Transaminases and Their Application in the Preparation of Optically Pure Chiral Amines

Abstract

The present invention discloses transaminases and their applications in the preparation of optically pure chiral amines. The amino acid sequence of the transaminase disclosed in the present invention is as shown in SEQ ID NO: 1, or compared with SEQ ID NO: 1, the amino acid sequence of the transaminase has one or more amino acid mutations at positions 65, 94, 132, 300, and 327, and the mutations are additions, deletions, or substitutions of amino acid residues. The present invention also discloses a nucleic acid molecule encoding the transaminase, a nucleic acid construct containing the nucleic acid molecule, a recombinant vector, and a host cell. The present invention also discloses an enzyme preparation containing the transaminase of the present invention. The present invention also discloses a method for preparing chiral amines using the transaminase. The enzyme provided by the present invention has advantages such as substrate specificity, enantioselectivity, and high conversion rate. The method for preparing optically pure chiral amines provided by the present invention has high reaction efficiency, stereoselectivity, and yield.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and relates to transaminases and their applications, especially in the biocatalytic preparation of optically pure chiral amines. Background Art

[0002] Optically pure chiral amines are important pharmaceutical and fine chemical intermediates. Currently, more than 70% of the synthesis of drugs and their derivatives uses chiral amines as intermediates. (R)-1-(3-fluorophenyl)ethylamine shown in Formula II below is a very important chiral amine and is used in the synthesis of many pharmaceutical intermediates.

[0003]

[0004] Currently, the methods for preparing (R)-1-(3-fluorophenyl)ethylamine mainly include chemical methods and biocatalytic methods. The conventional process of the chemical method is as follows. Usually, racemic 1-(3-fluorophenyl)ethylamine is used as the raw material, 3-methyl-2,4-acetylacetone is used to induce chirality, and then chemical resolution is carried out to improve chirality, but the total yield is less than 50%, and the enantiomeric excess S = 31. From the perspectives of economy and environmental protection, it is not suitable for large-scale industrial production (J. Am. Chem. Soc. 2017, 139, 1357 - 1359).

[0005]

[0006] Biocatalytic methods usually use transaminases to kinetically resolve racemic amines or to asymmetrically synthesize chiral amines from ketones. Compared with traditional chemical synthesis methods, enzymatic methods have the advantages of high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, and environmental friendliness. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that the existing enzymes have low substrate specificity, poor enantioselectivity, and low conversion rate, which do not meet the requirements of industrial production. Therefore, a reagent and method for preparing optically pure chiral amines with high reaction efficiency, stereoselectivity, and yield are provided.

[0008] To solve the above technical problem, in the first aspect of the present invention, a transaminase is provided. The amino acid sequence of the transaminase is as shown in SEQ ID NO:1, or compared with SEQ ID NO:1, the amino acid sequence of the transaminase has one or more amino acid mutations at positions 65, 94, 132, 300, and 327. The mutations are insertions, deletions, or substitutions of amino acid residues, and the amino acid residues at the remaining positions are the same as those in SEQ ID NO:1.

[0009] In one or more embodiments, the mutations are selected from the following group: substitution mutations at positions 65, 94, 132, 300 and a deletion mutation at position 327. Preferably, the mutations are selected from the following group: V65A, L94C, S132H and D300A.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the transaminase is as shown in SEQ ID NO:2.

[0011] To solve the above technical problems, a second aspect of the present invention provides a nucleic acid molecule, the polynucleotide sequence of which is selected from:

[0012] (1) a polynucleotide sequence encoding the transaminase according to any embodiment of the present invention; and

[0013] (2) the complementary sequence of the polynucleotide sequence in (1).

[0014] In a preferred embodiment of the present invention, the polynucleotide sequence is selected from the polynucleotide sequences shown in SEQ ID NO:3 or SEQ ID NO:4 or their complementary sequences.

[0015] To solve the above technical problems, a third aspect of the present invention provides a nucleic acid construct containing the nucleic acid molecule according to any embodiment of the present invention; preferably, the nucleic acid construct is an expression cassette.

[0016] To solve the above technical problems, a fourth aspect of the present invention provides a recombinant vector containing the nucleic acid molecule or nucleic acid construct according to any embodiment of the present invention; preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0017] To solve the above technical problems, a fifth aspect of the present invention provides a host cell containing the nucleic acid molecule, nucleic acid construct or recombinant vector according to any embodiment of the present invention, and / or expressing the transaminase according to any embodiment of the present invention; the host cell is conventional in the art, preferably, the host cell is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0018] To solve the above technical problems, a sixth aspect of the present invention provides an enzyme preparation containing the transaminase according to any embodiment of the present invention.

