Mutant transaminase with increased asymmetric reductive amination activity, as well as methods and uses related thereto

Abstract

The present invention relates to a mutant transaminase, a nucleic acid encoding the mutant transaminase, a vector containing the nucleic acid, a cell containing the mutant transaminase or nucleic acid, and a method for enzymatic reductive transamination of ketones and formation of primary amines in the presence of the mutant transaminase.

Description

[Technical Field]

[0001] The present invention relates to a mutant transaminase, a nucleic acid encoding the mutant transaminase, a vector containing the nucleic acid, a cell containing the mutant transaminase or nucleic acid, and a method for enzymatic reductive transamination of ketones and formation of primary amines in the presence of the mutant transaminase. [Background technology]

[0002] Transaminases (also called aminotransferases) catalyze aminotransfer reactions, i.e., the transfer of an amino group from an amine donor to an amine acceptor, particularly the amination of ketones accompanied by amine deamination, in which an NH2 group on one molecule or domain is exchanged for a carbonyl group on the other molecule or domain. Amine transaminases (ATAs) are ω-transaminases that catalyze the intermolecular transfer of amine groups between an amine donor and a carbonyl-containing amine acceptor (i.e., a ketone or aldehyde). ATAs use pyridoxal 5'-phosphate (PLP) as a cofactor, and the mechanism involves oxidative deamination and reductive amination half-reactions with PLP acting as an amine shuttle. Due to the reversibility of the transaminase reaction, ATA can be applied in the preparation of optically pure amines using two approaches: (1) kinetic resolution of racemic amines by oxidative deamination, in which one enantiomer is converted to the corresponding ketone while the desired amine enantiomer remains intact, or (2) asymmetric synthesis of primary amines by reductive amination of prochiral ketones. Common amine donors used in reductive amination reactions are alanine, 1-phenylethylamine (1-PEA), and isopropylamine (2-PrNH2). Isopropylamine is often preferred for industrial purposes because 2-PrNH2 is achiral and inexpensive, and equilibrium can be achieved by using excess 2-PrNH2 or by removing acetone byproducts formed under low pressure or slight heating.

[0003] Over the past decade, several (S) and (R) selective ATAs have been studied and engineered for the efficient synthesis of high-value chiral amines in the pharmaceutical and agrochemical industries. [Overview of the Initiative]

[0004] The objective was to develop mutants with increased reductive amination activity and stereoselectivity for a single enantiomer through enzymatic engineering for the synthesis of chiral amines in the pharmaceutical industry. In addition, stability in the presence of organic solvents was desirable, as these properties would allow for the removal of by-products by evaporation and the application of reactions in or within high concentrations of organic solvents, respectively. Furthermore, this would favor the formation of amine products by shifting the equilibrium, which could be achieved by carrying out the reaction in an organic solvent. To realize the latter approach, the goal was to develop high-performance mutants in organic solvents.

[0005] Remarkably, the mutant transaminase (derived from Ruegeria pomeroyi) contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, and the mutant transaminase has at least four amino acid substitutions at positions 61, 65, 266, and 419 relative to the amino acid sequence of SEQ ID NO: 1, and at least three of these substitutions are - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met(Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 must be substituted with Pro(Pro419) or Val(Val419). A mutant transaminase selected from the group consisting of the following is It was discovered that the transaminase exhibits increased asymmetric reductive amination activity compared to wild-type transaminases, particularly those from Ruegeria pomeroyi, and especially the transaminase of SEQ ID NO: 1 (see below).

[0006] As shown in the examples, a core mutational motif that increases the asymmetric reductive amination activity of the mutant transaminase has been identified. The mutant transaminase has at least four amino acid substitutions at positions 61, 65, 266 and 419 of the amino acid sequence of SEQ ID NO: 1, wherein at least three of these substitutions are - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met(Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 must be substituted with Pro(Pro419) or Val(Val419). A mutant transaminase selected from the group consisting of the following is

[0007] It was found that the transaminases exhibited increased asymmetric reductive amination activity compared to wild-type transaminases, particularly those of Ruegeria pomeroyi, especially the transamina of SEQ ID NO: 1. Single substitutions at position 61 (e.g., Leu), 65 (e.g., Gly, Ser, Ala, Met, Leu, Phe, Val, or Cys), 266, and 419 (e.g., Leu, Val, Pro, Ala, Cys, or Gly) slightly increased activity (see Tables 4, 6, 12, and 17), but combinations of four substitutions at the above positions resulted in a significant increase in activity (see Tables 5, 7, 13, and 18). Additional mutations in the transaminases (e.g., at positions 9, 62, 171, 198, 318, 320, 420, 421, and 464) can further increase enzyme activity.[実施例] See the tables in the section, specifically Tables 2, 3, 9, 11, and 16. [Modes for carrying out the invention]

[0008] Therefore, in the first aspect, the present invention relates to a mutant transaminase having increased asymmetric reductive amination activity compared to a wild-type transaminase, The mutant transaminase contains at least 80% of the same amino acid sequence as SEQ ID NO: 1 (transaminase from Ruegeria pomeroyi), The mutant transaminase has at least four amino acid substitutions at positions 61, 65, 266, and 419 of the amino acid sequence of SEQ ID NO: 1. Of these substitutions, at least three substitutions are - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met(Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 must be substituted with Pro(Pro419) or Val(Val419). This relates to mutant transaminases selected from a group consisting of the following.

[0009] The term "transaminase" (classified as EC2.6.1.XX by the International Biochemistry Commission on Enzymes, also known as aminotransferase) generally refers to an enzyme that catalyzes the transfer of an amine group from an amine donor to the carbonyl group of an amine acceptor (aminotransferase). Transaminases are pyridoxal 5'-phosphate-dependent (PLP-dependent) enzymes. The amine donor provides an amine acceptor with an amino group so that the desired amine is synthesized and the corresponding ketone is formed. Because transaminases often exhibit high stereoselectivity, the aminotransfer reaction can result in the desired amine by asymmetric reduction and / or the remaining amine donor as an enantiomerically enriched amine by cleavage via oxidative deamination. ATA is of particular interest in this invention because it can convert ketones to chiral amines. According to this invention, the mutant transaminase is preferably active as ATA. This means that the mutant transaminase can intermolecularly transfer an amine group from an amine donor to a carbonyl group-containing amine acceptor (i.e., a ketone or aldehyde) under appropriate conditions as detailed above and below.

[0010] As used herein, the term “wild-type transaminase” means any transaminase that is naturally occurring and unmutated. As used herein, the term “mutant transaminase” means any transaminase derived from a corresponding wild-type transaminase and having a modified amino acid sequence compared to such wild-type transaminase. For example, this may include the introduction, deletion, substitution, or post-translational mutation of one or more amino acids at one or more positions. Preferably, the mutant transaminase differs from the wild-type transaminase by amino acid substitution. Methods for causing mutations in the amino acid sequence, such as amino acid substitutions, are well known to those skilled in the art. For example, such mutations may have already been introduced at the nucleic acid level, resulting in the expression of a desired mutant amino acid sequence. Appropriate methods are well known to those skilled in the art and are also partially described below, for example, in the context of nucleic acids according to a second aspect of the present invention.

