WO2022143652A1 - 转氨酶及其在制备光学纯手性胺中的应用 - Google Patents

转氨酶及其在制备光学纯手性胺中的应用 Download PDF

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WO2022143652A1
WO2022143652A1 PCT/CN2021/142048 CN2021142048W WO2022143652A1 WO 2022143652 A1 WO2022143652 A1 WO 2022143652A1 CN 2021142048 W CN2021142048 W CN 2021142048W WO 2022143652 A1 WO2022143652 A1 WO 2022143652A1
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transaminase
seq
amino acid
nucleic acid
acid sequence
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French (fr)
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李�杰
孙丰来
唐大林
祝黛莲
郑晨抗
朱景仰
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上海合全药物研发有限公司
上海合全药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)

Definitions

  • the present invention relates to transaminase and its application, especially the application in biocatalytic preparation of optically pure chiral amine.
  • Optically pure chiral amines are a class of valuable pharmaceutical and fine chemical intermediates. At present, more than 70% of drugs are chiral amines and their derivatives are synthesized using chiral amines as intermediates.
  • (R)-1,1,1-trifluoropropylamine represented by the following formula II is a very important chiral amine, which is used in the synthesis of many pharmaceutical intermediates.
  • the methods for preparing (R)-1,1,1-trifluoropropylamine mainly include chemical method and biocatalysis method.
  • the general process of chemical method is shown below.
  • 1,1,1-trifluoroacetone is used as raw material
  • R- ⁇ -phenethylamine is used to induce chirality
  • chemical resolution is performed to increase the chirality value to 99%, but the total chirality value is increased to 99%.
  • the yield is less than 40%, which is not suitable for industrial large-scale production from the perspective of economy and environmental protection (J.Org.Chem., 1997, 62, 10, 3030-3031; Bioorganic & Medicinal Chemistry Letters, 2013, 23, 3947-3953).
  • Biological enzymatic catalysis usually utilizes transaminases to kinetically resolve racemic amines, or generate chiral amines through asymmetric synthesis of ketones.
  • transaminases to kinetically resolve racemic amines, or generate chiral amines through asymmetric synthesis of ketones.
  • enzymatic methods have the advantages of high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, and environmental friendliness.
  • the substrate specificity, enantioselectivity and/or conversion rates of the enzymes commonly used are not high enough for industrial production processes.
  • a first aspect of the present invention provides a transaminase, the amino acid sequence of the transaminase having the amino acid sequence at one or more of the 136th, 282nd and 306th positions compared with the amino acid sequence shown in SEQ ID NO: 1 Substitution mutations, the amino acid residues at the remaining positions are the same as SEQ ID NO:1.
  • the substitution mutation is selected from one or more of the following substitution mutations: I136W, T282S, and V306L.
  • amino acid sequence of the transaminase is shown in SEQ ID NO:2.
  • a second aspect of the present invention provides a nucleic acid molecule, the polynucleotide sequence of which is selected from:
  • the polynucleotide sequence is selected from the group consisting of: the polynucleotide sequence set forth in SEQ ID NO: 4 or its complement.
  • a third aspect of the present invention provides a nucleic acid construct comprising the nucleic acid molecule described in any of the embodiments of the present invention; preferably, the nucleic acid construct is an expression cassette.
  • 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.
  • 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; preferably, The host cells are selected from E. coli cells, insect cells, yeast cells and mammalian cells.
  • the sixth aspect of the present invention provides an enzyme preparation comprising the transaminase according to any one of the embodiments of the present invention.
  • the seventh aspect of the present invention provides the preparation method of the chiral amine shown in the following formula I:
  • the method comprises using a transaminase from Arthrobacter sp or a transaminase having at least 95% sequence identity to its amino acid sequence or any of the embodiments herein in the presence of a co-solvent and optionally a coenzyme
  • the transaminase mutant or its enzyme preparation catalyzes the reaction of the amino donor with the substrate shown in the following formula III, thereby preparing the chiral amine shown in the formula I:
  • R 1 is a halogenated C 1-4 alkyl group
  • R 2 is a C 1-4 alkyl group
  • the compound of formula I is (R)-1,1,1-trifluoropropylamine, and the compound of formula III is 1,1,1-trifluoroacetone.
  • the amino acid sequence of the transaminase of Arthrobacter sp is set forth in SEQ ID NO: 1, and the amino acid sequence is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1
  • the aminotransferases of sequence identity are those with NCBI accession numbers 3WWH_A, 3WWI_A, 5FR9_A or 3WWJ_A.
  • the co-solvent is selected from the group consisting of dimethyl sulfoxide, alcoholic solvents, toluene, or a combination thereof.
  • the amino donor is selected from the group consisting of: aromatic amines, aliphatic amines, amino acids, or combinations thereof.
  • the alcoholic solvent is isopropanol.
  • the aromatic amine is phenethylamine.
  • the aliphatic amine is an aliphatic amine with a carbon chain length of 2-6 carbon atoms, preferably isopropylamine.
  • the amino acid is alanine and/or aspartic acid.
  • the amount of transaminase used is 1-50% by weight of the substrate in the reaction system, such as 10-40% or 15-30%.
  • the reaction system contains a coenzyme, and the amount of the coenzyme is 0.1-5.0% by weight of the substrate, preferably 1-3%; the preferred coenzyme is pyridoxal-5-phosphate.
  • the amount of amino donor in the reaction system is 30% to 500% by weight of the substrate, such as 50% to 300%.
  • the pH of the reaction system is 6-10, preferably 7-9, more preferably 8-9, and more preferably 8.3-8.6.
  • the reaction temperature is 10°C to 50°C, preferably 20°C to 45°C, more preferably 20°C to 35°C.
  • the reaction time is 0.1 to 120 hours, such as 0.5 to 48 hours or 10 to 24 hours.
  • the cosolvent is dimethyl sulfoxide or isopropanol;
  • the amino donor is aliphatic amine, preferably isopropylamine, or aromatic amine, preferably R-phenethylamine;
  • transaminase It is a transaminase from Arthrobacter sp, more preferably a transaminase whose amino acid sequence is shown in SEQ ID NO: 1 or its enzyme preparation;
  • the compound of formula I is (R)-1,1,1-trifluoropropylamine;
  • the compound of formula III is 1,1,1-trifluoroacetone.