[0019] To solve the above technical problems, a seventh aspect of the present invention provides a method for preparing the chiral amine represented by formula I:

[0020]

[0021] Among them, the method includes using a transaminase from the genus Arthrobacter (Arthrobacter sp), a transaminase having at least 90% sequence identity with its amino acid sequence, a transaminase mutant described in any embodiment herein, or an enzyme preparation thereof to catalyze the reaction of an amino donor with a substrate represented by the following formula III in the presence of a cosolvent and an optional coenzyme, thereby preparing a chiral amine represented by formula I:

[0022]

[0023] Wherein, in formulas I and III, R 1 is a halogenated phenyl group, and R 2 is C 1-4 alkyl.

[0024] Preferably, the compound of formula I is (R)-1-(3-fluorophenyl)ethylamine, and the compound of formula III is 3-fluoroacetophenone.

[0025] In one or more embodiments, the amino acid sequence of the transaminase from the genus Arthrobacter (Arthrobacter sp) is as shown in SEQ ID NO:1, and the transaminase having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1 is the transaminase with NCBI accession number 3WWH_A, 3WWI_A, 5FR9_A or 3WWJ_A.

[0026] In one or more embodiments, the cosolvent can be conventional in the art, and the cosolvent is selected from: dimethyl sulfoxide, alcohol solvents, and toluene.

[0027] In one or more embodiments, the amino donor can be conventional in the art, and the amino donor is selected from: aromatic amines, aliphatic amines, and amino acids.

[0028] In a preferred embodiment of the present invention, the alcohol solvent is isopropanol.

[0029] In a preferred embodiment of the present invention, the aromatic amine is R-phenylethylamine.

[0030] In one or more embodiments, the aliphatic amine is an aliphatic amine having a carbon chain length of 2 to 6 carbon atoms, preferably isopropylamine or sec-butylamine.

[0031] In one or more embodiments, the amino acid is alanine and / or aspartic acid.

[0032] In one or more embodiments, the dosage of the transaminase is 1-50% of the weight of the substrate in the reaction system, such as 10-40% or 15-30%; preferably 15-20%.

[0033] In one or more embodiments, the reaction system contains a coenzyme, and the amount of the coenzyme used is 0.1-5.0% of the weight of the substrate, such as 1-3%; preferably 2-2.5%; the preferred coenzyme is pyridoxal 5'-phosphate (PLP).

[0034] In one or more embodiments, in the reaction system, the amount of the amino donor used is 600%-1240% of the weight of the substrate.

[0035] In one or more embodiments, the pH of the reaction system is 6-10, preferably 8-10, more preferably 8.9-9.1.

[0036] In one or more embodiments, the reaction temperature is 10°C-50°C, preferably 20°C-45°C, more preferably 28°C-35°C.

[0037] In one or more embodiments, the reaction time is 0.1-120 hours, such as 0.5-48 hours, 10-24 hours or 16-92 hours.

[0038] In one or more embodiments, in the method, the cosolvent is dimethyl sulfoxide or isopropanol; the amino donor is a fatty amine, preferably isopropylamine or sec-butylamine, or an aromatic amine preferably R-phenethylamine; the transaminase is a transaminase of the genus Arthrobacter sp, preferably the transaminase described in any one of the embodiments herein; the compound of formula I is (R)-1-(3-fluorophenyl)ethylamine; the compound of formula III is 3-fluoroacetophenone.

[0039] In one or more embodiments, in the method, the cosolvent is dimethyl sulfoxide; the amino donor is isopropylamine or sec-butylamine; the transaminase is a transaminase having an amino acid sequence as shown in SEQ ID NO:1; the compound of formula I is (R)-1-(3-fluorophenyl)ethylamine; the compound of formula III is 3-fluoroacetophenone.

[0040] In a preferred embodiment of the present invention, in the method, the cosolvent is dimethyl sulfoxide; the amino donor is isopropylamine or sec-butylamine; the transaminase is a transaminase having an amino acid sequence as shown in SEQ ID NO:2; the compound of formula I is (R)-1-(3-fluorophenyl)ethylamine; the compound of formula III is 3-fluoroacetophenone.

[0041] The present invention also provides the use of a transaminase from the genus Arthrobacter sp or its enzyme preparation and / or the transaminase described in any one of the embodiments herein or an enzyme preparation containing the same in improving the conversion rate of preparing optically pure chiral amines.