[0011] A suitable mutant transaminase according to the first embodiment may be derived from the wild-type transaminase of any organism. A preferred source is the Ruegeria species, in particular Ruegeria pomeroyi, also known as Silicibacter pomeroyi. Therefore, Ruegeria pomeroyi transaminase is particularly preferred (PDB code: 3HMU; see Sequence ID No. 1 below).

[0012] As detailed above, the transaminase of the present invention has increased asymmetric reductive amination activity compared to wild-type transaminase. Reductive amination, or the conversion of a carbonyl group to an amine via an iminium intermediate, is one of the most important reactions for synthesizing chiral amines (functional groups characterized by a significant proportion of biologically active small molecules). Since the stereocenters constituting the amine are often crucial in determining its biological activity, research is directed toward the development of asymmetric processes for reductive amination. Increased asymmetric reductive amination activity compared to each unmutated wild-type transaminase means that the mutant has increased reductive amination activity compared to the wild type and that the mutant is stereoselective, i.e., the formation of one stereoisomer is preferred over the formation of the other. (S)-selective ATAs have been known for 20 years. Therefore, the transaminase is preferably an amine transaminase (ATA). Therefore, the transaminases of the present invention are stereoselective with respect to the stereocenters constituting the amine, for example, (S)-selective or (R)-selective, preferably (S)-selective. Stereoselectivity can be biased, where the formation of one stereoisomer is more favorable than the other, or it can be complete, where only one stereoisomer is formed. Enantioselectivity is generally reported in the art as an enantiomer excess (ee) (typically in a percentage), calculated according to the formula [major enantiomer - secondary enantiomer] / [major enantiomer + secondary enantiomer]. Alternatively, stereoselectivity may be characterized using an enantiomer ratio or er(S:R). The enantiomer ratio is the ratio of the percentage of one enantiomer (e.g., (S)-enantiomer) in a mixture of enantiomers to the percentage of the other enantiomer (e.g., (R)-enantiomer).

[0013] As detailed above, the mutant transaminase according to the first aspect exhibits increased asymmetric reductive amination activity as compared to the wild-type transaminase. Methods for determining transaminase activity are well-known in the art and are described herein. Exemplary methods are also described in the Examples. Enzyme conversion is a measure of the activity of the enzyme. This activity can be determined in an enzyme assay that measures either the consumption of the substrate or the formation of the product over time. There are numerous different methods for measuring the concentration of the substrate and the product, and many enzymes can be assayed in several different ways known to those skilled in the art. To determine whether the mutant transaminase according to the first aspect exhibits increased transaminase activity as compared to the wild-type transaminase, the transaminase activities of both transaminases are measured using the same method. The conditions when measuring the activity are usually standardized. According to the present invention, a temperature of 40 °C to 70 °C, for example 50 °C, 60 °C or 70 °C (as in the Examples) and an appropriate pH value (for example pH 7) as well as a substrate concentration may be taken. Tests for stability in the presence of an organic solvent include the presence of an organic solvent, for example dimethyl sulfoxide (for example 20% to 40% in water), isopropyl acetate or any of the organic solvents used in the Examples. Suitable organic solvents are ethers, esters or hydrocarbons. Specific ethers are diethoxymethane or t-butyl methyl ether. Specific hydrocarbons include toluene, and specific esters include lower alkyl esters of acetic acid or propionic acid, for example ethyl acetate, isopropyl acetate, isobutyl acetate, isopropyl 2-methylpropionate or tert-butyl 2,2-dimethylpropionate. Preferred are the named esters, with isopropyl acetate being more specifically preferred.

[0014] For example, methods for determining the enzyme activity of transaminase may generally be based on fluorescence assays or colorimetric assays. HPLC-UV can likewise be used. The assay is usually carried out under well-controlled conditions including, for example, pH value, temperature, salts, buffers, and substrate concentration. Further, methods for determining the enzyme activity of transaminase may generally include detecting the concentration of the product formed or the reactant (educt) consumed.

[0015] A mutant transaminase according to a first aspect showing increased transaminase activity compared to wild-type transaminase shows, for example, an increase exceeding 1-fold of the transaminase activity. Those skilled in the art know statistical procedures for evaluating whether one value of enzyme activity is increased compared to another value, such as the Student's t-test or chi-square test. It is obvious to those skilled in the art that all background signals must be subtracted when analyzing the data.

[0016] The mutant transaminase of the present invention contains an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 1 (transaminase from Ruegeria pomeroyi). The terms “at least 80% identical” or “at least 80% sequence identity,” as used herein, mean that the sequence of the mutant transaminase according to the present invention has an amino acid sequence characterized in that, within a 100-amino acid stretch, at least 80 amino acid residues are identical to the sequence of the corresponding wild-type sequence. Sequence identity according to the present invention can be determined, for example, by a sequence alignment method that compares sequences. Sequence alignment methods are well known in the art and include, for example, various programs and alignment algorithms described in Pearson and Lipman (1988). Furthermore, the NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, and is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tbiastn, and tbiastx. The percentage of identity of the mutant according to the present invention with respect to the amino acid sequence of SEQ ID NO: 1 is typically characterized using NCBI Blast blastp in a standard configuration. Alternatively, sequence identity can be determined using the software GENEious in a standard configuration. In the present invention, the alignment results presented are derived from the software Geneious (version R8) using a global alignment protocol with free-end gap as the alignment type and Blosum62 as the cost matrix. According to the present invention, the mutant transaminase has at least four amino acid substitutions compared to the wild-type transaminase, with the amino acids substituted at positions 61, 65, 266, and 419.

[0017] The mutant transaminase of the first aspect of the present invention is - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met(Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 must be substituted with Pro(Pro419) or Val(Val419). It has at least three substitutions selected from the group consisting of the following.

[0018] In this invention, the location of the mutation is identified based on the amino acid sequence of Sequence ID No. 1, i.e., the transaminase of Ruegeria pomeroyi. The corresponding mutation sites of transaminases other than Sequence ID No. 1 can be identified by performing amino acid alignment as detailed above (for example, by using BLAST (Basic Local Alignment Search Tool), available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome with standard settings), or by structural comparison if available, and the corresponding amino acids can be identified. An example of a different wild-type transaminase is 3HMU (ATCC 700808), where the positions corresponding to positions 61, 65, 266, and 419 of Sequence ID No. 1 are positions 59, 63, 264, and 417, respectively.