  • the cosolvent is dimethyl sulfoxide;
  • the amino donor is isopropylamine or R-phenethylamine;
  • the transaminase is a transaminase from Arthrobacter sp, more preferably It is a transaminase or its enzyme preparation whose amino acid sequence is shown in SEQ ID NO: 1;
  • the compound of formula I is (R)-1,1,1-trifluoropropylamine;
  • the compound of formula III is 1,1,1-trifluoroacetone.
  • the cosolvent is isopropanol;
  • the amino donor is aliphatic amine, preferably isopropylamine;
  • the transaminase is a transaminase from Arthrobacter sp, more preferably an amino acid
  • the aminotransferase whose sequence is shown in SEQ ID NO: 1 or its enzyme preparation;
  • the compound of formula I is (R)-1,1,1-trifluoropropylamine;
  • the compound of formula III is 1,1,1-trifluoroacetone.
  • the co-solvent is dimethyl sulfoxide;
  • the amino donor is aliphatic amine, preferably isopropylamine;
  • the transaminase is the transaminase mutant described in any of the embodiments herein or an enzyme thereof formulation;
  • the compound of formula I is (R)-1,1,1-trifluoropropylamine;
  • the compound of formula III is 1,1,1-trifluoroacetone.
  • the present invention also provides a transaminase from Arthrobacter sp or its enzyme preparation and/or the transaminase mutant or its enzyme preparation according to any of the embodiments herein in increasing the conversion rate in the preparation of optically homochiral amines application.
  • Fig. 1 is the high performance liquid chromatographic method spectrum of (R)-1,1,1-trifluoropropylamine after the method of embodiment 1 is transformed.
  • Fig. 2 is the high-performance liquid chromatography collection of (R)-1,1,1-trifluoropropylamine after the method of embodiment 2 is transformed.
  • Fig. 3 is the high-performance liquid chromatography collection of (R)-1,1,1-trifluoropropylamine after the method of embodiment 3 is transformed.
  • Fig. 4 is the high-performance liquid chromatography collection of (R)-1,1,1-trifluoropropylamine after the method of embodiment 3 is transformed.
  • Fig. 5 is the high performance liquid chromatography of (R)-1,1,1-trifluoropropylamine after the method of embodiment 5 is transformed.
  • Figure 6 shows that the ee value obtained in Example 6 is 100%.
  • transaminase of the present invention can be used to prepare a chiral amine with the following formula I with very high conversion rate and chiral purity:
  • R 1 is a halogenated C 1-4 alkyl group
  • R 2 is a C 1-4 alkyl group
  • halogenated C 1-4 alkyl refers to C 1-4 alkyl substituted by halogen, wherein the halogen may include F, Cl, Br and I.
  • C 1-4 alkyl includes straight-chain and branched-chain alkyl groups, including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.
  • Examples of halogenated C1-4 alkyl groups include, but are not limited to, trifluoromethyl, pentafluoroethyl, trichloromethyl, and the like.
  • the chiral amine of the present invention is (R)-1,1,1-trifluoropropylamine represented by the following formula II:
  • the transaminase is preferably a transaminase derived from Arthrobacter sp.
  • Exemplary Arthrobacter-derived aminotransferases include aminotransferases having the amino acid sequence set forth in SEQ ID NO:1.
  • the aminotransferases of the present invention also include mutants of SEQ ID NO: 1, such as the amino acid sequence of which is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 85% of the amino acid sequence shown in SEQ ID NO: 1 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 identical to aminotransferases.
  • the sequence identity can be calculated using software commonly used in the art such as BLAST (from NCBI) with default parameters of the software.
  • the mutants of the transaminase of the present invention are obtained by performing one or more amino acid mutations on the amino acid sequence shown in SEQ ID NO: 1 and retain the transaminase activity possessed by SEQ ID NO: 1 (especially those possessed herein
  • the described function of preparing chiral amines is derived from SEQ ID NO: 1 mutants.
  • 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 3, such as amino acid residues. Substitution, insertion or deletion. Preferred mutations are substitutions.
  • exemplary transaminase mutants include, but are not limited to, transaminase with accession numbers 3WWH_A, 3WWI_A, 5FR9_A and 3WWJ_A.
  • particularly preferred transaminase mutants of the present invention include mutants resulting from mutations at one or more of positions 136, 282, and 306 of SEQ ID NO: 1, which The amino acid residues at the remaining positions of the mutant are identical to SEQ ID NO:1.
  • Preferred mutations are substitution mutations. In some embodiments, the substitutions are conservative substitutions.
  • the substitution at position 136 to wild-type I is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine acid, tryptophan or methionine, or substituted with an aromatic amino acid such as tyrosine, tryptophan or phenylalanine; preferably substituted with tryptophan.
  • a non-polar amino acid such as alanine, valine, leucine, isoleucine, proline, phenylalanine acid, tryptophan or methionine
  • an aromatic amino acid such as tyrosine, tryptophan or phenylalanine
  • the wild-type T substitution at position 282 is substituted with a polar uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine Amides, preferably substituted with asparagine, glutamine, tryptophan or threonine, more preferably substituted with tryptophan.
  • a polar uncharged amino acid such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine Amides, preferably substituted with asparagine, glutamine, tryptophan or threonine, more preferably substituted with tryptophan.
  • the substitution at position 306 to wild-type V is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine acid, tryptophan or methionine, or substituted with aliphatic non-polar amino acids such as alanine, leucine, valine or isoleucine; preferably substituted with leucine.
  • a non-polar amino acid such as alanine, valine, leucine, isoleucine, proline, phenylalanine acid, tryptophan or methionine
  • aliphatic non-polar amino acids such as alanine, leucine, valine or isoleucine
  • the substitution mutations are one, any two, or all three of the following substitution mutations: I136W, T282S, and V306L.
  • the amino acid sequence of the transaminase mutant is shown in SEQ ID NO:2.
  • the present invention also includes nucleic acid molecules whose polynucleotide sequences are the coding sequences of the transaminase mutants described in the present invention or their complements.
  • the polynucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO:4.
  • the present invention also includes nucleic acid constructs comprising the nucleic acid molecules of the present invention.
  • the nucleic acid construct is an expression cassette.
  • the expression frame may also contain transcription termination sequences and promoters.
  • the promoter may be various promoters known in the art as long as it is suitable for expressing the transaminase of the present invention in a 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.
  • the present invention also includes recombinant vectors.
