Transaminase and its application in the preparation of optically pure chiral amines

Abstract

The present invention discloses a transaminase and its use in preparing optically pure chiral amines. The amino acid sequence of the transaminase of the present invention has one or more mutations at positions 65, 318, 399, 419, 426, 465, and 466 compared to SEQ ID NO: 1, wherein the mutation is an addition, deletion, or substitution of an amino acid residue. The present invention also discloses a nucleic acid molecule encoding the transaminase, a nucleic acid construct comprising the nucleic acid molecule, a recombinant vector, and a host cell. The present invention also discloses an enzyme preparation comprising the transaminase of the present invention. The present invention also discloses a method for preparing chiral amines using the transaminase. In the industrial production process of optically pure chiral amines, the enzyme provided by the present invention has advantages such as substrate specificity, enantiomeric selectivity, 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 transaminase and its application, especially its application in biocatalytic preparation of optically pure chiral amines. Background Art

[0002] Optically pure chiral amines are a valuable class of pharmaceutical and fine chemical intermediates. Currently, over 70% of pharmaceuticals and their derivatives are synthesized using chiral amines as intermediates. (S)-4-bromo-α-methylbenzylamine, shown in Formula II below, is a very important chiral amine used in the synthesis of many pharmaceutical intermediates.

[0003]

[0004] Currently, the main methods for preparing (S)4-bromo-α-methylbenzylamine include chemical methods and biocatalytic methods. The general process of the chemical method is as follows: p-bromoacetophenone is used as the raw material, and (S)4-bromo-α-methylbenzylamine is produced under the catalytic action of ammonium formate. Specifically, when 5-10 equivalents of ammonium formate are added to 15-25% NH3 / methanol at a temperature of 60-85°C, the enantioselectivity of (S)4-bromo-α-methylbenzylamine is optimal. However, from an economic and environmental perspective, this method is not suitable for large-scale industrial production (Kadyrov R, Riermeier T H. Angew. Chem. Int. Ed. 2003, 42).

[0005]

[0006] Enzymatic methods typically utilize transaminases to kinetically resolve racemic amines or generate chiral amines through asymmetric synthesis of ketones. Compared to traditional chemical synthesis methods, enzymatic methods offer advantages such as 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 enzymes in the prior art have problems such as low substrate specificity, poor enantioselectivity and / or low conversion rate for substrates that have not been used in industry, and thus provide a reagent and method that can prepare optically pure chiral amines with high reaction efficiency, stereoselectivity and yield.

[0008] To solve the above technical problems, the first aspect of the present invention provides a transaminase, the amino acid sequence of which, compared with SEQ ID NO: 1, has one or more mutations at positions 65, 318, 399, 419, 426, 465 and 466, wherein the mutations are additions, 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 mutation is selected from the following group: substitution mutations at positions 65, 318, 399, 419 and 426 and deletion mutations at positions 465 and / or 466, preferably the mutation is selected from the following group: W65F, E318T, E399Y, R419V and I426V.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the transaminase is as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0011] In order to solve the above technical problems, the 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) A complementary sequence of the polynucleotide sequence described in (1).

[0014] In a preferred embodiment of the present invention, the polynucleotide sequence is a polynucleotide sequence such as SEQ ID NO: 6 or SEQ ID NO: 7, or a complementary sequence thereof.

[0015] To solve the above technical problems, the third aspect of the present invention provides a nucleic acid construct, which contains 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, the fourth aspect of the present invention provides a recombinant vector, which contains the nucleic acid molecule or nucleic acid construct described in 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, the fifth aspect of the present invention provides a host cell, which contains the nucleic acid molecule, nucleic acid construct or recombinant vector described in any embodiment of the present invention, and / or expresses the transaminase described in 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] In order to solve the above technical problems, the sixth aspect of the present invention provides an enzyme preparation, which contains the transaminase according to any embodiment of the present invention.

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

[0020]

[0021] The method comprises, in the presence of a cosolvent and an optional coenzyme, using a transaminase from Ruegeria arenilitoris sp or a transaminase having at least 95% sequence identity with its amino acid sequence or 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, thereby preparing a chiral amine represented by formula I:

[0022]

[0023] Wherein, in formula I and III, R1 is halogenated aromatic hydrocarbon, R2 is C 1-4 alkyl;

[0024] The substrates available for the reaction include p-bromoacetophenone, p-chloroacetophenone, p-cyanoacetophenone, methyl p-acetylbenzoate, m-fluoroacetophenone, and the like.

[0025] In a preferred embodiment of the present invention, the compound of formula I is (S)4-bromo-α-methylbenzylamine and the compound of formula III is p-bromoacetophenone.

[0026] In one or more embodiments, the amino acid sequence of the Ruegeria arenilitoris sp transaminase is as shown in SEQ ID NO: 1, and the transaminase having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 is the transaminase with NCBI accession number 3HMU_A or WP_011049154.1.

[0027] In one or more embodiments, the co-solvent can be conventional in the art, and the co-solvent is selected from the group consisting of dimethyl sulfoxide, alcohol solvents, and toluene. The alcohol solvent can be isopropyl alcohol.

[0028] 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. The aromatic amine can be phenylethylamine. The amino acid can be alanine and / or aspartic acid.

[0029] In a preferred embodiment of the present invention, the fatty amine is a fatty amine with a carbon chain length of 2 to 6 carbon atoms, preferably isopropylamine.

[0030] In one or more embodiments, the amount of transaminase used is 1-50% by weight of the substrate in the reaction system, such as 10-40% or 15-30%.

[0031] In one or more embodiments, the reaction system contains a coenzyme in an amount of 0.1-5.0%, preferably 1-3%, by weight of the substrate. A preferred coenzyme is pyridoxal-5-phosphate (PLP). As known to those skilled in the art, pyridoxal-5-phosphate is a universal coenzyme in transamination reactions. In transamination reactions, PLP and PMP (pyridoxamine phosphate) are mutually converted and can both serve as coenzymes according to the present invention.

[0032] In one or more embodiments, the amount of the amino donor in the reaction system is 200%-500%, such as 300%, based on the weight of the substrate.

[0033] In one or more embodiments, the pH of the reaction system is 6-10, preferably 7-9, more preferably 8-9, and most preferably 8.1-8.3.