[0019] In a preferred embodiment of the present invention, the mutant transaminase contains or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0020] In particular, the mutant transaminase has at least four amino acid substitutions at positions 61, 65, 266, and 419 of the amino acid sequence of SEQ ID NO: 1, and at least three of these substitutions are - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met(Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 must be substituted with Pro(Pro419) or Val(Val419). It is selected from the group consisting of the following.

[0021] In one embodiment of the present invention, the sequence of the mutant transaminase according to the present invention may include one or more combinations of deletions, substitutions, or additions as defined above, in addition to the substitutions specified herein.

[0022] In one embodiment, the sequence of the mutant transaminase according to the present invention may include one or more additional amino acid substitutions, in particular one or more conserved amino acid substitutions, in addition to the substitutions specified herein. A “conserved amino acid substitution” means the substitution of a residue with a different residue having a similar side chain, and therefore typically includes substituting an amino acid in a polypeptide with an amino acid within the same or similar defined amino acid class. As an example, and not an limitation, amino acids having aliphatic side chains may be substituted with other aliphatic amino acids (e.g., alanine, valine, leucine, and isoleucine); amino acids having hydroxyl side chains are substituted with other amino acids having hydroxyl side chains (e.g., serine and threonine); amino acids having aromatic side chains are substituted with other amino acids having aromatic side chains (e.g., phenylalanine, tyrosine, tryptophan, and histidine); amino acids having basic side chains are substituted with other amino acids having basic side chains (e.g., lysine and arginine); amino acids having acidic side chains are substituted with other amino acids having acidic side chains (e.g., aspartic acid or glutamic acid); and hydrophobic or hydrophilic amino acids are substituted with other hydrophobic or hydrophilic amino acids, respectively.

[0023] In one embodiment of the present invention, the mutant transaminase according to the present invention may include the addition of one or more amino acids, particularly the addition of a short chain (e.g., up to 10 amino acids) C-terminal amino acids or N-terminal amino acids.

[0024] Furthermore, the transaminase according to the present invention may be part of a larger molecule such as a fusion protein, or it may be immobilized on a solid support. A fusion protein is a protein made up of two or more originally distinct proteins joined together. Therefore, depending on the intended use of the transaminase, it may be combined with further proteins to form a fusion protein. Proteins may be fused via a linker or spacer, which increases the likelihood that the proteins will fold independently and behave as expected. In particular, if the linker allows for protein purification, the linker in protein fusion may be designed to have a cleavage site for a protease or chemical that allows for the release of the two distinct proteins. Preferably, the fusion protein of the present invention includes a tag, for example, to facilitate purification, to assist in proper protein folding, to prevent protein precipitation, or to alter chromatographic properties. In addition to or instead of this, the variant may be immobilized on a solid support. The solid support may be a resin, glass, metal, or nanomaterial, depending on the application. Immobilization techniques rely on effective protein bioconjugation chemistry and are known to those skilled in the art.

[0025] In addition to increased transaminase activity, mutant transaminases according to the first embodiment may exhibit even greater stability in the presence of organic solvents compared to wild-type transaminases.

[0026] The term "increased stability in the presence of organic solvents" compared to wild-type transaminase means that the mutant transaminase is less prone to loss of (enzyme) activity in the presence of organic solvents. In this invention, "in the presence of organic solvents" means that the amount of organic solvent is fairly large, i.e., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Note that there is generally a residual amount of water to form a hydration shell around the enzyme. This interaction between the enzyme surface and the surrounding water is essential for enzyme activity. This residual amount of water will typically be 1.0% to 5.0% by weight, particularly 1.4% to 3.4% by weight, more specifically 1.8% to 2.8% by weight of the total amount of solvent. Note that in this invention, the solvent is preferably single-phase. The organic solvent is one of those tested in the examples, e.g., dimethyl sulfoxide (e.g., 20% to 40% in water), ether, ester, or hydrocarbon. Certain ethers are diethoxymethane or t-butyl methyl ether. Certain hydrocarbons include toluene, and certain esters include lower alkyl esters of acetate or propionic acid, such as ethyl acetate, isopropyl acetate, isobutyl acetate, isopropyl 2-methylpropionate, or tert-butyl 2,2-dimethylpropionate. Preferred are designated esters, with isopropyl acetate being a more specific preference.

[0027] In a preferred embodiment of the present invention, the mutant transaminase of the first embodiment is -The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). -The amino acid at position 65 of sequence number 1 is substituted with Met(Met65), -The amino acid at position 266 of sequence number 1 is substituted with Val(Val266). - This is characterized by the substitution of the amino acid at position 419 of SEQ ID NO: 1 with Pro(Pro419) or Val(Val419).

[0028] In particular, the mutant transaminase of the first embodiment is -The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). -The amino acid at position 65 of sequence number 1 is substituted with Met(Met65), -The amino acid at position 266 of sequence number 1 is substituted with Val(Val266). - Is the amino acid at position 419 of SEQ ID NO: 1 substituted with Pro(Pro419)? or -The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). -The amino acid at position 65 of sequence number 1 is substituted with Met(Met65), -The amino acid at position 266 of sequence number 1 is substituted with Val(Val266). - This molecule is characterized by the substitution of the amino acid at position 419 of sequence number 1 with Val(Val419).

[0029] Mutants possessing the above mutations were found to be particularly suitable and exhibit the desired characteristics (see Examples).

[0030] Therefore, they were selected as a specific basis for further optimized variants into which additional mutations were introduced.

[0031] One mutation that has been shown to provide further beneficial effects (particularly for the variants characterized by mutations Leu61, Met65, Val266, and Val419) is the substitution of the amino acid at the position corresponding to position 426 of SEQ ID NO: 1 with Val(Val426). Therefore, in a preferred embodiment, the mutant transaminase of the present invention is characterized in that the amino acid at the position corresponding to position 426 of SEQ ID NO: 1 is substituted with Val(Val426).

[0032] Therefore, the mutant transaminase of the first embodiment is -The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). -The amino acid at position 65 of sequence number 1 is substituted with Met(Met65), -The amino acid at position 266 of sequence number 1 is substituted with Val(Val266). - The amino acid at position 419 of sequence number 1 is substituted with Pro(Pro419), and - Is the amino acid at position 426 of the sequence number substituted with Val(Val426)? or -The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61). -The amino acid at position 65 of sequence number 1 is substituted with Met(Met65), -The amino acid at position 266 of sequence number 1 is substituted with Val(Val266). - The amino acid at position 419 of sequence number 1 is substituted with Val(Val419), and -This sequence may be characterized by the substitution of the amino acid at position 426 of the sequence number with Val(Val426).