[0042] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0043] The reagents and raw materials used in the present invention are all commercially available.

[0044] The positive and progressive effects of the present invention are as follows: In the industrial production process of preparing optically pure chiral amines, the enzyme provided by the present invention has the advantages of high substrate specificity, enantioselectivity and conversion rate. The method for preparing optically pure chiral amines provided by the present invention has high reaction efficiency, stereoselectivity and yield. Description of the Drawings

[0045] Figure 1 It is the SFC chromatogram of (R)-1-(3-fluorophenyl)ethylamine after being transformed by the method of Example 1. The peak at t = 4.9 is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0046] Figure 2 It is the SFC chromatogram of (R)-1-(3-fluorophenyl)ethylamine after being transformed by the method of Example 2. The peak at t = 4.9 is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0047] Figure 3 It is the SFC chromatogram of (R)-1-(3-fluorophenyl)ethylamine after being transformed by the method of Example 3. The peak at t = 4.9 is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0048] Figure 4 It is the SFC chromatogram of (R)-1-(3-fluorophenyl)ethylamine after being transformed by the method of Example 3. The peak at t = 4.9 is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0049] Figure 5 It is the SFC chromatogram of (R)-1-(3-fluorophenyl)ethylamine after being transformed by the method of Example 5. The peak at t = 4.9 is the target compound (R)-1-(3-fluorophenyl)ethylamine. Detailed Embodiments

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form a preferred technical solution.

[0051] The present invention discovers that the chiral amine shown in the following formula I can be prepared with very high conversion rate and chiral purity by using the transaminase of the present invention:

[0052]

[0053] In the formula, R 1 is a halogenated C 1-4 phenyl, and R 2 is a C 1-4 alkyl.

[0054] In this text, halogenated C 1-4 alkyl refers to a phenyl group substituted by a halogen, where the halogen may include F, Cl, Br, and I. In this text, C 1-4 alkyl includes linear and branched alkyl groups, including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl, etc. Examples of halogenated C1-4 phenyl groups include but are not limited to trifluorophenyl, pentafluorophenyl, trichlorophenyl, etc.

[0055] In a particularly preferred embodiment, the chiral amine of the present invention is (R)-1-(3-fluorophenyl)ethylamine represented by the following formula II:

[0056]

[0057] In the present invention, the transaminase is preferably a transaminase from the genus Arthrobacter. Exemplary transaminases from the genus Arthrobacter include transaminases having the amino acid sequence shown in SEQ ID NO:1. The transaminases of the present invention also include mutants of SEQ ID NO:1, such as those having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1. The sequence identity can be calculated using commonly used software in the art, such as BLAST (from NCBI), with the software default parameters.

[0058] In some embodiments, the mutant of the transaminase of the present invention is a mutant derived from SEQ ID NO:1 obtained by one or more amino acid mutations to the amino acid sequence shown in SEQ ID NO:1 and retaining the transaminase activity possessed by SEQ ID NO:1 (especially having the function of preparing chiral amines described herein). The one or more amino acid mutations include amino acid mutations within 20, preferably within 15, more preferably within 10, more preferably within 8, more preferably within 5, more preferably within 4 amino acid mutations, such as substitution, insertion, or deletion of amino acid residues. Preferred mutations are substitution mutations or deletion mutations.

[0059] In the present invention, exemplary transaminase mutants include but are not limited to the transaminases with accession numbers 3WWH_A, 3WWI_A, 5FR9_A, and 3WWJ_A.

[0060] In some embodiments, particularly preferred transaminase mutants of the present invention include mutants obtained by mutating one or more of the positions at 65th, 94th, 132nd, 300th, and 327th positions of SEQ ID NO:1, and the amino acid residues at the remaining positions of the mutant are the same as those of SEQ ID NO:1. Preferred mutations are substitution mutations or deletion mutations. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution at the 65th position is that the wild-type V is substituted with a non-polar amino acid such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, and proline, or is substituted with an aromatic amino acid such as tyrosine, tryptophan, or phenylalanine; preferably substituted with alanine. In some embodiments, the substitution at the 132nd position is that the wild-type S is substituted with a basic amino acid such as lysine, arginine, and histidine, preferably substituted with arginine or histidine, more preferably substituted with histidine. In some embodiments, the substitution at the 300th position is that the wild-type D is substituted with a non-polar amino acid such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, or methionine, or is substituted with an aliphatic non-polar amino acid such as alanine, leucine, valine, or isoleucine; preferably substituted with alanine.