  • 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.
  • the recombinant vector may contain other regulatory elements, including but not limited to enhancers, multiple cloning sites, transcription terminators, resistance genes, and the like. Corresponding vector backbones with desired regulatory elements can be selected according to different purposes, and the nucleic acid molecules or nucleic acid constructs of the present invention can be cloned into the backbone to construct the recombinant vectors of the present invention.
  • Nucleic acid molecules, construct nucleic acid constructs and recombinant vectors can be prepared by methods known in the art, and expressed by conventional methods, thereby preparing the transaminases described herein.
  • the present invention also provides host cells containing the nucleic acid molecules, nucleic acid constructs and/or recombinant vectors described in any of the embodiments herein, or expressing the transaminases described in any of the embodiments herein.
  • host cells include E. coli cells, insect cells, yeast cells, and mammalian cells.
  • the method for preparing a chiral amine of the present invention includes the step of reducing the carbonyl group in the substrate represented by the following formula III to a chiral amino group using the transaminase of the present invention in a reaction system containing a coenzyme and an amino group donor:
  • R 1 and R 2 are as described above.
  • the amount of transaminase can be 1-50% by weight of the substrate in the reaction system, such as 10-40% or 15-30%.
  • Coenzymes can be various types of coenzymes conventionally used in the art in combination with transaminases, such as pyridoxal 5-phosphate (PLP).
  • the amount of coenzyme used can be a conventional amount, for example, it can be 0.1-5.0%, preferably 1-3% by weight of the substrate in the reaction system.
  • the amino donors in the reaction system can be 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 carbon chain lengths of 2-6 carbons Atoms of fatty amines, such as isopropylamine, amino acids, such as alanine (such as L-alanine) and aspartic acid (such as L-aspartic acid), etc.
  • the amount of the amino donor can be easily determined according to conventional reactions. Generally, depending on the type of amino donor, the amount of amino donor may be 30% to 500% by weight of the substrate, such as 50% to 300%.
  • the reaction system is a buffered saline solution system.
  • the pH of the reaction system is controlled by the buffer.
  • Commonly used buffers include, but are not limited to, phosphate buffer, triethanolamine-isopropylamine buffer, and the like.
  • the pH of the reaction system is 6-10, preferably 7-9, more preferably 8-9. In some embodiments, the pH of the reaction system is 8.3-8.6.
  • the reaction system may also contain a cosolvent.
  • Cosolvents commonly used in the preparation of chiral amines can be used in the present invention.
  • the co-solvent is an organic solvent, such as a co-solvent selected from the group consisting of dimethyl sulfoxide, toluene, alcoholic solvents, or a combination thereof.
  • the alcoholic solvent includes, but is not limited to, isopropanol.
  • the co-solvent is selected from dimethyl sulfoxide and isopropanol.
  • the co-solvent used should be miscible with water to further increase the solubility of the substrate.
  • the reaction temperature of the catalytic reaction of the present invention may be 10°C to 50°C, preferably 20°C to 45°C, and more preferably 20°C to 35°C. In some embodiments, the reaction temperature is room temperature, ie, 25 ⁇ 3°C.
  • the reaction time can be determined according to the amount of the reactants, and is usually 0.1 to 120 hours, for example, 0.5 to 48 hours or 10 to 24 hours.
  • the transaminase from Arthrobacter sp (especially the transaminase shown in SEQ ID NO: 1) is used to catalyze the carbonyl reduction reaction of the substrate when the amino group is , significantly higher conversions can also be achieved compared to the use of other co-solvents and amino donors.
  • the method of the present invention for preparing the chiral amine shown in formula I comprises, in the presence of a cosolvent, using the transaminase shown in SEQ ID NO: 1 to catalyze an amino donor with 1 , the reaction between 1,1-trifluoroacetone; wherein, the cosolvent is an alcohol solvent, preferably isopropanol; the amino donor is aliphatic amine, such as the carbon chain length is 2-6 carbon atoms of fatty amines, more preferably isopropylamine.
  • the amino donor is an aromatic amine, such as phenethylamine (eg, R-phenethylamine), and the solvent is dimethyl sulfoxide.
  • the method for preparing the chiral amine represented by the formula I of the present invention comprises, in the presence of dimethyl sulfoxide, using the transaminase shown in SEQ ID NO: 1 to catalyze R-phenethylamine with the formula The reaction between the substrates shown in III, or the transaminase shown in SEQ ID NO: 1 is used to catalyze the reaction between isopropylamine and the substrate shown in formula III in the presence of isopropanol.
  • the reaction system of the reaction also contains a coenzyme, such as pyridoxal phosphate.
  • a coenzyme such as pyridoxal phosphate.
  • the chiral amine is (R)-1,1,1-trifluoropropylamine, and the substrate is 1,1,1-trifluoroacetone.
  • the present invention also found that when using the transaminase mutants of the present invention, especially those obtained by mutating one or more of the positions 136, 282 and 306 of SEQ ID NO: 1 as described herein
  • the chiral amines of formula I are prepared with very high conversions from mutants of . Therefore, in some embodiments of the present invention, the method of the present invention for preparing the chiral amine of formula I comprises, in the presence of a cosolvent, using the transaminase mutant described in any of the embodiments herein to catalyze an amino donor reaction with the substrate.
  • the preferred co-solvent is dimethyl sulfoxide
  • the preferred amino donor is an aliphatic amine, such as isopropylamine.
  • the reaction system of the reaction also contains a coenzyme, such as pyridoxal phosphate.
  • the transaminase mutant is a transaminase whose amino acid sequence is shown in SEQ ID NO: 2.
  • the chiral amine is (R)-1,1,1-trifluoropropylamine, and the substrate is 1,1,1-trifluoroacetone.
  • the present invention also includes the aforementioned transaminase mutants, their coding sequences (nucleic acid molecules), nucleic acid constructs, recombinant vectors and host cells.
  • the present invention provides an enzyme preparation comprising the transaminase mutant of any of the embodiments of the present invention.
  • the enzyme preparation is a lyophilized powder.
  • the enzyme preparation is a buffer containing the transaminase mutant.
  • the buffer is a phosphate buffer, and the pH is 6-10, preferably 7-9, more preferably 8-9, and more preferably 8.3-8.6.
  • the phosphate buffer is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.