[0033] However, preferably, the mutant transaminase of the first embodiment is - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61), and - The amino acid at position 65 of SEQ ID NO: 1 is substituted with Met(Met65), and - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - Is the amino acid at position 419 of SEQ ID NO: 1 substituted with Pro(Pro419)? or - The amino acid at position 61 of sequence number 1 is substituted with Leu(Leu61), and - The amino acid at position 65 of SEQ ID NO: 1 is substituted with Met(Met65), and - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), and - The amino acid at position 419 of sequence number 1 is substituted with Val(Val419), and -This can be characterized by the substitution of the amino acid at position 426 of sequence number 1 with Val(Val426).

[0034] The main characteristic of the two mutants mentioned above (Leu61 / Met65 / Val266 / Pro419 and Leu61 / Met65 / Val266 / Val419 / Val426) is not only the increased activity in aqueous reactions. These mutants also exhibit activity in the presence of organic solvents, which is essentially absent in the wild type and many other investigated mutants that do not possess this set of mutations.

[0035] In the variants of the present invention as defined above, additional substitutions have been introduced and found to be beneficial, either individually or in combination with each other. These include: - The amino acid at position 9 of sequence number 1 is substituted with Tyr(Tyr9), and / or - The amino acid at position 62 of sequence number 1 is substituted with Ser(Ser62), and / or - The amino acid at position 65 of sequence number 1 is substituted with Phe(Phe65), Gly(Gly65), or Met(Met65), especially Met(Met65), and / or - The amino acid at position 171 of sequence number 1 is substituted with Phe(Phe171) or Trp(Trp171), and / or - The amino acid at position 198 of sequence number 1 is substituted with Leu(Leu198) or Met(Met198), especially Leu(Leu198), and / or - The amino acid at position 318 of sequence number 1 is substituted with Lys(Lys318), and / or - The amino acid at position 320 of SEQ ID NO: 1 is substituted with Cys(Cys320), Leu(Leu318), Lys(Lys320), or Met(Met320), especially Met(Met320), and / or - The amino acid at position 419 of sequence number 1 is substituted with Ala(Ala419), Cys(Cys419), Gly(Gly419), Pro(Pro419), or Val(Val419), and / or, - The amino acid at position 420 of sequence number 1 is substituted with Asn (Asn420), Asp (Asp420), Cys (Cys420), or Ser (Ser420), and / or - The amino acid at position 421 of sequence number 1 is substituted with Ser(Ser421), and / or - The amino acid at position 464 of the sequence number is substituted with Met(Met464).

[0036] Further mutations may or may not exist.

[0037] In particular, mutant transaminases are characterized as follows: - Mutant transaminases have substitutions Leu61, Met65, Val266 and Pro419, or - Mutant transaminases have substitutions Leu61, Met65, Val266, Val419 and Val426, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - Mutant transaminases have substitutions Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, or - Mutant transaminases have substitutions for Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426, and Met464.

[0038] More specifically, mutant transaminases are characterized as follows: - Mutant transaminases have substitutions Leu61, Met65, Val266, and Pro419, and the amino acids at positions 9, 62, 171, 198, 318, 320, 420, 421, 426, and 464 of SEQ ID NO: 1 are unsubstituted. - Mutant transaminases have substitutions Leu61, Met65, Val266, Val419 and Val426, and the amino acids at positions 9, 62, 171, 198, 318, 320, 420, 421 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, and the amino acids at positions 9, 198, 320, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, and the amino acids at positions 9, 198, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, and the amino acids at positions 9, 62, 198, 318 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions of Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, and the amino acids at positions 198, 320, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions of Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, and the amino acids at positions 198, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions of Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, and the amino acids at positions 62, 198, 318 and 464 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, and the amino acids at positions 9, 320 and 426 of SEQ ID NO: 1 are unsubstituted, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, and the amino acids at positions 9 and 426 of SEQ ID NO: 1 are unsubstituted, or -The mutant transaminase has substitutions at Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426 and Met464, and the amino acids at positions 9, 62 and 318 of SEQ ID NO: 1 are unsubstituted.

[0039] More specifically, mutant transaminases are characterized as follows: - Mutant transaminases have substitutions Leu61, Met65, Val266 and Pro419, or - Mutant transaminases have substitutions Leu61, Met65, Val266, Val419 and Val426, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - Mutant transaminases have substitutions Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - Mutant transaminases have substitutions Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, or - Mutant transaminases have substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, or - Mutant transaminases have substitutions Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426 and Met464, This substitution is the only substitution in the mutant transaminase relative to the amino acid sequence of SEQ ID NO: 1.

[0040] In a particularly preferred embodiment of the present invention, the mutant transaminase of the first aspect of the present invention is as defined above, i) consisting of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, particularly 100%, identical to any of the amino acid sequences of SEQ ID NOs: 2 to 12, or ii) comprising the same.

[0041] As used herein, the term “sequence identity” refers to the percentage of characters that exactly match between two different sequences. For example, the term “at least 85% identical to the amino acid sequence of SEQ ID NO: 2” means, as used herein, that the amino acid sequence of the mutant transaminase of the present invention has an amino acid sequence characterized in that, within a stretch of 100 amino acids, at least 85 amino acid residues are identical to the sequence of the corresponding sequence of SEQ ID NO: 2. Sequence identity according to the present invention can be determined, for example, by the method described above.

[0042] The mutant transaminase of the first embodiment exhibits substantially increased reductive amination activity compared to wild-type transaminase, particularly in the presence of organic solvents and amine donors. In the presence of organic solvents and amine donors, the increase may be at least twofold, but can reach levels of 50 times or more.

[0043] The mutant transaminase of the first embodiment may also exhibit increased stability in aqueous organic media, particularly in aqueous media containing 4.0% v / v to 40.0% v / v organic solvents that create a monophase environment, such as dimethyl sulfoxide.

[0044] The mutant transaminase of the first embodiment may further exhibit increased stability in the presence of an organic solvent and residual water. The presence of residual water, typically 1.0% to 5.0% by weight, particularly 1.4% to 3.4% by weight, and more specifically 1.8% to 2.8% by weight, of the total amount of solvent, is important for the activity of the mutant transaminase.

[0045] Suitable organic solvents are ethers, esters, or hydrocarbons. Certain ethers include diethoxymethane or t-butyl methyl ether. Certain hydrocarbons include toluene, and certain esters include lower alkyl esters of acetic acid or propionic acid, such as ethyl acetate, isopropyl acetate, isobutyl acetate, isopropyl 2-methylpropionate, or tert-butyl 2,2-dimethylpropionate. Preferred are designated esters, with isopropyl acetate being a more specific preference.

[0046] The mutant transaminase of the first embodiment may be capable of reductive amination from ketones to primary amines, particularly from prochiral ketones to chiral primary amines. In addition, or alternatively, the mutant transaminase may exhibit reductive amination activity at temperatures of 40°C to 70°C, particularly 50°C to 70°C.