[0061] In preferred embodiments, the mutations are selected from the group consisting of: V65A, L94C, S132H, and D300A.

[0062] In further preferred embodiments, the amino acid sequence of the transaminase mutant is as shown in SEQ ID NO:2.

[0063] The present invention also includes nucleic acid molecules, the polynucleotide sequences of which are the coding sequences of the transaminase or its mutants of the present invention and their complementary sequences. In some embodiments, the polynucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:3 or as shown in SEQ ID NO:4.

[0064] The present invention further includes nucleic acid constructs containing the nucleic acid molecules of the present invention. In some embodiments, the nucleic acid construct is an expression cassette. In addition to the nucleic acid molecule, the expression cassette may further contain a transcription termination sequence and a promoter. The promoter can be various promoters well-known in the art as long as it is suitable for expressing the transaminase of the present invention in the desired host. Those skilled in the art can select an appropriate promoter according to the host cell used to construct the expression cassette of the present invention and the recombinant vector described below.

[0065] The present invention also includes a recombinant vector. The recombinant vector may contain the nucleic acid molecule or nucleic acid construct described in any of the embodiments herein. The recombinant vector can be a recombinant cloning vector or a recombinant eukaryotic expression vector. Other regulatory elements may be contained in the recombinant vector, including but not limited to enhancers, multiple cloning sites, transcription terminators, resistance genes, etc. A corresponding vector backbone with the required regulatory elements can be selected according to different purposes, and the nucleic acid molecule or nucleic acid construct of the present invention can be cloned into the backbone to construct the recombinant vector of the present invention.

[0066] The nucleic acid molecule, nucleic acid construct and recombinant vector can be prepared by methods well-known in the art, and expressed by conventional methods to prepare the transaminase described herein.

[0067] In some embodiments, the present invention also provides a host cell containing the nucleic acid molecule, nucleic acid construct and / or recombinant vector described in any of the embodiments herein, or expressing the transaminase described in any of the embodiments herein. Any host known in the art suitable for expressing the target protein can be used in the present invention. Exemplary host cells include Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0068] The method for preparing chiral amines of the present invention includes the step of reducing the carbonyl group in the substrate shown in Formula III to a chiral amino group using the transaminase of the present invention in a reaction system containing a coenzyme and an amino donor:

[0069]

[0070] wherein, R 1 and R 2 are as described above.

[0071] The dosage of the transaminase can be 1-50% of the weight of the substrate in the reaction system, such as 10-40% or 15-30%; preferably 15-20%.

[0072] The coenzyme can be various coenzymes conventionally used in combination with transaminases in the art, such as pyridoxal 5'-phosphate (PLP). The dosage of the coenzyme can be a conventional dosage. For example, it can be 0.1-5.0% of the weight of the substrate in the reaction system, such as 1-3%; preferably 2-2.5%.

[0073] The amino donor in the reaction system can be various amino donors commonly used in the art for preparing chiral amines, including but not limited to aromatic amines, such as phenethylamine; aliphatic amines, such as aliphatic amines with a carbon chain length of 2-6 carbon atoms, such as isopropylamine; amino acids, such as alanine (such as L-alanine) and aspartic acid (such as L-aspartic acid), etc. Generally, in the reaction system, the dosage of the amino donor can be determined according to the conventional reaction. Generally, depending on the type of the amino donor, the dosage of the amino donor can be 600%-1240% of the weight of the substrate.

[0074] In the present invention, the reaction system is a buffered saline solution system. The pH of the reaction system is controlled by a buffer solution. Commonly used buffer solutions include, but are not limited to, phosphate buffer solution, triethanolamine-isopropylamine buffer solution, etc. Preferably, the pH of the reaction system is 6-10, more preferably 8-10. In some embodiments, the pH of the reaction system is 8.9-9.1.

[0075] The reaction system may also contain a cosolvent. Cosolvents commonly used in the preparation of chiral amines can all be used in the present invention. Generally, the cosolvent is an organic solvent, for example, selected from: dimethyl sulfoxide, toluene, and alcohol solvents. The alcohol solvents include, but are not limited to, isopropanol. Preferably, the cosolvent is selected from dimethyl sulfoxide and isopropanol. The cosolvent used should be miscible with water to further increase the solubility of the substrate.