[0034] In one or more embodiments, the reaction temperature is 10-50°C, preferably 20-45°C, and more preferably 40°C.

[0035] In one or more embodiments, the reaction time is 0.1-120 hours, such as 0.5-72 hours or 10-42 hours.

[0036] In one or more embodiments, in the method, the cosolvent is dimethyl sulfoxide; the amino donor is a fatty amine, preferably isopropylamine; the transaminase is a transaminase from Ruegeria arenilitoris sp, preferably the transaminase described in any embodiment herein; the compound of formula I is (S) 4-bromo-α-methylbenzylamine; and the compound of formula III is p-bromoacetophenone.

[0037] In one or more embodiments, in the method, the cosolvent is dimethyl sulfoxide; the amino donor is isopropylamine; the transaminase is a transaminase having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3; the compound of formula I is (S) 4-bromo-α-methylbenzylamine; and the compound of formula III is p-bromoacetophenone.

[0038] The present invention also provides use of a transaminase from Ruegeria arenilitoris sp or an enzyme preparation thereof and / or a transaminase or an enzyme preparation containing the same as described in any embodiment herein in improving the conversion rate of preparing optically pure chiral amines.

[0039] 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.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The present invention has the following positive effects: in the industrial production of optically pure chiral amines, the enzyme provided by the present invention exhibits advantages such as substrate specificity, enantioselectivity, and high conversion rate. Furthermore, the enzyme provided by the present invention can tolerate more stringent conditions, such as higher reaction temperatures or reaction pH values. The method for preparing optically pure chiral amines provided by the present invention exhibits high reaction efficiency, stereoselectivity, and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the HPLC spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 1. The peak at t=5.575 is the target compound (S)4-bromo-α-methylbenzylamine.

[0043] Figure 2 This is the supercritical fluid chromatography spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 2. The peak at t=1.081 is the target compound (S)4-bromo-α-methylbenzylamine.

[0044] Figure 3 This is the HPLC spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 3. The peak at t=5.502 is the target compound (S)4-bromo-α-methylbenzylamine.

[0045] Figure 4 This is the supercritical fluid chromatography spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 4. The peak at t=1.071 is the target compound (S)4-bromo-α-methylbenzylamine.

[0046] Figure 5 This is the HPLC spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 5. The peak at t=5.148 is the target compound (S)4-bromo-α-methylbenzylamine.

[0047] Figure 6 This is the HPLC spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 6. The peak at t=4.67 is the target compound (S)4-bromo-α-methylbenzylamine.

[0048] Figure 7 This is the HPLC spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 7. The peak at t=4.67 is the target compound (S)4-bromo-α-methylbenzylamine.

[0049] Figure 8 This is the supercritical fluid chromatography spectrum of (S)4-bromo-α-methylbenzylamine after conversion using the method of Example 8. The peak at t=1.069 is the target compound (S)4-bromo-α-methylbenzylamine. DETAILED DESCRIPTION

[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 embodiments) can be combined with each other to form a preferred technical solution.

[0051] The present invention has found that the transaminase of the present invention can be used to prepare a chiral amine having the following formula I with very high conversion rate and chiral purity:

[0052]

[0053] In the formula, R1 is a halogenated aromatic hydrocarbon, R2 is C 1-4 alkyl.

[0054] Herein, halogenated aromatic hydrocarbons refer to aromatic hydrocarbons substituted by halogens, wherein the halogens may include F, Cl, Br and I. Herein, C 1-4 The alkyl group includes straight chain and branched chain alkyl groups, including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl and tert-butyl, etc. Examples of halogenated aromatic hydrocarbons include but are not limited to side chain halogenated aromatic hydrocarbons or aromatic ring halogenated aromatic hydrocarbons.

[0055] In a particularly preferred embodiment, the chiral amine of the present invention is (S)4-bromo-α-methylbenzylamine represented by the following formula II:

[0056]

[0057] In the present invention, the transaminase is preferably a transaminase from Ruegeria arenilitoris sp. Exemplary transaminases from Ruegeria include transaminases having an amino acid sequence as shown in SEQ ID NO: 1. The transaminase of the present invention also includes mutants of SEQ ID NO: 1, such as transaminases having an amino acid sequence with the amino acid sequence as shown in SEQ ID NO: 1 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. The sequence identity can be calculated using software commonly used in the art, such as BLAST (from NCBI) using the software default parameters.

[0058] In some embodiments, the transaminase mutant of the present invention is a mutant derived from SEQ ID NO: 1 that retains the transaminase activity of SEQ ID NO: 1 (especially the function of preparing chiral amines as described herein) obtained by subjecting the amino acid sequence of SEQ ID NO: 1 to one or more amino acid mutations. The one or more amino acid mutations include no more than 20, preferably no more than 15, more preferably no more than 10, more preferably no more than 8, more preferably no more than 5, and more preferably no more than 3 amino acid mutations, such as substitutions, insertions, or deletions 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, transaminase with accession number 3HMU_A or WP_011049154.1.

[0060] In some embodiments, particularly preferred transaminase mutants of the present invention include mutants obtained by mutation at one or more key positions of SEQ ID NO: 1, including positions 65, 318, 399, 419, 426, 465, and 466. Preferred mutations are substitution mutations or deletion mutations. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the wild-type W at position 65 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, or phenylalanine, or with an aromatic amino acid, such as tyrosine, tryptophan, or phenylalanine; preferably, it is substituted with phenylalanine. In some embodiments, the wild-type E at position 318 is substituted with a polar, uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine, preferably with asparagine, glutamine, tryptophan, or threonine, and more preferably with threonine. In some embodiments, the wild-type substitution at position 399 is substituted with a polar uncharged amino acid such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine, or with an aromatic amino acid such as tyrosine, tryptophan, or phenylalanine; preferably, with tyrosine. In some embodiments, the wild-type substitution at position 419 is substituted with a non-polar amino acid such as alanine, valine, leucine, isoleucine, proline, or phenylalanine, or with an aliphatic non-polar amino acid such as alanine, leucine, valine, or isoleucine; preferably, with valine. In some embodiments, the wild-type substitution at position 426 is substituted with a non-polar amino acid such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, or methionine, or with an aliphatic non-polar amino acid such as alanine, leucine, valine, or isoleucine; preferably, with valine.