[0047] The ketones suitable for application in reductive amination are those of formula I TIFF2026528779000001.tif27170 (in the formula, R 1 and R 2 These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R 1 and R 2 These can be exemplified by the fact that they, together with the carbon atoms to which they are bonded, form monocyclic or polycyclic carbocyclic or heterocyclic rings, which may be substituted. The substituent that may be replaced is selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl or cyano.

[0048] In a preferred embodiment, the ketone is a prochiral ketone, and R 1 and R 2 are different.

[0049] In a more preferred embodiment, R 1 is optionally substituted C 1~12 alkyl, R 2 is optionally substituted aryl, arylalkyl, heterocyclyl or heterocyclylalkyl, or R 1 and R 2 together with the carbon atom to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring, The substituent that may be substituted is selected from C 1~12 alkyl, C 1~12 alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl or cyano.

[0050] The substitution itself may be further substituted with the substitutions outlined above.

[0051] Examples of suitable ketones are cyclohexanone, 2 - hexanone, acetophenone, benzylacetone, 3 - acetylpyridine, 3 - propionylpyridine, 5 - acetyl - 2 - chloropyridine, 3 - acetyl - 5 - chloropyridine, 3 - acetyl - 2 - chloropyridine, 5 - acetyl - 2 - methoxy - pyridine, 1 - quinolin - 3 - yl - ethanone, 2 - acetylpyridine, indan - 1 - one or 1 - piperidin - 3 - yl - ethanone.

[0052] A preferred example is the prochiral ketone of formula X. The prochiral ketone of formula X is an intermediate in the synthesis of pralcetinib, a kinase inhibitor indicated for the treatment of non-small cell lung cancer (NSCLC). TIFF2026528779000002.tif43170

[0053] The terms used herein with respect to the ketone of formula I have the following meanings:

[0054] The term "halogen" refers to fluoro, chloro, bromo, or iodine, especially chlorine or fluorine.

[0055] The term "alkyl" refers to a monovalent linear or branched saturated hydrocarbon group with 1 to 12 carbon atoms. In certain embodiments, alkyl groups have 1 to 7 carbon atoms, and in more specific embodiments, 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. 1 The most preferred alkyl group is methyl.

[0056] The term "alkoxy" refers to a group in which an alkyl group, as defined above, is bonded to an oxygen radical. Examples of alkoxys include methoxy, ethoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0057] The term "aryl" refers to monocyclic or bicyclic systems of monovalent aromatic carbocyclic compounds containing 6 to 10 carbocyclic atoms. Examples of aryl moieties include phenyl and naphthyl.

[0058] The term "arylalkyl" refers to an aryl-substituted alkyl group, and the definitions of aryl and alkyl are outlined above.

[0059] The term "aryloxy" refers to a group in which an aryl group, as defined above, is bonded to an oxygen radical. Suitable examples are phenoxy or naphthyloxy.

[0060] The terms "monocyclic or polycyclic ring" refer to compounds characterized by the closure of one or more rings of atoms, primarily carbon. These ring substructures include cycloalkanes, aromatics, and other cyclic types. "Poly" literally means "many," but it also includes bicyclic, tricyclic, tetracyclic, and so on.

[0061] The term "carbocyclic" refers to saturated, partially unsaturated, or unsaturated cyclic compounds in which all ring members are carbon atoms, such as cyclohexane, decalin, or 1,2-dihydronaphthalene. The compounds may be aromatic or non-aromatic. Simple aromatic rings consist only of conjugated planar ring systems. Typical simple aromatic compounds are benzene, indole, and cyclotetradecaheptaene. Polycyclic aromatic hydrocarbons contain only carbon and hydrogen and consist of multiple aromatic rings. Examples include naphthalene, anthracene, phenanthrene, and indane.

[0062] The term "heterocyclyl" refers to a saturated, partially unsaturated, or unsaturated 5- to 6-membered monocyclic ring or an 8- to 10-membered bicyclic ring that can contain one, two, or three heteroatoms selected from nitrogen, oxygen, and / or sulfur. The ring system may be aromatic or non-aromatic. Typical heterocyclyl residues are pyridyl, pyridinyl, pyrazolyl, pyrimidinyl, benzimidazolyl, quinolinyl and isoquinolinyl, thienyl, furyl, pyrrolyl, pyrazolyl, isoxazolyl, oxazolyl, thiazolyl, or imidazolyl.

[0063] The term "heterocyclylalkyl" refers to an alkyl group that has been heterocyclil-substituted, and the definitions of heterocyclyl and alkyl are outlined above.

[0064] The optionally substituted substituents of the ketone of formula I may be selected from alkyl, alkoxy, aryl, aryloxy, halogen, hydroxyl, or cyano when used herein. The preferred ones and examples outlined above also constitute substitutions.

[0065] In a second aspect, the present invention relates to a nucleic acid encoding a mutant transaminase of the first aspect, which is optionally contained in a vector.

[0066] As used herein, the term “nucleic acid” generally refers to any nucleotide molecule that encodes the mutant transaminase of the present invention and may be of variable length. Examples of nucleic acids of the present invention include, but are not limited to, plasmids, vectors, or any type of DNA and / or RNA fragment that can be isolated by standard molecular biology procedures, including, for example, ion-exchange chromatography. Nucleic acids of the present invention may be used for transfection or transduction of specific cells or organisms.

[0067] The nucleic acid molecules of the present invention may be in the form of RNA, for example mRNA or cRNA, or in the form of DNA, for example, including cDNA and genomic DNA, and may be obtained, for example, by cloning, or by a combination of multiple chemical synthesis techniques. DNA may be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA may be a coding strand, also known as a sense strand, or a non-coding strand, also called an antisense strand. As used herein, nucleic acid molecules also refer, among other things, to DNA which is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, hybrid molecules including DNA and RNA which may be single-stranded or more typically double-stranded or triple-stranded, or mixtures of single-stranded and double-stranded regions. Furthermore, as used herein, nucleic acid molecules refer to a triple-stranded region which includes RNA or DNA or both RNA and DNA.

[0068] Furthermore, nucleic acids may contain one or more unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples. Such nucleic acids may also contain modifications, for example, in the ribose-phosphate backbone, to increase the stability and half-life of such molecules in a physiological environment. Thus, DNA or RNA having a backbone modified for stability or other reasons is a “nucleic acid molecule” as its characteristics are intended herein. Naturally, a wide variety of modifications have been made to DNA and RNA, serving many useful purposes known to those skilled in the art. As used herein, the term nucleic acid molecule encompasses such chemically, enzymatically, or metabolically modified forms of nucleic acid molecules, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, in particular.

[0069] Furthermore, the nucleic acid molecule encoding the mutant transaminase of the present invention can be functionally ligated to any desired sequence, such as a regulatory sequence, a leader sequence, a heterologous marker sequence, or a heterologous coding sequence, using standard techniques such as standard cloning techniques, to produce a fusion protein.