[0076] The reaction temperature of the catalytic reaction of the present invention can be 10°C to 50°C, preferably 20°C to 45°C, more preferably 28°C to 35°C. In some embodiments, the reaction temperature is room temperature, that is, 25±3°C. The reaction time can be determined according to the amount of the reactants, and generally can be 0.1 to 120 hours, such as 0.5 to 48 hours, 10 to 24 hours, or 16 to 92 hours.

[0077] The present invention discovers that when a specific cosolvent and an amino donor are selected, when using a transaminase from the genus Arthrobacter sp (especially the transaminase shown in SEQ ID NO:1) to catalyze the carbonyl reduction reaction of the substrate to an amino group, compared with using other cosolvents and amino donors, significantly higher conversion rates can also be achieved. Therefore, in some embodiments of the present invention, the method for preparing the chiral amine shown in formula I of the present invention includes using the transaminase shown in SEQ ID NO:1 to catalyze the reaction between the amino donor and 3-fluorobenzophenone in the presence of a cosolvent; wherein, the cosolvent is dimethyl sulfoxide; the amino donor is a fatty amine, such as a fatty amine with a carbon chain length of 2-6 carbon atoms, more preferably isopropylamine or sec-butylamine. In some embodiments, the amino donor is an aromatic amine, such as phenethylamine (such as R-phenethylamine), and the solvent is dimethyl sulfoxide. In a preferred embodiment, the method for preparing the chiral amine shown in formula I of the present invention includes using the transaminase shown in SEQ ID NO:1 to catalyze the reaction between R-phenethylamine and the substrate shown in formula III in the presence of dimethyl sulfoxide, or using the transaminase shown in SEQ ID NO:1 to catalyze the reaction between isopropylamine and the substrate shown in formula III in the presence of isopropanol. Preferably, the reaction system of the reaction also contains a coenzyme, such as pyridoxal phosphate (PLP). Preferably, the chiral amine is (R)-1-(3-fluorophenyl)ethylamine, and the substrate is 3-fluorobenzophenone.

[0078] The present invention also found that when using the transaminase mutants of the present invention, especially the mutants obtained by mutating at multiple positions among positions 65, 94, 132, 300 and 327 of SEQ ID NO:1 described herein, to prepare the chiral amine of formula I, the conversion rate is very high. Therefore, in some embodiments of the present invention, the method for preparing the chiral amine of formula I of the present invention includes using the transaminase mutant described in any embodiment herein to catalyze the reaction between the amino donor and the substrate in the presence of a cosolvent. The preferred cosolvent is dimethyl sulfoxide, and the preferred amino donor is a fatty amine, such as isopropylamine or sec-butylamine. Preferably, the reaction system of the reaction further contains a coenzyme, such as pyridoxal phosphate (PLP). In a particularly preferred embodiment, the transaminase mutant is a transaminase having an amino acid sequence as shown in SEQ ID NO:2. Preferably, the chiral amine is (R)-1-(3-fluorophenyl)ethylamine, and the substrate is 3-fluorobenzophenone.

[0079] The present invention also includes the transaminase mutants described above, their coding sequences (nucleic acid molecules), nucleic acid constructs, recombinant vectors, and host cells.

[0080] In some embodiments, the present invention provides an enzyme product containing the transaminase mutant described in any embodiment of the present invention. In some embodiments, the enzyme product is a freeze-dried powder. In some embodiments, the enzyme product is a buffer solution containing the transaminase mutant. Preferably, the buffer solution is a phosphate buffer solution with a pH of 6-10, preferably 7-9, more preferably 8-9, and even more preferably 8.3-8.6. In some embodiments, the phosphate buffer solution is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0081] The present invention also provides the use of the transaminase mutant, its coding sequence (nucleic acid molecule), nucleic acid construct, recombinant vector, and host cell described in any of the above embodiments in the preparation of a reagent for preparing the chiral amine of formula I. In some embodiments, the reagent is the enzyme product described in any embodiment herein.

[0082] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0083] Preparation Example

[0084] Using the sequence of SEQ ID NO:1 (the coding sequence is as shown in SEQ ID NO:3) as the parent, it was subjected to directed evolution and modification using strategies such as rolling circle PCR, iterative saturation mutagenesis, and combinatorial mutagenesis. Then, the mutants were transformed into competent Escherichia coli BL21(DE3) cells and evenly spread on an LB agar plate containing 50 μg / ml kanamycin, and placed in a 37°C incubator for static culture for 18 h. The mutants on the transformed plate were picked with toothpicks into a 96-well plate and cultured overnight in a shaker at 37°C and 220 rpm. 50 μl of the bacterial liquid was pipetted from the wells of the primary plate into the corresponding wells of the secondary plate, and after culturing at 37°C and 220 rpm for 2 - 3 h, IPTG with a final concentration of 0.2 mM was added, and cultured at 30°C for 20 h to obtain the corresponding mutants for high-throughput screening. Combining SFC detection for re-screening, mutants with significantly improved activity and stability were identified for gene sequencing. The sequencing result is as shown in SEQ ID NO:2, and its coding sequence is as shown in SEQ ID NO:4.