  • the present invention also provides the transaminase mutant described in any of the foregoing embodiments, its coding sequence (nucleic acid molecule), nucleic acid construct, recombinant vector and reagents used in the preparation of host cells for preparing the chiral amine shown in formula I applications in .
  • the agent is the enzyme preparation of any of the embodiments herein.
  • SEQ ID NO: 3 the coding sequence is shown in SEQ ID NO: 3
  • SEQ ID NO: 3 the mother parent
  • the mutants on the transformed plates were toothpicked into 96-well plates and incubated overnight at 37°C in a shaker at 220 rpm. Pipette 50 microliters of bacterial solution from the holes of the primary plate into the corresponding holes of the secondary plate.
  • the conversion rate was 21.3% as detected by GC.
  • the conversion rate was 64.8% as detected by GC.
  • reaction flask In an 8mL reaction flask, add 4mL of I80X buffer, then add 30mg of transaminase (amino acid sequence shown in SEQ ID NO: 1) lyophilized powder and 2mg of pyridoxal phosphate (PLP) and stir well, add 100mg of 1,1 , 1-trifluoroacetone and 100 mg of dimethyl sulfoxide, control the reaction temperature to be 30 ° C, and continuously stir for 4 hours to make it fully react.
  • transaminase amino acid sequence shown in SEQ ID NO: 1
  • PDP pyridoxal phosphate
  • the conversion rate was 90.2% as detected by GC.
  • the conversion rate was 66.2% as detected by GC.

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Abstract

提供一种转氨酶及其在制备光学纯手性胺中的应用。所述转氨酶的氨基酸序列与SEQ ID NO：1所示的氨基酸序列相比在第136位、第282位和第306位中的一个或多个位置上具有取代突变，其余位置上的氨基酸残基与SEQ ID NO：1相同。还提供使用所述转氨酶制备手性胺的方法。采用所述转氨酶能提高转化率。

Description

转氨酶及其在制备光学纯手性胺中的应用 技术领域
本发明涉及转氨酶及其应用，尤其是在生物催化制备光学纯手性胺的应用。
背景技术
光学纯手性胺是一类具有重要价值的医药及精细化工中间体。目前，超过70％的药物都是手性胺及其衍生物的合成都是以手性胺作为中间体。下式II所示的(R)-1，1，1-三氟丙胺即为一种非常重要的手性胺，应用于很多医药中间体的合成。
Figure PCTCN2021142048-appb-000001
目前，制备(R)-1，1，1-三氟丙胺的方法主要有化学法和生物催化法。化学法的常规流程如下所示，通常以1，1，1-三氟丙酮为原料，使用R-α-苯乙胺诱导出手性，然后进行化学拆分提高手性值到99％，但总收率小于40％，从经济和环保角度出发不适合工业化大规模生产(J.Org.Chem.，1997，62，10，3030-3031；Bioorganic&Medicinal Chemistry Letters，2013，23，3947-3953)。
Figure PCTCN2021142048-appb-000002
生物酶催化法通常利用转氨酶，通过动力学拆分消旋胺，或者通过酮的不对称合成生成手性胺。与传统的化学合成方法相比，酶法具有反应效率高、立 体选择性好、反应条件温和、低能耗、环境友好等优点。但是，通常使用的酶底物特异性、对映体选择性和/或转化率对于工业化的生产过程还不够高。
因此，本领域仍然需要一种能以高的反应效率、立体选择性以及收率来制备光学纯手性胺的试剂和方法。
发明内容
本发明第一方面提供一种转氨酶，所述转氨酶的氨基酸序列与SEQ ID NO：1所示的氨基酸序列相比在第136位、第282位和第306位中的一个或多个位置上具有取代突变，其余位置上的氨基酸残基与SEQ ID NO：1相同。
在一个或多个实施方案中，所述取代突变选自下述取代突变中的一种或多种：I136W、T282S和V306L。
在一个或多个实施方案中，所述转氨酶的氨基酸序列如SEQ ID NO：2所示。
本发明第二方面提供一种核酸分子，其多核苷酸序列选自：
(1)编码本发明任一实施方案所述的转氨酶的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列。
在一个或多个实施方案中，所述多核苷酸序列选自：SEQ ID NO：4所示的多核苷酸序列或其互补序列。
本发明第三方面提供一种核酸构建体，其含有本发明任一实施方案所述的核酸分子；优选地，所述核酸构建体为表达框。