[0061] In a preferred embodiment, the mutation is selected from the group consisting of W65F, E318T, E399Y, R419V and I426V.

[0062] In a further preferred embodiment, the amino acid sequence of the transaminase mutant is as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0063] The present invention also includes a nucleic acid molecule whose polynucleotide sequence is the coding sequence of the transaminase mutant of the present invention or its complementary sequence. In some embodiments, the polynucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0064] The present invention also includes a nucleic acid construct comprising the nucleic acid molecule 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 also contain a transcription termination sequence and a promoter. The promoter may be any of various promoters 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 based on 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 recombinant vectors. The recombinant vectors may contain the nucleic acid molecules or nucleic acid constructs described in any of the embodiments herein. The recombinant vectors may be recombinant cloning vectors or recombinant eukaryotic expression vectors. The recombinant vectors may contain other regulatory elements, including but not limited to enhancers, multiple cloning sites, transcription terminators, resistance genes, and the like. Depending on the purpose, a corresponding vector backbone containing the desired regulatory elements may be selected, and the nucleic acid molecules or nucleic acid constructs of the present invention may be cloned into the backbone to construct the recombinant vectors of the present invention.

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

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

[0068] The method for preparing a chiral amine of the present invention comprises the step of using the transaminase of the present invention in a reaction system containing a coenzyme and an amino donor to reduce the carbonyl group in the substrate represented by the following formula III to a chiral amino group:

[0069]

[0070] Wherein, R1 and R2 are as described above.

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

[0072] The coenzyme can be any coenzyme conventionally used in combination with transaminase in the art, such as pyridoxal-5-phosphate (PLP). The amount of the coenzyme can be conventional, for example, 0.1-5.0%, preferably 1-3%, of the weight of the substrate in the reaction system.

[0073] The amino donor in the reaction system can be any of the various amino donors commonly used in the art to prepare 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, and amino acids such as alanine (e.g., L-alanine) and aspartic acid (e.g., L-aspartic acid). Generally, the amount of the amino donor used in the reaction system can be easily determined based on conventional reaction methods. Typically, depending on the type of amino donor, the amount of the amino donor used can be 200% to 500%, such as 300%, 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. Commonly used buffers include, but are not limited to, phosphate buffer, triethanolamine-isopropylamine buffer, and the like. Preferably, the reaction system has a pH of 6-10, preferably 7-9, and more preferably 8-9. In some embodiments, the reaction system has a pH of 8.1-8.3.

[0075] The reaction system may also contain a cosolvent. Any cosolvent commonly used in the preparation of chiral amines can be used in the present invention. Typically, the cosolvent is an organic solvent, for example, selected from dimethyl sulfoxide, toluene, and alcoholic solvents. Alcoholic 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 10-50° C., preferably 20-45° C., and more preferably 40° C. In some embodiments, the reaction temperature is room temperature, i.e., 25±3° C. The reaction time can be determined depending on the amount of reactants and can generally be 0.1-120 hours, for example, 0.5-72 hours or 10-42 hours.

[0077] The present invention has found that when a specific cosolvent and amino donor are selected, a transaminase from Ruegeria arenilitoris sp (particularly the transaminase shown in SEQ ID NO: 1) catalyzes the reduction of the carbonyl group of the substrate to the amino group, and a significantly higher conversion rate can be achieved compared to the use of other cosolvents and amino donors. Therefore, in some embodiments of the present invention, the method for preparing the chiral amine represented by Formula I comprises using the transaminase shown in SEQ ID NO: 1 to catalyze the reaction between the amino donor and p-bromoacetophenone in the presence of a cosolvent; wherein the cosolvent is an alcohol solvent, preferably isopropanol; and the amino donor is an aliphatic amine, such as an aliphatic amine with a carbon chain length of 2-6 carbon atoms, more preferably isopropylamine. 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 of the present invention for preparing the chiral amine of Formula I comprises catalyzing the reaction between isopropylamine and a substrate of Formula III using the transaminase of SEQ ID NO: 1 in the presence of dimethyl sulfoxide. Preferably, the reaction system further comprises a coenzyme, such as pyridoxal phosphate. Preferably, the chiral amine is (S)-4-bromo-α-methylbenzylamine, and the substrate is p-bromoacetophenone.

[0078] The present invention also found that when the transaminase mutant of the present invention, especially the mutant obtained by mutation at one or more key positions of SEQ ID NO: 1 at positions 65, 318, 399, 419, 426, 465 and 466, is used to prepare the chiral amine of formula I, the substrate recognition ability is higher. Therefore, in some embodiments of the present invention, the method of preparing the chiral amine of formula I of the present invention comprises catalyzing the reaction between an amino donor and a substrate using the transaminase mutant described in any embodiment of the present invention in the presence of a cosolvent. The preferred cosolvent is dimethyl sulfoxide, and the preferred amino donor is an aliphatic amine, such as isopropylamine. Preferably, the reaction system of the reaction further contains a coenzyme, such as pyridoxal phosphate. In a particularly preferred embodiment, the transaminase mutant is a transaminase having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, the chiral amine is (S) 4-bromo-α-methylbenzylamine, and the substrate is p-bromoacetophenone.

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

[0080] In some embodiments, the present invention provides an enzyme preparation comprising the transaminase mutant described in any embodiment of the present invention. In some embodiments, the enzyme preparation is a lyophilized powder. In some embodiments, the enzyme preparation is a buffer containing the transaminase mutant. Preferably, the buffer is a phosphate buffer having a pH of 6-10, preferably 7-9, more preferably 8-9, and more preferably 8.1-8.3. In some embodiments, the phosphate buffer is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[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 represented by Formula I. In some embodiments, the reagent is the enzyme preparation described in any of the embodiments herein.