[0085] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the present invention. The methods, materials, and reagents used in the embodiments are conventional methods in the art and materials and reagents available from commercial sources, unless otherwise specified.

[0086] The parameters for SFC detection used in the present invention are as follows:

[0087]

[0088] Example 1

[0089] Prepare the basic phosphate buffer: Weigh 27.8 g of dipotassium hydrogen phosphate trihydrate and 10.6 g of potassium dihydrogen phosphate, add 200 mL of purified water, stir at room temperature until the solid dissolves, adjust the pH to 6.9 - 7.1, and add distilled water to a final volume of 2 L.

[0090] Prepare the basic isopropylamine buffer: Add 280 mL of the basic phosphate buffer, 84 mL of isopropylamine, and 40 mL of 85% phosphoric acid to a 500 mL glass bottle to obtain the basic isopropylamine buffer.

[0091] Add 3 mL of the basic isopropylamine buffer to an 8 mL reaction flask and adjust the pH to between 8.8 and 9.1. Add 20 mg of freeze-dried transaminase (the amino acid sequence is as shown in SEQ ID NO:1) and 2 mg of pyridoxal 5'-phosphate (PLP) to the reaction solution, stir until the solids are completely dissolved, then add 50 mg of the substrate and 100 μL of dimethyl sulfoxide, and stir at 28 - 32°C for 16 h to allow sufficient reaction.

[0092] The conversion rate detected by SFC is 14.3%, and the ee value > 99%. As Figure 1 And Table 1 shows that the peak at t = 4.9 min in the spectrogram is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0093] Table 1

[0094]

[0095] Example 2

[0096] Add 28 mL of basic phosphate buffer solution, 9.8 mL of sec-butylamine, and 2.2 - 3.0 mL of 85% phosphoric acid to a 100 mL jacket, and adjust the pH to be between 8.5 and 9.0. Add 400 mg of freeze-dried transaminase (amino acid sequence as shown in SEQ ID NO: 2) and 20 mg of PLP to the reaction solution. After stirring until the solids are completely dissolved, add 1 g of the substrate, and react in a sealed manner at 28 - 32 °C for 64 hours to allow it to react fully.

[0097] The conversion rate detected by SFC is 69.4%, and the ee value > 99%. As Figure 2 And Table 2 shows that the peak at t = 4.9 min in the spectrogram is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0098] Table 2

[0099]

[0100] Example 3

[0101] Add 20 mL of basic phosphate buffer solution, 7.5 mL of sec-butylamine, and 2.5 mL of 85% phosphoric acid to a 50 mL jacket, adjust the pH to be between 8.9 and 9.1, and stir well.

[0102] Add 200 mg of freeze-dried transaminase (amino acid sequence as shown in SEQ ID NO: 2) and 20 mg of PLP to the reaction solution. After stirring until the solids are completely dissolved, add 0.5 g of the substrate, react in an open manner at 28 - 32 °C for 16 hours, then add 0.25 g of the substrate, react in an open manner at 28 - 32 °C for 8 hours, add 0.25 g of the substrate, and stir for 68 hours to allow it to react fully.

[0103] The conversion rate detected by SFC is 96.9%, and the ee value > 99%. As Figure 3 And Table 3 shows that the peak at t = 4.9 min in the spectrogram is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0104] Table 3

[0105]

[0106] Example 4

[0107] Add 1200 mL of purified water, 240 mL of sec-butylamine, and 105 mL of 85% phosphoric acid into a 2000 mL jacket, and adjust the pH to be between 8.9 and 9.1.

[0108] Add 6 g of freeze-dried powder of transaminase (amino acid sequence as shown in SEQ ID NO: 2) and 1 g of PLP into the reaction solution. After stirring until the solids are completely dissolved, add 40 g of substrate and 40 mL of dimethyl sulfoxide, and stir open to the air at 28 - 35 °C for 40 hours to allow sufficient reaction.