[0070] The nucleic acid of the present invention may be contained in an expression vector, and the nucleic acid is operably linked to a promoter sequence that can promote nucleic acid expression in a host cell.

[0071] In a third aspect, the present invention relates to cells comprising a mutant transaminase of the first aspect of the present invention and / or a nucleic acid of the second aspect of the present invention. The cells may be referred to as host cells and comprise the transaminase of the first aspect of the present invention (optionally as part of a fusion protein linked to another molecule) or the nucleic acid of the second aspect of the present invention (optionally contained in an expression vector). Culturing host cells according to the present invention is a standard procedure known to those skilled in the art. That is, the nucleic acid encoding the mutant transaminase of the present invention can be introduced into a suitable host cell, and the respective proteins can be produced by recombinant means. These host cells may be any type of suitable cell that can be cultured under culture conditions, preferably bacterial cells such as Escherichia coli (E. coli). After the proteins have been expressed in the respective host cells, the cells can be harvested and serve as starting materials for the preparation of cell extracts containing the protein of interest. Cell extracts containing the protein of interest may be obtained by cell lysis. Methods for preparing cell extracts by either chemical or mechanical cell lysis are well known to those skilled in the art and are not limited to, but include, for example, hypotonic salt treatment, homogenization, or sonication. In preferred embodiments of the present invention, host cells, particularly E. coli cells containing the mutant transaminase according to the first embodiment, are spray-dried and used in the methods of the present invention described below.

[0072] In a further embodiment, the present invention relates to a method for enzymatic reductive transamination of a ketone and formation of a primary amine in the presence of the mutant transaminase of the present invention, as described above. Preferably, the ketone is a prochiral ketone and the primary amine is a chiral primary amine.

[0073] As mentioned above, the appropriate ketone is given by formula I TIFF2026528779000003.tif27170 (in the formula, R 1 and R 2 These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R 1 and R 2 These have (which, together with the carbon atoms to which they are bonded, form a monocyclic or polycyclic carbocyclic or heterocyclic carbocyclic ring, which may be substituted), The substituents that may be substituted are selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano. The resulting primary amine is given by formula II TIFF2026528779000004.tif27170 (in the formula, R 1 and R 2 These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R 1 and R 2 These have (which, together with the carbon atoms to which they are bonded, form a monocyclic or polycyclic carbocyclic or heterocyclic carbocyclic ring, which may be substituted), The substituents that may be substituted are selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano.

[0074] The preferred embodiments described above also apply to primary amines of formula II.

[0075] Enzymatic reductive amino group transfer of a preferred prochiral ketone of formula X yields a chiral primary amine of formula XI. TIFF2026528779000005.tif41170

[0076] Enzymatic reductive amination is typically carried out in the presence of an organic solvent.

[0077] However, the presence of residual water, typically 1.0% to 5.0% by weight, particularly 1.4% to 3.4% by weight, and more specifically 1.8% to 2.8% by weight, is important for enzyme activity.

[0078] The organic solvent, along with residual water, preferably forms a single-phase system also known as a microaqueous reaction system (MARS).

[0079] Therefore, organic solvents are selected according to their ability to create such a monophase environment.

[0080] Suitable organic solvents are ethers, esters, or hydrocarbons. Certain ethers are diethoxymethane or t-butyl methyl ether. Certain hydrocarbons include toluene, and certain esters include lower alkyl esters of acetic acid or propionic acid, such as ethyl acetate, isopropyl acetate, isobutyl acetate, isopropyl 2-methylpropionate, or tert-butyl 2,2-dimethylpropionate. Preferred organic solvents are designated esters, with isopropyl acetate being a more specific preference.

[0081] Alternatively, though less desirable, enzymatic reductive amination may be carried out in an aqueous organic medium, particularly in an aqueous medium containing 4.0% v / v to 40.0% v / v of an organic solvent, such as dimethyl sulfoxide, to create a monophase environment.

[0082] Aminotransfer reactions require a suitable amine donor, which can be easily separated from the desired primary amine during its conversion in each ketone.

[0083] The amine donor may be a primary aliphatic amine. In principle, primary aliphatic amines such as isopropylamine or phenylethylamine are used. The preferred amine donor is isopropylamine.

[0084] The amine donor is used in an amount of 2 to 20 equivalents, preferably 4 to 8 equivalents, and more preferably 4 to 6 equivalents.

[0085] Transaminase mutant enzymes are typically supplied in a dry formulation that allows for a defined water content and ensures high activity in microaqueous reaction systems (MARS).

[0086] The transaminase mutant enzyme can be used as a freeze-dried or spray-dried enzyme powder, as an enzyme immobilized product, or as whole cells such as E. coli cells containing the overexpressed transaminase. Whole E. coli cells are preferred for higher stability.

[0087] The substrate-to-enzyme ratio (s / e weight ratio) is usually 1 to 5, preferably 2 to 3. In the case of E. coli cell powder, the s / e weight ratio is usually 1 to 5, preferably 3 to 4.

[0088] The reaction temperature depends on the stability of the transaminase mutant enzyme preparation and the reaction environment. As a general rule, the reaction temperature is selected between 40°C and 70°C.

[0089] For enzyme powders, the reaction temperature can be selected from 40°C to 65°C, preferably 45°C to 60°C, and more preferably about 50°C or 55°C. For whole E. coli (E. coli) cell powders, the reaction temperature can be selected from 40°C to 70°C, preferably 55°C to 65°C, and more preferably about 60°C or 65°C.

[0090] The ketone substrate load (by weight) can be selected from 1% to 15%, preferably 2% to 10%, and more preferably about 5%.

[0091] The reaction equilibrium can be further shifted to the formation of the product (primary amine) by the in-situ removal of the product and / or the successive removal of ketone by-products (formed from the amine donor).

[0092] For example, a primary amine of formula II can be precipitated as an insoluble salt, such as a hydrochloride salt, and / or ketone by-products can be removed by distillation.

[0093] The isolation of the obtained primary amine can be achieved according to methods well known to those skilled in the art.

[0094] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0095] The present invention is not limited to the specific methodologies, protocols, and reagents described herein, for this reason, as these may vary. Any method and materials similar to or equivalent to those described herein may be used in carrying out the present invention, but preferred methods and materials are described herein. Furthermore, the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention.

[0096] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. Similarly, the words “comprise,” “contain,” and “encompass” are to be interpreted inclusively, not exclusively. Likewise, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. The term “plural” refers to two or more.

[0097] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific modifications described should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it should be understood that such equivalent embodiments are included herein.