[0082] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0083] Preparation Example

[0084] Using SEQ ID NO:1 (coding sequence as shown in SEQ ID NO:5) as the parent, directed evolution was performed using roller-wheel PCR, iterative saturation mutagenesis, and / or combinatorial mutagenesis strategies. Mutants were then transformed into competent Escherichia coli BL21(DE3) cells and evenly plated onto LB agar plates supplemented with 50 μg / ml kanamycin. The plates were then incubated in a 37°C incubator for 18 hours. Mutants from the transformed plates were picked with a toothpick and transferred to 96-well plates and incubated overnight at 37°C and 220 rpm in a shaker. Fifty microliters of bacterial culture were pipetted from the wells of the primary plate into the corresponding wells of the secondary plate. After incubation at 37°C and 220 rpm for 2-3 hours, IPTG was added to a final concentration of 0.2 mM and incubated at 30°C for 20 hours to obtain the corresponding mutants for high-throughput screening. Rescreening was performed using HPLC and SFC assays to identify mutants with significantly improved activity and stability for sequencing. The sequencing results are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the coding sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

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

[0086] In the examples of the present invention, two detection methods were used: HPLC and SFC.

[0087] Among them, HPLC detection is more suitable for production applications, has a longer analysis time, can detect various by-products, impurities, etc., and can ultimately confirm the conversion rate of the enzyme.

[0088] SFC testing is more suitable for enzyme screening, with shorter analysis times and rapid reflection of reaction conversion and chirality. It is used to compare enzyme selectivity and activity, and has significant advantages in comparing the activity of mutant enzymes. The HPLC and SFC parameters used in this invention are as follows:

[0089] HPLC parameters:

[0090] Instrument: Shimadzu LC-20A

[0091] Column: Xbridge C18

[0092] Mobile phase: 0.05% TFA (trifluoroacetic acid) in H2O: 0.05% TFA (trifluoroacetic acid) in ACN 85:15

[0093] Flow rate: 1 mL / min

[0094] Wavelength: 230nm

[0095] SFC parameters:

[0096]

[0097] Buffer preparation:

[0098] I80X Buffer: Weigh 27.8 g of potassium phosphate dibasic trihydrate and 10.6 g of potassium dihydrogen phosphate into a 2 L glass bottle. Add 1.3 L of purified water to the bottle and stir at room temperature until the solids dissolve. Add 171 mL of isopropylamine to the bottle, maintain the temperature at 20-30°C, and add 35% HCl to adjust the pH to 8.0. Add purified water to bring the volume up to 2 L.

[0099] I95X Buffer: Weigh 27.8 g of potassium phosphate dibasic trihydrate and 10.6 g of potassium dihydrogen phosphate into a 2 L glass bottle. Add 1.3 L of purified water to the bottle and stir at room temperature until the solids dissolve. Add 176 mL of isopropylamine to the bottle, maintain the temperature at 20-30°C, and add 35% HCl to adjust the pH to 9.5. Add purified water to bring the volume up to 2 L.

[0100] The difference between I80X buffer and I95X buffer lies in the pH. Using a higher pH is to increase the environmental pressure of the reaction and evaluate whether the screened enzyme can withstand a higher pH. On this basis, further evolution can be carried out to facilitate the screening of points with higher conversion rates under more stringent conditions.

[0101] Example 1

[0102] Prepare reaction buffer: Measure 350 mL of purified water into a 1000 mL jacketed bottle, add 2.78 g of dipotassium phosphate trihydrate and 1.06 g of potassium dihydrogen phosphate, and stir until completely dissolved. Measure 135 mL of isopropylamine and add 52 mL of 85% phosphoric acid. Adjust the pH to 8.1-8.3. Maintain the temperature at 20-30°C and stir until the solids are completely dissolved.

[0103] Add 0.25g of pyridoxal phosphate (PLP) to the vial and slowly add 4.0g of lyophilized transaminase (amino acid sequence shown in SEQ ID NO: 1). Mix 80mL of dimethyl sulfoxide (DMSO) with 20g of p-bromoacetophenone until the solution is clear, then add the solution to the vial. After reacting at 40°C for 48 hours, add 1g of enzyme and 25mg of pyridoxal phosphate (PLP) (dissolved in 10mL of reaction buffer), and then add once every 12 hours (a total of 2 additions).

[0104] After 72 hours of reaction, the conversion rate was 91.9% as determined by HPLC. Figure 1 As shown in Table 1, the peak at t=5.575 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0105] Table 1

[0106] Chl 230nm 4nm

[0107] peak Component name Retention time Peak area Peak area% Peak height Resolution tailing factor Theoretical plates 1 SM1 5.575 5802358 91.921 557269 0.0 1.8 44241.520 2 SM0 7.838 509942 8.079 127357 11.4 1.4 432677.097 total 6312300 100.000 684626

[0108] Example 2

[0109] To an 8 mL reaction vial, add 5.9 mL of I80X buffer, 10 mg of transaminase (amino acid sequence as shown in SEQ ID NO: 2) lyophilized powder, and 2.4 mg of pyridoxal phosphate (PLP). After thorough stirring, add 120 mg of p-bromoacetophenone and 360 μL of dimethyl sulfoxide (DMSO). The reaction was controlled at 45°C and shaken at 1000 rpm for 18 hours to allow for sufficient reaction.

[0110] After the reaction was completed, the SFC test showed that the conversion rate was 55% and the ee value was >99%. Figure 2 As shown in Table 2, the peak at t = 1.081 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0111] Table 2

[0112] Peak name Retention time (RT) Peak area % Peak Area Peak height 1 SM 0.934 114935 44.13 99059 2 Product 1.081 145504 55.87 38417

[0113] Example 3

[0114] Prepare reaction buffer: Measure 720 mL of purified water into a 2000 mL jacketed flask. Add 6.95 g of dipotassium hydrogen phosphate trihydrate and 2.65 g of potassium dihydrogen phosphate. Stir until the solids are completely dissolved. Add 214 mL of isopropylamine and 89 mL of 85% phosphoric acid. Adjust the pH to 8.1-8.3 and maintain the temperature at 20-25°C.

[0115] Add 1.0 g of pyridoxal phosphate (PLP) to the bottle, and slowly add 5.0 g of lyophilized transaminase (amino acid sequence as shown in SEQ ID NO: 2) to the bottle. Control the temperature at 20-25°C and stir until the solid dissolves. Dissolve 50 g of p-bromoacetophenone in 150 mL of dimethyl sulfoxide (DMSO) and slowly add the solution dropwise to the bottle over 4 hours. Maintain the temperature at 40°C and stir under a nitrogen stream for 38 hours.