[0109] The conversion rate detected by SFC is 94.5%, and the ee value > 99%. As Figure 4 shown in Table 4, the peak at t = 4.9 min in the chromatogram is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0110] Table 4

[0111]

[0112] Example 5

[0113] Add 1200 mL of purified water, 240 mL of sec-butylamine, and 105 mL of 85% phosphoric acid into a 2000 mL jacket, and adjust the pH to be between 8.9 and 9.1.

[0114] Add 6 g of freeze-dried powder of transaminase (amino acid sequence as shown in SEQ ID NO: 1) and 1 g of PLP into the reaction solution. After stirring until the solids are completely dissolved, add 40 g of substrate and 40 mL of dimethyl sulfoxide, and stir open to the air at 28 - 35 °C for 40 hours to allow sufficient reaction.

[0115] The conversion rate detected by SFC is 27.1%, and the ee value > 99%. As Figure 5 shown in Table 5, the peak at t = 4.9 min in the chromatogram is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0116] Table 5

[0117]

[0118] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. SEQUENCE LISTING <110> Shanghai ChemPartner Pharmaceutical R & D Co., Ltd. Shanghai ChemPartner Pharmaceutical Co., Ltd. <120> Transaminase and its application in the preparation of optically pure chiral amines <130> P21018741C <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> Arthrobacter sp <400> 1 Met Ser Phe Ser Ala Asp Thr Ser 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 Arg Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp 50 55 60 Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Leu Arg Ile 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 Phe Val Ser Val Ser Ile Thr 115 120 125 Arg Gly Tyr Ser Thr Thr Pro Trp Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Ala 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 Val Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser 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 Leu Ala Glu Leu 260 265 270 Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly 290 295 300 Pro Leu Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Ser Ser Ser Pro Leu Thr Pro Val Gln Tyr 325 330 <210> 2 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Transaminase mutant <220> <221> MISC_FEATURE <222> (327)..(327) <223> X represents amino acid deletion <400> 2 Met Ser Phe Ser Ala Asp Thr Ser 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 Arg Ile Glu Gly Ala Phe Val Pro Pro 35 40 45 Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp 50 55 60 Ala Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu 65 70 75 80 Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Cys Arg Ile 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 Phe Val Ser Val Ser Ile Thr 115 120 125 Arg Gly Tyr His Thr Thr Pro Trp Glu Arg Asp Ile Thr Lys His Arg 130 135 140 Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe 145 150 155 160 Asp Arg Ile Arg Asp Gly Val His Ala 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 Val Gln Glu Thr His Asp Arg Gly Phe Glu 195 200 205 Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser 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 Leu Ala Glu Leu 260 265 270 Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp 275 280 285 Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Ala Gly Val Pro Gly 290 295 300 Pro Leu Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu 305 310 315 320 Ser Ser Ser Pro Leu Thr Xaa Val Gln Tyr 325 330 <210> 3 <211> 993 <212> DNA <213> Arthrobacter sp <400> 3 atgtccttct ctgctgacac ctctgaaatc gtttacaccc acgacaccgg tctggactac 60 atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgcagctagg 120 atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggttac 180 ctgcactctg acgttaccta caccgttttc cacgtttgga acggcaacgc tttccgtctg 240 gacgaccaca tcgaacgtct gttctctaac gctgaatctc tgcgtatcat cccgccgctg 300 acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360 gctttcgttt ctgtttctat cacccgtggt tactctacga ccccgtggga acgtgacatc 420 accaaacacc gtccgcaggt ttacatgtac gctgttccgt accagtggat cgttccgttc 480 gaccgtatcc gtgacggtgt tcacgctatg gttgctcagt ctgttcgtcg taccccgcgt 540 tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctgttcag 600 gaaacccacg accgtggttt cgaagctccg ctgctgctgg acggtgacgg tctgctggct 660 gaaggttctg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720 gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780 gaagctatcc tggctgacat caccctggct gaactgctgg acgctgacga agttctgggt 840 tgcaccaccg ctggtggtgt ttggccattc gtttctgttg acggtaaccc gatctctgac 900 ggtgttccgg gtccgcttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960 tcttcttctc cgcttacccc ggttcagtac taa 993 <210> 4 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Transaminase Mutant <400> 4 atgtccttct ctgctgacac ctctgaaatc gtttacaccc acgacaccgg tctggactac 60 atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgcagctagg 120 atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggttac 180 ctgcactctg acgcgaccta caccgttttc cacgtttgga acggtaacgc tttccgtctg 240 gacgaccaca tcgaacgtct gttctctaac gctgaatctt gccgtatcat cccgccgctg 300 acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360 gctttcgttt ctgtttctat cacccgtggt taccatacga ccccgtggga acgtgacatc 420 accaaacacc gtccgcaggt ttacatgtac gctgttccgt accagtggat cgttccgttc 480 gaccgtatcc gtgacggtgt tcacgctatg gttgctcagt ctgttcgtcg taccccgcgt 540 tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctgttcag 600 gaaacccacg accgtggttt cgaagctccg ctgctgctgg acggtgacgg tctgctggct 660 gaaggttctg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720 gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780 gaagctatcc tggctgacat caccctggct gaactgctgg acgctgacga agttctgggt 840 tgcaccaccg ctggtggtgt ttggccgttc gtttctgttg acggtaaccc gatctctgcc 900 ggtgttccgg gtccgcttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960 tcttcttctc cgcttaccgt tcagtactaa 990