本发明第四方面提供一种重组载体，其含有本发明任一实施方案所述的核酸分子或核酸构建体；优选地，所述重组载体为重组克隆载体或重组表达载体。
本发明第五方面提供一种宿主细胞，其含有本发明任一实施方案所述的核酸分子、核酸构建体或重组载体，和/或表达本发明任一实施方案所述的转氨酶；优选地，所述宿主细胞选自大肠杆菌细胞、昆虫细胞、酵母细胞和哺乳动物细胞。
本发明第六方面提供一种酶制剂，所述酶制剂含有本发明任一实施方案所述的转氨酶。
本发明第七方面提供下式I所示的手性胺的制备方法：
Figure PCTCN2021142048-appb-000003
其中，所述方法包括，在助溶剂和任选的辅酶的存在下，使用来自节杆菌属(Arthrobacter sp)的转氨酶或与其氨基酸序列具有至少95％的序列同一性的转氨酶或本文任一实施方案所述的转氨酶突变体或其酶制剂催化氨基供体与下式III所示的底物反应，从而制备得到式I所示的手性胺：
Figure PCTCN2021142048-appb-000004
其中，式I和III中，R 1为卤代C 1-4烷基，R 2为C 1-4烷基；
优选地，式I化合物为(R)-1，1，1-三氟丙胺，式III化合物为1，1，1-三氟丙酮。
在一个或多个实施方案中，所述节杆菌属(Arthrobacter sp)的转氨酶的氨基酸序列如SEQ ID NO：1所示，所述与SEQ ID NO：1所示的氨基酸序列具有至少97％的序列同一性的转氨酶为NCBI登陆号为3WWH_A、3WWI_A、5FR9_A或3WWJ_A的转氨酶。
在一个或多个实施方案中，所述助溶剂选自：二甲基亚砜，醇类溶剂，甲苯，或其组合。
在一个或多个实施方案中，所述氨基供体选自：芳香胺、脂肪胺、氨基酸、或其组合。
在一个或多个实施方案中，所述醇类溶剂为异丙醇。
在一个或多个实施方案中，所述芳香胺为苯乙胺。
在一个或多个实施方案中，所述脂肪胺为碳链长度为2-6个碳原子的脂肪胺，优选为异丙胺。
在一个或多个实施方案中，所述氨基酸为丙氨酸和/或天冬氨酸。
在一个或多个实施方案中，转氨酶的用量是反应体系中底物重量的1-50％，如10-40％或15-30％。
在一个或多个实施方案中，反应体系中含有辅酶，辅酶的用量为底物重量的0.1-5.0％，优选1-3％；优选的辅酶为5-磷酸吡哆醛。
在一个或多个实施方案中，反应体系中，氨基供体的用量为底物重量的30％到500％，如50％到300％。
在一个或多个实施方案中，反应体系的pH6～10，优选为7～9，更优选为8～9，更优选为8.3～8.6。
在一个或多个实施方案中，反应温度为10℃～50℃，优选为20℃～45℃，更优选为20℃～35℃。
在一个或多个实施方案中，反应时间为0.1～120小时，例如0.5～48小时或10～24小时。
在一个或多个实施方案中，所述方法中，助溶剂为二甲亚砜或异丙醇；氨基供体为脂肪胺，优选为异丙胺，或芳香胺，优选R-苯乙胺；转氨酶为来自节杆菌属(Arthrobacter sp)的转氨酶，更优选为氨基酸序列如SEQ ID NO：1所示的转氨酶或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
在一个或多个实施方案中，所述方法中，助溶剂为二甲亚砜；氨基供体为异丙胺或R-苯乙胺；转氨酶为来自节杆菌属(Arthrobacter sp)的转氨酶，更优选为氨基酸序列如SEQ ID NO：1所示的转氨酶或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
在一个或多个实施方案中，所述方法中，助溶剂为异丙醇；氨基供体为脂肪胺，优选为异丙胺；转氨酶为来自节杆菌属(Arthrobacter sp)的转氨酶，更优选为氨基酸序列如SEQ ID NO：1所示的转氨酶或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
在一个或多个实施方案中，所述方法中，助溶剂为二甲亚砜；氨基供体为脂肪胺，优选为异丙胺；转氨酶为本文任一实施方案所述的转氨酶突变体或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
本发明还提供来自节杆菌属(Arthrobacter sp)的转氨酶或其酶制剂和/或本文任一实施方案所述的转氨酶突变体或其酶制剂在提高制备光学纯手性胺 时的转化率中的应用。
附图说明
图1是实施例1的方法转化后的(R)-1，1，1-三氟丙胺的高效液相色谱法图谱。t＝5.13处峰为目标化合物(R)-1，1，1-三氟丙胺。
图2是实施例2的方法转化后的(R)-1，1，1-三氟丙胺的高效液相色谱法图谱。t＝5.13处峰为目标化合物(R)-1，1，1-三氟丙胺。
图3是实施例3的方法转化后的(R)-1，1，1-三氟丙胺的高效液相色谱法图谱。t＝7.07处峰为目标化合物(R)-1，1，1-三氟丙胺。
图4是实施例3的方法转化后的(R)-1，1，1-三氟丙胺的高效液相色谱法图谱。t＝5.13处峰为目标化合物(R)-1，1，1-三氟丙胺。
图5是实施例5的方法转化后的(R)-1，1，1-三氟丙胺的高效液相色谱法图谱。t＝5.13处峰为目标化合物(R)-1，1，1-三氟丙胺。
图6显示实施例6获得的ee值为100％。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本发明发现，利用本发明所述的转氨酶可以以非常高的转化率和手性纯度制备得到具有下式I所示的手性胺：
Figure PCTCN2021142048-appb-000005
式中，R 1为卤代C 1-4烷基，R 2为C 1-4烷基。
本文中，卤代C 1-4烷基指被卤素取代的C 1-4烷基，其中，卤素可包括F、Cl、Br和I。本文中，C 1-4烷基包括直链和支链烷基，包括但不限于甲基、乙基、丙基、异丙基、正丁基、异丁基和叔丁基等。卤代C1-4烷基的例子包括但不限于三氟甲基、五氟乙基、三氯甲基等。
在特别优选的实施方案中，本发明所述的手性胺为下式II所示的(R)-1，1，1-三氟丙胺：
Figure PCTCN2021142048-appb-000006
本发明中，转氨酶优选为来自节杆菌属(Arthrobacter sp)的转氨酶。示例性的来自节杆菌属的转氨酶包括具有SEQ ID NO：1所示的氨基酸序列的转氨酶。本发明的转氨酶还包括SEQ ID NO：1的突变体，如其氨基酸序列与SEQ ID NO：1所示的氨基酸序列具有至少70％、优选至少75％、优选至少80％、优选至少85％、优选至少90％，更优选至少95％、更优选至少96％、更优选至少97％、更优选至少98％、更优选至少99％的序列相同性的转氨酶。所述序列相同性可采用本领域常用的软件如BLAST(来自NCBI)以软件默认参数计算得到。