[0116] After 38 hours of reaction, the conversion rate was 92.1% as determined by HPLC. Figure 3 As shown in Table 3, the peak at t=5.502 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0117] Table 3

[0118] Chl 230nm 4nm

[0119] peak Retention time Peak area Peak area% Peak height Resolution tailing factor 1 5.502 13246564 92.051 800601 0.00 1.94 2 7.858 1143931 7.949 318129 9.29 1.34 total 14390495 100.000 1118730

[0120] Example 4

[0121] To an 8 mL reaction vial, add 2 mL of I95X buffer, 0.6 mg of transaminase (amino acid sequence as shown in SEQ ID NO: 3) lyophilized powder, and 0.92 mg of pyridoxal phosphate (PLP). After thorough stirring, add 46 mg of p-bromoacetophenone and 138 μL of dimethyl sulfoxide (DMSO). The reaction was controlled at 50°C and shaken at 1000 rpm for 20 hours to allow for sufficient reaction.

[0122] After the reaction was completed, SFC detection showed that the conversion rate was 66.4% and the ee value was >99%. Figure 4 As shown in Table 4, the peak at t = 1.071 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0123] Table 4

[0124] Peak name Retention time (RT) Peak area % Peak Area Peak height 1 SM 0.946 166203 33.6222 150183 2 Product 1.071 328121 66.3778 72787

[0125] Example 5

[0126] Prepare reaction buffer: add 750 mL of purified water to a 2000 mL jacketed bottle, add 344 mL of isopropylamine and 135 mL of 85% phosphoric acid, control the pH at 9.4-9.6, and the temperature at 44-46°C.

[0127] Add 0.1g of pyridoxal phosphate (PLP) to the bottle, and slowly add 1.0g of transaminase (amino acid sequence as shown in SEQ ID NO: 3) lyophilized powder to the bottle. Control the temperature at 20-25°C and stir until the solid dissolves. Dissolve 50g of p-bromoacetophenone in 150mL of dimethyl sulfoxide (DMSO), raise the temperature to 37-45°C, and slowly add the p-bromoacetophenone and dimethyl sulfoxide (DMSO) mixed solution dropwise to the bottle. Maintain the temperature at 45°C and stir under a nitrogen stream for 42 hours.

[0128] After 42 hours of reaction, the conversion rate was 82% as determined by HPLC. Figure 5 As shown in Table 5, the peak at t=5.148 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0129] Table 5

[0130] Chl 230nm 4nm

[0131] peak Retention time Peak area Peak area% Peak height Resolution tailing factor 1 5.148 4965030 82.857 970514 0.00 1.57 2 6.986 1027276 17.143 249514 13.40 1.42 total 5992306 100.000 1220028

[0132] Example 6

[0133] Prepare reaction buffer: add 15 mL of purified water to a 40 mL glass bottle, add 6.85 mL of isopropylamine and 2.5 mL of 85% phosphoric acid, control the pH at 9.4-9.6, and the temperature at 44-46°C.

[0134] Add 0.02g of pyridoxal phosphate (PLP) to the vial and slowly add 200mg of lyophilized transaminase (amino acid sequence as shown in SEQ ID NO: 2) to the vial. Control the temperature at 20-25°C and stir until the solid dissolves. Dissolve 1.0g of p-bromoacetophenone in 3mL of dimethyl sulfoxide (DMSO). Raise the temperature to 37-45°C and slowly add the p-bromoacetophenone and DMSO mixed solution dropwise to the vial. Maintain the temperature at 45°C and stir for 2 hours.

[0135] After 2 hours of reaction, the conversion rate was 50.49% as determined by HPLC. Figure 6 As shown in Table 6, the peak at t=4.67 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0136] Table 6

[0137] Chl 230nm 4nm

[0138] peak Retention time Peak area Peak area% Peak height Resolution tailing factor 1 4.675 1611150 50.490 404958 0.00 1.61 2 6.990 1579902 49.510 512078 21.58 1.40 total 3191052 100.000 917036

[0139] Example 7

[0140] Prepare reaction buffer: add 15 mL of purified water to a 40 mL glass bottle, add 6.85 mL of isopropylamine and 2.5 mL of 85% phosphoric acid, control the pH at 9.4-9.6, and the temperature at 44-46°C.

[0141] Add 0.02g of pyridoxal phosphate (PLP) to the vial and slowly add 200mg of lyophilized transaminase (amino acid sequence as shown in SEQ ID NO: 3) to the vial. Control the temperature at 20-25°C and stir until the solid dissolves. Dissolve 1.0g of p-bromoacetophenone in 3mL of dimethyl sulfoxide (DMSO). Raise the temperature to 37-45°C and slowly add the p-bromoacetophenone and DMSO mixed solution dropwise to the vial. Maintain the temperature at 45°C and stir for 2 hours.

[0142] After 2 hours of reaction, the conversion rate was 61.92% as determined by HPLC. Figure 7 As shown in Table 7, the peak at t = 4.67 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0143] Table 7

[0144] Chl 230nm 4nm

[0145] peak Retention time Peak area Peak area% Peak height Resolution tailing factor 1 4.671 1968553 61.919 462957 0.00 1.66 2 7.000 1210709 38.081 396606 21.09 1.40 total 3179262 100.000 859563

[0146] Example 8

[0147] To an 8 mL reaction vial, add 2 mL of I95X buffer, 0.6 mg of transaminase (amino acid sequence as shown in SEQ ID NO: 4) lyophilized powder, and 0.92 mg of pyridoxal phosphate (PLP). After thorough stirring, add 46 mg of p-bromoacetophenone and 138 μL of dimethyl sulfoxide (DMSO). The reaction was controlled at 50°C and shaken at 1000 rpm for 20 hours to allow for sufficient reaction.

[0148] After the reaction was completed, SFC detection showed that the conversion rate was 5.30% and the ee value was >99%. Figure 8 As shown in Table 8, the peak at t = 1.069 is the target compound (S) 4-bromo-α-methylbenzylamine.

[0149] Table 8

[0150] Peak name Retention time (RT) Peak area % Peak Area Peak height 1 SM 0.960 353633 94.70 328035 2 Product 1.069 19790 5.30 4593

[0151] Example result analysis:

[0152] In the above examples, the experimental conditions of Examples 1, 3, and 5 are the reaction conditions that can minimize the cost of large-scale production and can be used as simulation data for scale-up production experiments. Comparing these three examples, the most prominent difference is the different enzyme dosage (in the production of this compound, enzymes account for the majority of the cost).