[0098] Example 1: Enzyme Production Culture, protein expression, cell harvesting, or cell lysis Culture on an automated platform: Bacterial cells were cultured in 96-well microtiter plates (ThermoFisher Scientific) using the Tecan Fluent automated platform. Preliminary cultures were grown from fresh single transformants in 160 μL of LB medium supplemented with 50 mg / L kanamycin and incubated at 30°C and 850 rpm for 20 hours. 10 microliters (10 μL) of overnight pre-culture was inoculated into 180 μL of ZYM-5052 auto-inducing medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L glycerol, 0.55 g / L glucose monohydrate, 2.1 g / L lactose monohydrate, 10.6 g / L sodium phosphate dibase, 3.4 g / L potassium phosphate monobase, 2.15 g / L ammonium chloride, 0.59 g / L sodium chloride, 0.663 g / L ammonium sulfate, 2 mM magnesium sulfate) supplemented with 50 mg / L kanamycin, without trace components. The culture was grown at 20°C and 800 rpm for 22 hours. Automated cell collection was performed by plate centrifugation at 4°C and 4060 rpm for 30 minutes, followed by removal of the supernatant by manually inverting the plate. The cell pellet was stored at -20°C for use in the reductive amination reaction.

[0099] Manual culture in 96-deep-well plates: Bacterial cells were cultured in 96-deep-well plates (DWP) equipped with V-bottom and conical auxiliary substrates. All plates contained strains with negative control plasmids as well as parent enzymes or positive controls. Preliminary cultures were initiated by inoculating fresh single transformants into LB medium containing 50 mg / L kanamycin, followed by incubation at 30°C and shaking at 300 rpm for 18 hours (Duetz system, Kuhner shaker). Primary culture in DWPs was initiated by inoculating 8 μL of the preliminary culture into 500 μL of ZYM-5052 autoinducing medium without trace components containing 50 mg / L kanamycin. The cultures were incubated at 20°C, 300 rpm for 20 hours at no humidity (Duetz system, Kuhner shaker). Cells were pelletized by centrifugation at 4°C, 3300 g for 20 minutes.

[0100] Preparation of biocatalysts for reactions in aqueous media: Cell pellets after one freeze-thaw cycle were used for reductive amination. Alternatively, cells were lysed by adding 0.2 ml of 10 mM HEPES buffer (pH 7) containing 1 mg / mL chicken egg white lysozyme, 0.75 mg / mL polymyxin B, and 0.2 mg / mL DNase I. The cell suspension was incubated at 30°C with shaking at 300 rpm for 1 hour (Duetz system, Kuhner shaker), followed by centrifugation at 4°C and 3300 g for 30 minutes. The freshly extracted supernatant was used for reductive amination in aqueous buffer.

[0101] Example 2: Screening of enzyme activity and selectivity Microscale reactions in aqueous media (4-40% DMSO): Biocatalytic reactions in aqueous media were carried out in 2 mL Eppendorf tubes or 96-DWP with a total reaction volume of 0.2 mL. Samples contained 0.2% or 1% [w / v] substrate, 4-40% [v / v] DMSO, 0.5-2 M isopropylamine hydrochloride, 2 mM pyridoxal 5'-phosphate monohydrate, and 0.2 M HEPES buffer (pH 7). To accurately compare the tested generated enzyme variants with their parent enzymes, the biomass or lysate concentrations were adjusted according to the stage of development. Unless otherwise specified, samples were incubated at 50°C in a ventilated hood with shaking at 1000 rpm (Eppendorf thermomixer) or 300 rpm (Kuhner Kelvin+ shaker). The reaction mixture was quenched with acetonitrile in a sample-solvent ratio of 1:2 or 1:5 (final acetonitrile concentration of 50–62.5% in water), mixed for 5–10 minutes on a thermomix or by pipetting, and centrifuged at room temperature, 13500 g for 2 minutes (tube) or at 10°C, 3330 g for 10 minutes (DWP). The supernatant was transferred from the tube to a glass vial or measured in plate form using either chiral or accelerated achiral HPLC.

[0102] Microscale reactions in organic media: Small-scale reactions were carried out in 2 mL Eppendorf tubes with a final volume of 0.5 mL or 1 mL. Samples contained 1% or 2% [w / v] substrate, 5–9 equivalents of isopropylamine free base, and water-saturated isopropyl acetate. First, the organic media was prepared by mixing 0.3 mL of water containing 1 M potassium phosphate buffer (pH 7), 5 M isopropylamine hydrochloride, and 2 m pyridoxal 5'-phosphate monohydrate with 10 mL of isopropyl acetate for 1 hour. In later stages, it was observed that the reaction proceeded without buffer or without the addition of pyridoxal 5'-phosphate monohydrate, so isopropyl acetate was saturated with water alone. Lyophilized cells or lyophilized enzyme lysates were used in ratios appropriate for each developmental round. Samples were incubated at 50–70°C with shaking. After the determined reaction time, 100 μL of the reaction mixture was diluted with 900 μL of dimethylacetamide and centrifuged. The supernatant was analyzed using the corresponding HPLC-UV method.

[0103] Substrate and product: Using the ketone substrate 1-(6-(4-fluoro-1H-pyrazole-1-yl)pyridine-3-yl)ethane-1-one of formula X, The obtained chiral primary amine is (S)-1-(6-(4-fluoro-1H-pyrazole-1-yl)pyridine-3-yl)ethane-1-amine of formula XI. TIFF2026528779000007.tif42170

[0104] Chiral HPLC analysis of aqueous reactions (4-40% DMSO): Samples from reactants in aqueous medium were measured by HPLC-UV at 269 nm on an Agilent 1290 HPLC instrument coupled to a DAD and equipped with a Daicel CrownPak CR-I(+) chiral column (3 mm, 150 mm, 5 μm). Flow rate and column temperature were adjusted to 0.8 mL / min and 30 °C. The injection volume was 1 μL. Mobile phase A consisted of 95% [v / v] Millipore water + 5% [v / v] Acetonitrile + 0.25% [v / v] Trifluoroacetic acid (TFA), and mobile phase B consisted of Acetonitrile + 0.25% [v / v] TFA. The method was isocratic: 0-4.5 min, 30% B. Conversion rates were calculated from product formation using the calibration curve of XI and the initial concentration of X. Accordingly, the increment multiplier relative to the wild-type (FIOWT) or parent (FIOP) value was calculated as the peak area of ​​XI (variant) / the peak area of ​​XI (wild-type or parent).

[0105] Chiral HPLC analysis of organic reactions: Samples from reactants in organic media were measured using an HPLC-UV system and a column with the same characteristics as above, but with the following modifications: flow rate and column temperature were adjusted to 1 mL / min and 25°C. Injection volume was 1–5 μL. Mobile phase A consisted of 0.31% (v / v) perchloric acid in Millipore water, and mobile phase B consisted of acetonitrile. The method was isocratic: 0–10 minutes, 20% B. Conversion rates were calculated from the relative areas of X and XI.