在一些实施方案中，本发明转氨酶的突变体为对SEQ ID NO：1所示的氨基酸序列进行一个或多个氨基酸突变所得的保留了SEQ ID NO：1所具备的转氨酶活性(尤其是具备本文所述的制备手性胺的功能)的由SEQ ID NO：1衍生得到的突变体。所述一个或多个氨基酸突变包括20个以内、优选15个以内、更优选10个以内、更优选8个以内、更优选5个以内、更优选3个以内的氨基酸突变，如氨基酸残基的取代、插入或缺失。优选的突变是取代。
本发明中，示例性的转氨酶突变体包括但不限于登陆号为3WWH_A、3WWI_A、5FR9_A以及3WWJ_A的转氨酶。
在一些实施方案中，本发明特别优选的转氨酶突变体包括在SEQ ID NO：1第136位、第282位以及第306位中的一个或多个位置上发生了突变所得到的突变体，该突变体其余位置上的氨基酸残基与SEQ ID NO：1相同。优选的突变是取代突变。在一些实施方案中，取代是保守性取代。在一些实施方案中，第136位上的取代为野生型的I被取代为非极性氨基酸，如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸或蛋氨酸，或者被取代为芳香族氨基酸，如酪氨酸、色氨酸或苯丙氨酸；优选被取代为色氨酸。在一些实施方 案中，第282位上的取代为野生型的T被取代为极性不带电荷的氨基酸，如丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺或谷氨酰胺，优选被取代为天冬酰胺、谷氨酰胺、色氨酸或苏氨酸，更优选被取代为色氨酸。在一些实施方案中，第306位上的取代为野生型的V被取代为非极性氨基酸，如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸或蛋氨酸，或者被取代为脂肪族非极性氨基酸，如丙氨酸、亮氨酸、缬氨酸或异亮氨酸；优选被取代为亮氨酸。
在优选的实施方案中，所述取代突变为以下取代突变中的一种、任意两种或全部三种：I136W、T282S和V306L。在进一步优选的实施方案中，所述转氨酶突变体的氨基酸序列如SEQ ID NO：2所示。
本发明也包括核酸分子，其多核苷酸序列为本发明所述转氨酶突变体的编码序列或其互补序列。在一些实施方案中，所述核酸分子的多核苷酸序列如SEQ ID NO：4所示。
本发明还包括核酸构建体，其含有本发明所述的核酸分子。在一些实施方案中，所述核酸构建体为表达框。表达框内除所述核酸分子外，还可含有转录终止序列和启动子。启动子可以是本领域周知的各种启动子，只要其适于在期望的宿主中表达本发明的转氨酶即可。本领域技术人员可根据所用的宿主细胞选择适当的启动子，构建本发明的表达框以及下文所述的重组载体。
本发明还包括重组载体。该重组载体可含有本文任一实施方案所述的核酸分子或核酸构建体。该重组载体可以是重组克隆载体或重组真核表达载体。重组载体中可含有其它调节元件，包括但不限于增强子、多克隆位点、转录终止子、抗性基因等。可根据不同目的选择具有所需调节元件的相应的载体骨架，将本发明的核酸分子或核酸构建体克隆入所述骨架中，从而构建得到本发明的重组载体。
可采用本领域周知的方法制备核酸分子、构建核酸构建体和重组载体，并采用常规的方法表达，从而制备得到本文所述的转氨酶。
在一些实施方案中，本发明还提供宿主细胞，其含有本文任一实施方案所述的核酸分子、核酸构建体和/或重组载体，或表达本文任一实施方案所述的转 氨酶。本领域已知的任何适用于表达目标蛋白的宿主均可用于本发明，示例性的宿主细胞包括大肠杆菌细胞、昆虫细胞、酵母细胞和哺乳动物细胞。
本发明制备手性胺的方法包括含辅酶和氨基供体的反应体系中使用本发明的转氨酶将下式III所示的底物中的羰基还原为手性氨基的步骤：
Figure PCTCN2021142048-appb-000007
式中，R 1和R 2如前文所述。
转氨酶的用量可以是反应体系中底物重量的1-50％，如10-40％或15-30％。
辅酶可以是本领域常规与转氨酶结合使用的各类辅酶，如5-磷酸吡哆醛(PLP)。辅酶的用量可以是常规用量，例如，可以是反应体系中底物重量的0.1-5.0％，优选1-3％。
反应体系中的氨基供体可以是本领域常用用来制备手性胺的各类氨基供体，包括但不限于芳香胺，如苯乙胺，脂肪胺，如碳链长度为2-6个碳原子的脂肪胺，如异丙胺，氨基酸，如丙氨酸(如L-丙氨酸)和天冬氨酸(如L-天冬氨酸)等。通常，反应体系中，氨基供体的用量可根据常规的反应容易确定。通常，根据氨基供体种类的不同，氨基供体的用量可为底物重量的30％到500％，如50％到300％。
本发明中，反应体系为缓冲盐水溶液体系。通过缓冲液来对反应体系的pH进行控制。常用的缓冲液包括但不限于磷酸盐缓冲液、三乙醇胺-异丙胺缓冲液等。优选地，反应体系的pH6～10，优选为7～9，更优选为8～9。在一些实施方式中，反应体系的pH为8.3～8.6。
反应体系中还可含有助溶剂。手性胺制备中常用的助溶剂都可用于本发明。通常，助溶剂为有机溶剂，例如选自下组的助溶剂：二甲基亚砜，甲苯，醇类溶剂，或其组合。所述醇类溶剂包括但不限于异丙醇。较佳地，所述助溶剂选自二甲基亚砜和异丙醇。所使用的助溶剂应当与水可混溶，以进一步增加底物的溶解性。
本发明的催化反应的反应温度可为10℃～50℃，优选为20℃～45℃，更优选为20℃～35℃。在一些实施方案中，反应温度为室温，即25±3℃。反应时间可视反应物的量确定，通常可为0.1～120小时，例如0.5～48小时或10～24小时。
本发明发现，当选用特定的助溶剂和氨基供体时，使用来自节杆菌属(Arthrobacter sp)的转氨酶(尤其是SEQ ID NO：1所示的转氨酶)催化底物的羰基还原反应为氨基时，相较于使用其它助溶剂和氨基供体，也能取得显著较高转化率。因此，在本发明的一些实施方案中，本发明制备所述式I所示的手性胺的方法包括，在助溶剂的存在下使用SEQ ID NO：1所示的转氨酶催化氨基供体与1，1，1-三氟丙酮之间的反应；其中，所述助溶剂为醇类溶剂，优选为异丙醇；所述氨基供体为脂肪胺，如碳链长度为2-6个碳原子的脂肪胺，更优选为异丙胺。在一些实施方案中，所述氨基供体为芳香胺，如苯乙胺(如R-苯乙胺)，所述溶剂为二甲亚砜。在优选的实施方案中，本发明制备所述式I所示的手性胺的方法包括，在二甲亚砜的存在下使用SEQ ID NO：1所示的转氨酶催化R-苯乙胺与式III所示底物之间的反应，或在异丙醇的存在下使用SEQ ID NO：1所示的转氨酶催化异丙胺与式III所示底物之间的反应。优选地，所述反应的反应体系中还含有辅酶，如磷酸吡哆醛。优选地，所述手性胺为(R)-1，1，1-三氟丙胺，所述底物为1，1，1-三氟丙酮。
