctcaaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900 ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960 ccttcttctc tgctgacccc ggtacagtac 990

Claims

1. A transaminase, characterized in that The amino acid residue differences between the amino acid sequence of the transaminase and the amino acid sequence shown in SEQ ID NO: 1 are selected from the following groups of amino acid residue differences, as shown in the following table: 。 2. The transaminase according to claim 1, wherein The nucleotide residue differences between the nucleotide sequence encoding the transaminase and the nucleotide sequence shown in SEQ ID NO: 2 are selected from the following group of nucleotide residue differences, as shown in the following table: Among them, 150, 152 and 155 in the nucleotide residue difference column respectively indicate that the encoded amino acid residues correspond to positions 150, 152 and 155 of SEQ ID NO: 1; the three nucleotide residues after 150, 152 and 155 are codons that are different from the original codons.

3. An isolated nucleic acid, characterized in that It encodes the transaminase according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: It comprises the isolated nucleic acid according to claim 3.

5. The recombinant expression vector according to claim 4, wherein The skeleton of the recombinant expression vector is plasmid pET21a.

6. A transformant, characterized in that The method comprises introducing the isolated nucleic acid according to claim 3 or the recombinant expression vector according to claim 4 or 5 into a host.

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

8. The transformant according to claim 7, wherein The Escherichia coli is Escherichia coli BL21.

9. A method for preparing the transaminase according to claim 1 or 2, characterized in that: The method comprises culturing the transformant according to any one of claims 6 to 8 under conditions suitable for expressing the transaminase.

10. An enzyme preparation, characterized in that It comprises the transaminase according to claim 1 or 2.

11. The enzyme preparation according to claim 10, wherein The enzyme preparation includes a cofactor for the transaminase.

12. The enzyme preparation according to claim 11, wherein The cofactor of the transaminase is pyridoxal phosphate.

13. An enzyme combination, characterized in that It comprises two or more of the transaminases according to claim 1 or 2.

14. A method for preparing (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)-1-butanone, characterized in that: The method comprises the step of catalyzing the substrate 1-morpholine-4-(2,4,5-trifluorophenyl)-1,3-butanedione to obtain (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone in a reaction solvent using the transaminase according to claim 1 or 2, the enzyme preparation according to any one of claims 10 to 12, or the enzyme combination according to claim 13 in the presence of an amino donor.

15. The method according to claim 14, wherein The reaction solvent is isopropanol and water; and / or, the amino donor is isopropylamine hydrochloride; and / or, the molar ratio of the amino donor to the substrate is 1:1 to 10:1; and / or, the concentration of the substrate is 5 to 100 g / L; And / or, the mass ratio of the transaminase to the substrate is 1:1 to 6:1; wherein the transaminase exists in the form of bacteria or protein; And / or, the prepared reaction system further includes a cofactor for transaminase; and / or, the reaction temperature is 30-60° C.; And / or, the reaction speed is 100-300 rpm.

16. The method according to claim 15, wherein The concentration of the substrate is 10 g / L; and / or, the mass ratio of the transaminase to the substrate is 3:1; and / or, the cofactor is pyridoxal phosphate; and / or, the reaction temperature is 45° C.; And / or, the reaction speed is 200 rpm.

17. The method according to claim 15 or 16, wherein The concentration of the cofactor is 0.5~5 mM.

18. The method according to claim 17, wherein The concentration of the cofactor was 1 mM.

19. A method for preparing sitagliptin or sitagliptin phosphate, characterized in that: The method comprises the step of preparing (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone according to the method according to any one of claims 14 to 18.

20. Use of the transaminase according to claim 1 or 2, the enzyme preparation according to any one of claims 10 to 12, or the enzyme combination according to claim 13 in the preparation of (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone, sitagliptin, or sitagliptin phosphate.

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