[0153] Calculating the conversion yield per unit enzyme amount in these three examples, 1g of enzyme powder with the sequence of SEQ ID NO:1 can produce 3.06g of product, 1g of enzyme powder with the sequence of SEQ ID NO:2 can produce 9.1g of product, and 1g of enzyme powder with the sequence of SEQ ID NO:3 can produce 41g of product. These data fully demonstrate that the enzyme with the sequence of SEQ ID NO:3 has a substrate recognition and conversion ability far superior to the enzymes with the sequences of SEQ ID NO:1 and SEQ ID NO:2.

[0154] Examples 6 and 7 more intuitively demonstrate the difference between the enzyme with the sequence of SEQ ID NO: 2 and the enzyme with the sequence of SEQ ID NO: 3. That is, under the same reaction conditions, the conversion rate of the enzyme with the sequence of SEQ ID NO: 3 is higher than that of the enzyme with the sequence of SEQ ID NO: 2.

[0155] Examples 2, 4, and 8 are reaction conditions used for enzyme screening. By comparing Example 2 and Example 4, it can be seen that the conversion rate of the enzyme with the sequence of SEQ ID NO: 3 is higher than the conversion rate of the enzyme with the sequence of SEQ ID NO: 2, and the enzyme with the sequence of SEQ ID NO: 3 can tolerate higher reaction temperatures and reaction pH values ​​than the enzyme with the sequence of SEQ ID NO: 2, that is, the enzyme with the sequence of SEQ ID NO: 3 can tolerate more harsh conditions.