本发明还发现，当使用本发明的转氨酶突变体，尤其是本文所述的在SEQ ID NO：1第136位、第282位以及第306位中的一个或多个位置上发生了突变所得到的突变体制备式I的手性胺时，其转化率非常高。因此，在本发明的一些实施方案中，本发明制备所述式I所示的手性胺的方法包括，在助溶剂的存在下使用本文任一实施方案所述的转氨酶突变体催化氨基供体与底物之间的反应。优选的助溶剂为二甲亚砜，优选的氨基供体为脂肪胺，如异丙胺。优选地，所述反应的反应体系中还含有辅酶，如磷酸吡哆醛。在特别优选的实施方案中，所述转氨酶突变体为氨基酸序列如SEQ ID NO：2所示的转氨酶。优选地，所述手性胺为(R)-1，1，1-三氟丙胺，所述底物为1，1，1-三氟丙酮。
本发明也包括前文所述的转氨酶突变体、其编码序列(核酸分子)、核酸 构建体、重组载体以及宿主细胞。
在一些实施方案中，本发明提供一种酶制品，其含有本发明任一实施方案所述的转氨酶突变体。在一些实施方案中，所述酶制品为冻干粉末。在一些实施方案中，所述酶制品为含有所述转氨酶突变体的缓冲液。优选地，所述缓冲液为磷酸盐缓冲液，pH为6～10，优选为7～9，更优选为8～9，更优选为8.3～8.6。在一些实施方案中，所述磷酸盐缓冲液为磷酸氢二钾-磷酸二氢钾缓冲液。
本发明还提供前文任一实施方案所述的转氨酶突变体、其编码序列(核酸分子)、核酸构建体、重组载体以及宿主细胞在制备用于制备所述式I所示的手性胺的试剂中的应用。在一些实施方案中，所述试剂为本文任一实施方案所述的酶制品。
下面将对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
制备例
用SEQ ID NO：1(编码序列如SEQ ID NO：3所示)的序列为母本，采用滚轮PCR，迭代饱和突变，以及组合突变等策略对其进行定向进化改造，然后将突变体转化到大肠杆菌BL21(DE3)感受态细胞中，并均匀涂布在50微克/ml的卡那霉素的LB琼脂平板上，放置在37℃培养箱中静止培养18h。将转化的平板上的突变体用牙签挑选到96孔板中，于37℃、220rpm摇床中培养过夜。从一级板的孔洞中吸取50微升菌液接入二级版的相应孔洞中，于37℃、220rpm培养2～3h后，加入终浓度为0.2mM的IPTG，30℃培养20h得到相应的突变体进行高通量筛选。结合HPLC和GC检测复筛，鉴别获活性和稳性的显著提高的突变体进行基因测序。测序结果如SEQ ID NO：2所示，其编码序列如SEQ ID NO：4所示。
SEQ ID NO：1，野生型转氨酶
Figure PCTCN2021142048-appb-000008
SEQ ID NO：2，转氨酶突变体
Figure PCTCN2021142048-appb-000009
SEQ ID NO：3，野生型转氨酶的编码序列
Figure PCTCN2021142048-appb-000010
Figure PCTCN2021142048-appb-000011
SEQ ID NO：4，转氨酶突变体的编码序列
Figure PCTCN2021142048-appb-000012
Figure PCTCN2021142048-appb-000013
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并不意图限制本发明。实施例中所用到的方法、材料和试剂，除非另有说明，否则为本领域常规的方法，以及可从市售途径获得的材料和试剂。
实施例1
配制I80X缓冲液：称取27.8g三水合磷酸氢二钾以及10.6g磷酸二氢钾倒入2L玻璃瓶中。量取纯化水1300mL置于瓶中，室温搅拌，直至固体溶解。量取异丙胺171mL置于瓶中，保持温度在20-30℃，加入35％HCl，调节pH＝8.0。加入纯化水定容至2L。
在8mL反应瓶中，加入4mL I80X缓冲液，再加入30mg转氨酶(氨基酸序列如SEQ ID NO：1所示)冻干粉和2mg磷酸吡哆醛(PLP)，搅拌充分后，加入100mg的1，1，1-三氟丙酮与100mg二甲基亚砜，控制反应温度为30℃，连续搅拌4小时，使其充分反应。
待反应结束后，经GC检测，转化率为21.3％。如图1显示，t＝5.13处峰为目标化合物1，1，1-三氟丙胺。
实施例2
配制I80X缓冲液：称取27.8g三水合磷酸氢二钾以及10.6g磷酸二氢钾倒入2L玻璃瓶中。量取纯化水1300mL置于瓶中，室温搅拌，直至固体溶解。量取异丙胺171mL置于瓶中，保持温度在20-30℃，加入35％HCl，调节pH＝8.0。加入纯化水定容至2L。
在8mL反应瓶中，加入4mL I80X缓冲液，再加入30mg转氨酶(氨基酸序列如SEQ ID NO：2所示)冻干粉和2mg磷酸吡哆醛(PLP)，搅拌充分后，加入100mg的1，1，1-三氟丙酮与100mg二甲基亚砜，控制反应温度为30℃， 连续搅拌4小时，使其充分反应。
待反应结束后，经GC检测，转化率为64.8％。如图2显示，t＝5.13处峰为目标化合物1，1，1-三氟丙胺。
实施例3
配制I80X缓冲液：称取27.8g三水合磷酸氢二钾以及10.6g磷酸二氢钾倒入2L玻璃瓶中。量取纯化水1300mL置于瓶中，室温搅拌，直至固体溶解。量取R-苯乙胺242g置于瓶中，保持温度在20-30℃，加入35％HCl，调节pH＝8.0。加入纯化水定容至2L。
在8mL反应瓶中，加入4mL I80X缓冲液，再加入30mg转氨酶(氨基酸序列如SEQ ID NO：1所示)冻干粉和2mg磷酸吡哆醛(PLP)搅拌充分后，加入100mg的1，1，1-三氟丙酮与100mg二甲基亚砜，控制反应温度为30℃，连续搅拌4小时，使其充分反应。
待反应结束后，经GC检测，转化率为90.2％。如图3显示，t＝7.07处峰为目标化合物1，1，1-三氟丙胺。
实施例4
配制I80X缓冲液：称取27.8g三水合磷酸氢二钾以及10.6g磷酸二氢钾倒入2L玻璃瓶中。量取纯化水1300mL置于瓶中，室温搅拌，直至固体溶解。量取异丙胺171mL置于瓶中，保持温度在20-30℃，加入35％HCl，调节pH＝8.0。加入纯化水定容至2L。