Claims

1. An aminotransferase, characterized in that the amino acid sequence of the aminotransferase is as shown in SEQ ID NO:

2.

2. A nucleic acid molecule, characterized in that its polynucleotide sequence encodes the aminotransferase according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that the polynucleotide sequence is as shown in SEQ ID NO:

4.

4. A nucleic acid construct, characterized in that it contains the nucleic acid molecule according to claim 2 or 3.

5. The nucleic acid construct according to claim 4, characterized in that the nucleic acid construct is an expression cassette.

6. A recombinant vector, characterized in that it contains the nucleic acid molecule according to claim 2 or 3 or the nucleic acid construct according to claim 4 or 5.

7. The recombinant vector according to claim 6, characterized in that the recombinant vector is a recombinant cloning vector.

8. The recombinant vector according to claim 6, characterized in that the recombinant vector is a recombinant expression vector.

9. A host cell, characterized in that it contains the nucleic acid molecule according to claim 2 or 3, the nucleic acid construct according to claim 4 or 5, or the recombinant vector according to any one of claims 6 to 8.

10. A host cell, characterized in that it expresses the aminotransferase according to claim 1.

11. An enzyme preparation, characterized in that the enzyme preparation contains the aminotransferase according to claim 1.

12. A method for preparing a chiral amine represented by formula I: （I） wherein, the method includes, in the presence of a cosolvent and a coenzyme, using the aminotransferase according to claim 1 or the enzyme preparation according to claim 11 to catalyze the reaction of an amino donor with a substrate represented by formula III, thereby preparing the chiral amine represented by formula I: (III) wherein, in formula I and III, the compound of formula I is (R)-1-(3-fluorophenyl)ethylamine, and the compound of formula III is 3-fluoroacetophenone.

13. The method according to claim 12, characterized in that the cosolvent is dimethyl sulfoxide; and / or, the amino donor is isopropylamine or sec-butylamine.

14. The method according to claim 12, characterized in that the dosage of the aminotransferase is 1-50% of the weight of the substrate in the reaction system; the reaction system contains a coenzyme, and the dosage of the coenzyme is 0.1-5.0% of the weight of the substrate; in the reaction system, the dosage of the amino donor is 600%-1240% of the weight of the substrate; the pH of the reaction system is 8-10; the reaction temperature is 10°C to 50°C; the reaction time is 0.1-120 hours.

15. The method according to claim 14, characterized in that the method satisfies one or more of the following conditions: the dosage of the aminotransferase is 10-40% of the weight of the substrate in the reaction system; the dosage of the coenzyme is 1-3% of the weight of the substrate; the reaction temperature is 20°C to 45°C; and, the reaction time is 10-24 hours.

16. The method according to claim 15, characterized in that the method satisfies one or more of the following conditions: the dosage of the aminotransferase is 15-20% of the weight of the substrate in the reaction system; the dosage of the coenzyme is 2-2.5% of the weight of the substrate; the coenzyme is pyridoxal 5'-phosphate; The pH of the reaction system is 8.9 to 9.1; and, The reaction temperature is 28 to 35 °C.

17. The method according to claim 14, characterized in that the dosage of the transaminase is 15-30% of the weight of the substrate in the reaction system; and / or, the reaction time is 16 to 92 hours.

18. Use of the transaminase according to claim 1 or an enzyme preparation containing the same in improving the conversion rate of preparing optically pure chiral amine; the optically pure chiral amine is (R)-1-(3-fluorophenyl)ethylamine.

Images