[0156] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. SEQUENCE LISTING <110> Shanghai Hequan Pharmaceutical Research and Development Co., Ltd. Shanghai Hequan Pharmaceutical Co., Ltd. <120> Transaminase and its application in the preparation of optically pure chiral amines <130> P21018747C <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> Wild-type transaminase <400> 1 Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln 1 5 10 15 Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala 20 25 30 Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp 35 40 45 Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu 50 55 60 Trp Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala 65 70 75 80 Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr 85 90 95 Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala 100 105 110 Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala 115 120 125 Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly 130 135 140 Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly 145 150 155 160 Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala 165 170 175 Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp 180 185 190 Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg 195 200 205 Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val 210 215 220 Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val 225 230 235 240 Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr 245 250 255 Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr 260 265 270 Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met 275 280 285 Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser 290 295 300 Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn 305 310 315 320 His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg 340 345 350 Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp 355 360 365 His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile 370 375 380 Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro 385 390 395 400 Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu 405 410 415 Ile Met Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val 420 425 430 Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser 435 440 445 Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser 450 455 460 Glu Gly 465 <210> 2 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> transformation <400> 2 Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln 1 5 10 15 Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala 20 25 30 Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp 35 40 45 Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu 50 55 60 Phe Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala 65 70 75 80 Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr 85 90 95 Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala 100 105 110 Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala 115 120 125 Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly 130 135 140 Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly 145 150 155 160 Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala 165 170 175 Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp 180 185 190 Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg 195 200 205 Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val 210 215 220 Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val 225 230 235 240 Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr 245 250 255 Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr 260 265 270 Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met 275 280 285 Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser 290 295 300 Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn 305 310 315 320 His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg 340 345 350 Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp 355 360 365 His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile 370 375 380 Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro 385 390 395 400 Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu 405 410 415 Ile Met Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val 420 425 430 Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser 435 440 445 Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser 450 455 460 Glu Gly 465 <210> 3 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> transformation <400> 3 Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln 1 5 10 15 Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala 20 25 30 Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp 35 40 45 Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu 50 55 60 Phe Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala 65 70 75 80 Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr 85 90 95 Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala 100 105 110 Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala 115 120 125 Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly 130 135 140 Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly 145 150 155 160 Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala 165 170 175 Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp 180 185 190 Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg 195 200 205 Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val 210 215 220 Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val 225 230 235 240 Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr 245 250 255 Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr 260 265 270 Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met 275 280 285 Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser 290 295 300 Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Thr Phe Asn 305 310 315 320 His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg 340 345 350 Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp 355 360 365 His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile 370 375 380 Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Tyr Pro 385 390 395 400 Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu 405 410 415 Ile Met Val His Val Gly Asp Arg Met Val Ile Ser Pro Pro Leu Val 420 425 430 Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser 435 440 445 Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser 450 455 460 <210> 4 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> transformation <400> 4 Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln 1 5 10 15 Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala 20 25 30 Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp 35 40 45 Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu 50 55 60 Phe Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala 65 70 75 80 Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr 85 90 95 Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala 100 105 110 Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala 115 120 125 Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly 130 135 140 Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly 145 150 155 160 Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala 165 170 175 Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp 180 185 190 Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg 195 200 205 Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val 210 215 220 Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val 225 230 235 240 Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr 245 250 255 Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr 260 265 270 Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met 275 280 285 Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser 290 295 300 Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Thr Phe Asn 305 310 315 320 His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg 340 345 350 Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp 355 360 365 His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile 370 375 380 Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Tyr Pro 385 390 395 400 Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu 405 410 415 Ile Met Val His Ser Gly Asp Arg Met Val Ile Ser Pro Pro Leu Val 420 425 430 Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser 435 440 445 Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser 450 455 460 <210> 5 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of wild-type transaminase <400> 5 atgtccctgg caactatcac caaccacatg ccgactgcgg agctgcaggc tctggatgcg 60 gctcaccatc tgcacccgtt ctctgcgaac aacgcactgg gtgaggaggg tacgcgtgtg 120 attacccgtg ctcgtggcgt gtggctgaac gactcagaag gcgaggagat tctggacgct 180 atggcaggcc tgtggtgcgt taacatcggt tatggtcgtg atgaactggc tgaggttgcg 240 gctcgtcaga tgcgcgaact gccttactac aataccttct tcaaaaccac tcatgtacca 300 gcaatcgctc tggcccagaa actggctgaa ctggctccgg gcgatctgaa ccatgtattc 360 tttgcgggcg gcggttccga agcaaacgac accaatatcc gaatggttcg tacttactgg 420 cagaacaaag gccagccgga aaaaactgta atcatctctc gtaaaaacgc ttatcacggc 480 agcactgtag cgtcttccgc gctgggcggt atggccggta tgcacgcgca gagcggtctg 540 atcccggacg tgcaccatat caaccagccg aattggtggg cagagggtgg tgacatggac 600 ccagaagagt tcggtctggc tcgtgcgcgt gagctggagg aggctatctt agaactgggt 660 gaaaaccgtg tggccgcatt tatcgccgag ccagtacagg gtgctggtgg cgttatcgta 720 gccccggact cctactggcc ggaaatccag cgaatttgtg acaagtacga tatcctgctg 780 atcgccgatg aagtcatttg cggtttcggt cgtaccggta attggttcgg tacgcagacg 840 atgggtatcc gcccgcacat catgaccatc gcaaaaggtc tgtccagcgg ctacgctccg 900 attggtggtt ctatcgtctg tgatgaagtc gcgcacgtga tcggcaaaga tgagttcaat 960 catggttaca cctatagcgg tcacccggtt gctgcggcgg tcgccttgga gaacctgcgt 1020 atcctggaag aagaaaatat tctggatcac gtacgtaacg tggcggcccc gtatctgaaa 1080 gaaaaatggg aagcgctgac tgaccatccg cttgtgggtg aagctaaaat cgtcggcatg 1140 atggcctcta tcgctctgac gccgaacaaa gcaagccgcg cgaaattcgc atccgaaccg 1200 ggcaccatcg gttacatctg tcgtgaacgc tgtttcgcta acaatctgat catgcgtcac 1260 gttggcgacc gcatgattat ctccccgcca cttgtcatca ccccggcgga aatcgatgaa 1320 atgtttgtac gcattcgcaa aagcctggac gaggcgcagg cggagattga gaaacagggt 1380 ctgatgaaat ccgaaggttg a 1401 <210> 6 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of transaminase mutant <400> 6 atgtccctgg caactatcac caaccacatg ccgactgcgg agctgcaggc tctggatgcg 60 gctcaccatc tgcacccgtt ctctgcgaac aacgcactgg gtgaggaggg tacgcgtgtg 120 attacccgtg ctcgtggcgt gtggctgaac gactcagaag gcgaggagat tctggacgct 180 atggcaggcc tgttttgcgt taacatcggt tatggtcgtg atgaactggc tgaggttgcg 240 gctcgtcaga tgcgcgaact gccttactac aataccttct tcaaaaccac tcatgtacca 300 gcaatcgctc tggcccagaa actggctgaa ctggctccgg gcgatctgaa ccatgtattc 360 tttgcgggcg gcggttccga agcaaacgac accaatatcc gaatggttcg tacttactgg 420 cagaacaaag gccagccgga aaaaactgta atcatctctc gtaaaaacgc ttatcacggc 480 agcactgtag cgtcttccgc gctgggcggt atggccggta tgcacgcgca gagcggtctg 540 atcccggacg tgcaccatat caaccagccg aattggtggg cagagggtgg tgacatggac 600 ccagaagagt tcggtctggc tcgtgcgcgt gagctggagg aggctatctt agaactgggt 660 gaaaaccgtg tggccgcatt tatcgccgag ccagtacagg gtgctggtgg cgttatcgta 720 gccccggact cctactggcc ggaaatccag cgaatttgtg acaagtacga tatcctgctg 780 atcgccgatg aagtcatttg cggtttcggt cgtaccggta attggttcgg tacgcagacg 840 atgggtatcc gcccgcacat catgaccatc gcaaaaggtc tgtccagcgg ctacgctccg 900 attggtggtt ctatcgtctg tgatgaagtc gcgcacgtga tcggcaaaga tgagttcaat 960 catggttaca cctatagcgg tcacccggtt gctgcggcgg tcgccttgga gaacctgcgt 1020 atcctggaag aagaaaatat tctggatcac gtacgtaacg tggcggcccc gtatctgaaa 1080 gaaaaatggg aagcgctgac tgaccatccg cttgtgggtg aagctaaaat cgtcggcatg 1140 atggcctcta tcgctctgac gccgaacaaa gcaagccgcg cgaaattcgc atccgaaccg 1200 ggcaccatcg gttacatctg tcgtgaacgc tgtttcgcta acaatctgat catgcgtcac 1260 gttggcgacc gcatgattat ctccccgcca cttgtcatca ccccggcgga aatcgatgaa 1320 atgtttgtac gcattcgcaa aagcctggac gaggcgcagg cggagattga gaaacagggt 1380 ctgatgaaat ccgaaggttg a 1401 <210> 7 <211> 1395 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of transaminase mutant <400> 7 atgtccctgg caactatcac caaccacatg ccgactgcgg agctgcaggc tctggatgcg 60 gctcaccatc tgcacccgtt ctctgcgaac aacgcactgg gtgaggaggg tacgcgtgtg 120 attacccgtg ctcgtggcgt gtggctgaac gactcagaag gcgaggagat tctggacgct 180 atggcaggcc tgttttgcgt taacatcggt tatggtcgtg atgaactggc tgaggttgcg 240 gctcgtcaga tgcgcgaact gccttactac aataccttct tcaaaaccac tcatgtacca 300 gcaatcgctc tggcccagaa actggctgaa ctggctccgg gcgatctgaa ccatgtattc 360 tttgcgggcg gcggttccga agcaaacgac accaatatcc gaatggttcg tacttactgg 420 cagaacaaag gccagccgga aaaaactgta atcatctctc gtaaaaacgc ttatcacggc 480 agcactgtag cgtcctccgc gctgggcggt atggccggta tgcacgcgca gagcggtctg 540 atcccggacg tgcaccatat caaccagccg aattggtggg cagagggtgg tgacatggac 600 ccagaagagt tcggtctggc tcgtgcgcgt gagctggagg aggctatctt agaactgggt 660 gaaaaccgtg tggccgcatt tatcgccgag ccagtacagg gtgctggtgg cgttatcgta 720 gccccggact cctactggcc ggaaatccag cgaatttgtg acaagtacga tatcctgctg 780 atcgccgatg aagtcatttg cggtttcggt cgtaccggta attggttcgg tacgcagacg 840 atgggtatcc gcccgcacat catgaccatc gcaaaaggtc tgtccagcgg ctacgctccg 900 attggtggtt ctatcgtctg tgatgaagtc gcgcacgtga tcggcaaaga tacgttcaat 960 catggttaca cctatagcgg tcacccggtt gctgcggcgg tcgccttgga gaacctgcgt 1020 atcctggaag aagaaaatat tctggatcac gtacgtaacg tggcggcccc gtatctgaaa 1080 gaaaaatggg aagcgctgac tgaccatccg cttgtgggtg aagctaaaat cgtcggcatg 1140 atggcctcta tcgctctgac gccgaacaaa gcaagccgcg cgaaattcgc atcctatccg 1200 ggcaccatcg gttacatctg tcgtgaacgc tgtttcgcta acaatctgat catggttcac 1260 gttggcgacc gcatggttat ctccccgcca cttgtcatca ccccggcgga aatcgatgaa 1320 atgtttgtac gcattcgcaa aagcctggac gaggcgcagg cggagattga gaaacagggt 1380 ctgatgaaat cctga 1395 <210> 8 <211> 1395 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of transaminase mutant <400> 8 atgtccctgg caactatcac caaccacatg ccgactgcgg agctgcaggc tctggatgcg 60 gctcaccatc tgcacccgtt ctctgcgaac aacgcactgg gtgaggaggg tacgcgtgtg 120 attacccgtg ctcgtggcgt gtggctgaac gactcagaag gcgaggagat tctggacgct 180 atggcaggcc tgttttgcgt taacatcggt tatggtcgtg atgaactggc tgaggttgcg 240 gctcgtcaga tgcgcgaact gccttactac aataccttct tcaaaaccac tcatgtacca 300 gcaatcgctc tggcccagaa actggctgaa ctggctccgg gcgatctgaa ccatgtattc 360 tttgcgggcg gcggttccga agcaaacgac accaatatcc gaatggttcg tacttactgg 420 cagaacaaag gccagccgga aaaaactgta atcatctctc gtaaaaacgc ttatcacggc 480 agcactgtag cgtcctccgc gctgggcggt atggccggta tgcacgcgca gagcggtctg 540 atcccggacg tgcaccatat caaccagccg aattggtggg cagagggtgg tgacatggac 600 ccagaagagt tcggtctggc tcgtgcgcgt gagctggagg aggctatctt agaactgggt 660 gaaaaccgtg tggccgcatt tatcgccgag ccagtacagg gtgctggtgg cgttatcgta 720 gccccggact cctactggcc ggaaatccag cgaatttgtg acaagtacga tatcctgctg 780 atcgccgatg aagtcatttg cggtttcggt cgtaccggta attggttcgg tacgcagacg 840 atgggtatcc gcccgcacat catgaccatc gcaaaaggtc tgtccagcgg ctacgctccg 900 attggtggtt ctatcgtctg tgatgaagtc gcgcacgtga tcggcaaaga tacgttcaat 960 catggttaca cctatagcgg tcacccggtt gctgcggcgg tcgccttgga gaacctgcgt 1020 atcctggaag aagaaaatat tctggatcac gtacgtaacg tggcggcccc gtatctgaaa 1080 gaaaaatggg aagcgctgac tgaccatccg cttgtgggtg aagctaaaat cgtcggcatg 1140 atggcctcta tcgctctgac gccgaacaaa gcaagccgcg cgaaattcgc atcctatccg 1200 ggcaccatcg gttacatctg tcgtgaacgc tgtttcgcta acaatctgat catggttcac 1260 agtggcgacc gcatggttat ctccccgcca cttgtcatca ccccggcgga aatcgatgaa 1320 atgtttgtac gcattcgcaa aagcctggac gaggcgcagg cggagattga gaaacagggt 1380 ctgatgaaat cctga 1395