在8mL反应瓶中，加入4mL I80X缓冲液，再加入30mg转氨酶(氨基酸序列如SEQ ID NO：1所示)冻干粉和2mg磷酸吡哆醛(PLP)搅拌充分后，加入100mg的1，1，1-三氟丙酮与100mg异丙醇，控制反应温度为30℃，连续搅拌4小时，使其充分反应。
待反应结束后，经GC检测，转化率为66.2％。如图4显示，t＝5.13处峰为目标化合物1，1，1-三氟丙胺。
实施例5
量取纯化水216mL置于500mL夹套瓶中，加入1.56g三水合磷酸氢二钾以及0.6g磷酸二氢钾，搅拌至完全溶解。称取异丙胺6.3g加入瓶中，控制温度在20-30℃用35％盐酸调节pH至8.3-8.6。然后，向瓶中加入0.24g磷酸吡哆醛和2.4g转氨酶(氨基酸序列如SEQ ID NO：2所示)。将12g的1，1，1-三氟丙酮与12g二甲基亚砜混合后，向瓶中滴加。待滴加完毕后，升温至25℃，反应16小时。如图5所示，经GC检测，转化率为99.9％。如图6所示，ee值为100％。
综上所述，上述各实施例仅为本发明的较佳实施例而已，并不用以限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，皆应包含在本发明的保护范围内。

Claims (17)

  1. 一种转氨酶，其特征在于，所述转氨酶的氨基酸序列与SEQ ID NO：1所示的氨基酸序列相比在第136位、第282位和第306位中的一个或多个位置上具有取代突变，其余位置上的氨基酸残基与SEQ ID NO：1相同。
  2. 如权利要求1所述的转氨酶，其特征在于，所述取代突变选自下述取代突变中的一种或多种：I136W、T282S和V306L。
  3. 如权利要求2所述的转氨酶，其特征在于，所述转氨酶的氨基酸序列如SEQ ID NO：2所示。
  4. 一种核酸分子，其多核苷酸序列选自：
    (1)编码权利要求1-3中任一项所述的转氨酶的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列；
    优选地，(1)所述的多核苷酸序列如SEQ ID NO：4所示。
  5. 一种核酸构建体，其含有权利要求4所述的核酸分子；优选地，所述核酸构建体为表达框。
  6. 一种重组载体，其含有权利要求4所述的核酸分子或权利要求5所述的核酸构建体；优选地，所述重组载体为重组克隆载体或重组表达载体。
  7. 一种宿主细胞，其含有权利要求4所述的核酸分子、权利要求5所述的核酸构建体或权利要求6所述的重组载体，和/或表达权利要求1-3中任一项所述的转氨酶；优选地，所述宿主细胞选自大肠杆菌细胞、昆虫细胞、酵母细胞和哺乳动物细胞。
  8. 一种酶制剂，所述酶制剂含有权利要求1-3中任一项所述的转氨酶。
  9. 下式I所示的手性胺的制备方法：
    Figure PCTCN2021142048-appb-100001
    其中，所述方法包括，在助溶剂和任选的辅酶的存在下，使用来自节杆菌 属(Arthrobacter sp)的转氨酶或与其氨基酸序列具有至少95％的序列同一性的转氨酶或权利要求1-3中任一项所述的转氨酶或权利要求8所述的酶制剂催化氨基供体与下式III所示的底物反应，从而制备得到式I所示的手性胺：
    Figure PCTCN2021142048-appb-100002
    其中，式I和III中，R 1为卤代C 1-4烷基，R 2为C 1-4烷基；
    优选地，式I化合物为(R)-1，1，1-三氟丙胺，式III化合物为1，1，1-三氟丙酮。
  10. 如权利要求9所述的方法，其特征在于，所述节杆菌属(Arthrobacter sp)的转氨酶的氨基酸序列如SEQ ID NO：1所示，所述与SEQ ID NO：1所示的氨基酸序列具有至少95％的序列同一性的转氨酶为NCBI登陆号为3WWH_A、3WWI_A、5FR9_A或3WWJ_A的转氨酶。
  11. 如权利要求9所述的方法，其特征在于，所述方法具有以下一项或多项特征：
    所述助溶剂选自：二甲基亚砜，醇类溶剂，甲苯，或其组合；和
    所述氨基供体选自：芳香胺、脂肪胺、氨基酸或其组合。
  12. 如权利要求11所述的方法，其特征在于，
    所述醇类溶剂为异丙醇；
    所述芳香胺为苯乙胺；
    所述脂肪胺为碳链长度为2-6个碳原子的脂肪胺，优选为异丙胺；
    所述氨基酸为丙氨酸和/或天冬氨酸。
  13. 如权利要求9所述的方法，其特征在于，所述方法具有以下一项或多项特征：
    转氨酶的用量是反应体系中底物重量的1-50％，如10-40％或15-30％；
    反应体系中含有辅酶，辅酶的用量为底物重量的0.1-5.0％，优选1-3％；优选的辅酶为5-磷酸吡哆醛；
    反应体系中，氨基供体的用量为底物重量的30％到500％，如50％到300％。
  14. 如权利要求9所述的方法，其特征在于，所述方法具有以下一项或多项特征：
    反应体系的pH值为6～10，优选为7～9，更优选为8～9，更优选为8.3～8.6；
    反应温度为10℃～50℃，优选为20℃～45℃，更优选为20℃～35℃；
    反应时间为0.1～120小时，例如0.5～48小时或10～24小时。
  15. 如权利要求9-14中任一项所述的方法，其特征在于，所述方法中，助溶剂为二甲亚砜或异丙醇；氨基供体为脂肪胺，优选为异丙胺，或芳香胺，优选R-苯乙胺；转氨酶为来自节杆菌属(Arthrobacter sp)的转氨酶，更优选为氨基酸序列如SEQ ID NO：1所示的转氨酶或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
  16. 如权利要求9-14中任一项所述的方法，其特征在于，所述方法中，助溶剂为二甲亚砜；氨基供体为脂肪胺，优选为异丙胺；转氨酶为权利要求1-3中任一项所述的转氨酶或其酶制剂；式I化合物为(R)-1，1，1-三氟丙胺；式III化合物为1，1，1-三氟丙酮。
  17. 来自节杆菌属(Arthrobacter sp)的转氨酶或其酶制剂和/或权利要求1-3中任一项所述的转氨酶或其酶制剂在提高制备光学纯手性胺时的转化率中的应用。
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