Claims

1. A transaminase, characterized in that The amino acid sequence of the transaminase is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. A nucleic acid molecule, the polynucleotide sequence of which encodes the transaminase according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The polynucleotide sequence is shown in SEQ ID NO: 6 or SEQ ID NO:

7. A nucleic acid construct comprising the nucleic acid molecule according to claim 2 or 3.

5. The nucleic acid construct according to claim 4, wherein The nucleic acid construct is an expression cassette. A recombinant vector comprising 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, wherein The recombinant vector is a recombinant cloning vector.

8. The recombinant vector according to claim 6, wherein The recombinant vector is a recombinant expression vector.

9. A host cell comprising 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. A host cell expressing the transaminase according to claim 1. An enzyme preparation comprising the transaminase according to claim 1 .

12. A method for preparing a chiral amine represented by the following formula I: （I）； in, The preparation method comprises using the transaminase 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 the following formula III in the presence of a cosolvent and a coenzyme, thereby preparing a chiral amine represented by formula I: (III); Wherein, the compound of formula I is (S)4-bromo-α-methylbenzylamine; and the compound of formula III is p-bromoacetophenone.

13. The preparation method according to claim 12, wherein The cosolvent is dimethyl sulfoxide; and / or the amino donor is isopropylamine.

14. The preparation method according to claim 12, wherein The amount of transaminase used is 1-50% of the weight of the substrate in the reaction system; The reaction system contains coenzyme, and the amount of coenzyme used is 0.1-5.0% of the weight of the substrate; In the reaction system, the amount of the amino donor is 200%-500% of the weight of the substrate; Reaction system pH 6-10; The reaction temperature is 10-50°C; and / or The reaction time is 0.1-120 hours.

15. The preparation method according to claim 14, wherein The amount of transaminase used is 10-40% of the weight of the substrate in the reaction system; The reaction system contains a coenzyme, the amount of which is 1-3% of the weight of the substrate; the coenzyme is pyridoxal 5-phosphate (PLP); In the reaction system, the amount of amino donor used was 300% of the weight of the substrate; The pH of the reaction system is 7-9; The reaction temperature is 20-45°C; and / or The reaction time is 0.5-72 hours.

16. The preparation method according to claim 14, wherein The amount of transaminase used is 15-30% of the weight of the substrate in the reaction system; and / or the reaction time is 10-42 hours.

17. The preparation method according to claim 15, wherein The reaction system has a pH of 8-9; and / or The reaction temperature was 40°C.

18. The preparation method according to claim 17, wherein The reaction system pH is 8.1~8.

3.

19. Use of the transaminase or an enzyme preparation containing the transaminase according to claim 1 in the preparation of optically pure chiral amines; the optically pure chiral amine is (S) 4-bromo-α-methylbenzylamine.

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