[0106] Accelerated achiral UHPLC-UV analysis of aqueous reactions (4-40% DMSO): Samples from reactions in aqueous media were analyzed on an Agilent 1290 HPLC-DAD / MS instrument equipped with InfinityLab Poroshell 120 EC-C18 guard columns (2.1 mm, 5 mm, 1.9 μm). The guard columns were assembled as standard analytical columns for DAD analysis without MS detection. Flow rate and column temperature were adjusted to 1 mL / min and 30°C. The injection volume was 1 μL. Mobile phase A consisted of 95% [v / v] Millipore water + 5% [v / v] Acetonitrile + 0.25% [v / v] TFA, and mobile phase B consisted of Acetonitrile + 0.25% [v / v] TFA. The following gradients were applied: 0–0.1 min, 0% B; 0.1–0.25 min, 100% B; 0.25–0.34 min, 100% B, 0.34–0.35 min, 0% B. The enantioselectivity of the engineered enzyme hit was continuously verified using the chiral HPLC-UV method, as previously described and reported as the enantiomeric excess (ee) of product X. Conversion rates, product titers, and FIOWT / FIOP values ​​were also calculated as previously described.

[0107] result: A series of promising mutation sites were identified during seven rounds of transaminase optimization. For further analysis, top variants were selected, primarily based on performance after 20 hours. The following 12 enzyme variants, as well as the wild-type transaminase of SEQ ID NO: 1, were further investigated: -M61L_W65M_I266V_R419V -W65G_R419V_I426V -M61L_W65M_I266V_R419P -M61L_W65G_R419V_I426V -M61L_W65F_I266V_D349Y_R419P_I426V -M61L_W65M_I266V_R419V_I426V -W65F_R419P_I426V -W65C_R419P_I426V -W65F_R419P -W65M_I266V_R419P_I426V -W65M_R419V_I426V -M61L_W65F_I266V_R419P_I426V_K463T

[0108] In the above variants, the numbers indicate the amino acid positions in SEQ ID NO: 1. The letter before the number specifies the amino acid of the wild-type transaminase in SEQ ID NO: 1, and the letter after the number indicates the amino acid used for the substitution of the wild-type amino acid.

[0109] Two of the best enzymes were selected as parent enzymes. Among the R419V-containing variants, M61L_W65M_I266V_R419V_I426V (LMVVV, SEQ ID NO: 3) yielded the highest conversion level at 30% DMSO after 20 hours (relative to biomass concentration). It was also the fastest and one of the most tolerant to 40% DMSO. Among the R419P-containing variants, M61L_W65M_I266V_R419P (LMVPI, SEQ ID NO: 2) yielded the highest conversion level at 30% DMSO after 20 hours (relative to biomass concentration).

[0110] The following shows the results of experiments using various transaminase variants with one or more substitutions: [Table 1] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. ee(XI) > 99.5%. [Table 2] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, 17 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 3] [Table 4] The aqueous reaction mixture contained 0.2% [w / v]X, 34% [v / v] lysate, 1M isopropylamine hydrochloride, and 4% [v / v] DMSO, and was incubated at 50°C for 2 hours. ee(XI) > 99.5%. [Table 5] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, resuspended in 200 μL of buffer, 50 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 6] The aqueous reaction mixture contained 0.2% [w / v]X, 34% [v / v] lysate, 1M isopropylamine hydrochloride, and 4% [v / v] DMSO, and was incubated at 50°C for 2 hours. In all cases, ee(XI) > 99.5%. [Table 7] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, resuspended in 200 μL of buffer, 50 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 8] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 9] The organic reaction mixture contained 1% [w / v]X, lyophilized cells (approximately 17 mg of CWW derived from 0.5 mL of culture in a deep-well plate), water-saturated isopropyl acetate, and 5 equivalents of isopropylamine free base, and was incubated at 60°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 10] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 11] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, 17 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 12] The aqueous reaction mixture contained 0.2% [w / v]X, 34% [v / v] lysate, 1M isopropylamine hydrochloride, and 4% [v / v] DMSO, and was incubated at 50°C for 2 hours. In all cases, ee(XI) > 99.5%. [Table 13] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, resuspended in 200 μL of buffer, 50 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 14] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 15] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 16] The organic reaction mixture contained 1% [w / v]X, lyophilized cells (approximately 17 mg of CWW derived from 0.5 mL of culture in a deep-well plate), water-saturated isopropyl acetate, and 5 equivalents of isopropylamine free base, and was incubated at 60°C for 20 hours. In all cases, ee(XI) > 99.5%. [Table 17] The aqueous reaction mixture contained 0.2% [w / v]X, 34% [v / v] lysate, 1M isopropylamine hydrochloride, and 4% [v / v] DMSO, and was incubated at 50°C for 2 hours. In all cases, ee(XI) > 99.5%. [Table 18] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (cells manually cultured in 0.5 mL, OD600 approximately 10, resuspended in 200 μL of buffer, 50 μL of which was used), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%.

[0111] Variants with a mutation at position 419 and other mutations at positions 61, 65, 266, and 426 (combinatorial site mutagenesis): See Tables 2, 3, 9, and 16. [Table 19] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. In all cases, ee(XI) > 99.5%.

[0112] Variants with a mutation at position 420 and at least one further mutation (combinatorial site mutagenesis): See Tables 2, 3, 9, and 16. [Table 20] The aqueous reaction mixture contained 1% [w / v]X, single freeze-thawed cells (robot-cultured cells resuspended in 50 μL of buffer, and 40 μL of the same), 2 M isopropylamine hydrochloride, and 30% [v / v] DMSO, and was incubated at 50°C for 20 hours. ee(XI) > 99.5%.

[0113] Variants with a mutation at position 421 and at least one further mutation (combinatorial site mutagenesis): See Tables 2, 3, 9, and 16. [Table 21] The aqueous reaction mixture contained 0.2% [w / v]X, 34% [v / v] lysate, 1M isopropylamine hydrochloride, and 4% [v / v] DMSO, and was incubated at 50°C for 2 hours. In all cases, ee(XI) > 99.5%.

[0114] Variants with a mutation at position 426 and at least one further mutation (combinatorial site mutagenesis): See Tables 5, 7, 9, 11, and 16.

[0115] Variants with a mutation at position 464 and at least one further mutation (combinatorial site mutagenesis): See Table 11.

[0116] Example 3: Aqueous and organic reactions using substrate X 3.1 1 ml MARS / 1% [w / w] substrate loading: The reaction mixture contained X (10 mg, 48.7 μmol, 1 equivalent), solvent (1 ml), isopropylamine (20 μl, 0.2 mmol, 5 equivalents), potassium phosphate buffer (30 μl, 1 M pH 7.2, containing 5 M isopropylamine hydrochloride and 2 mM PLP), and lyophilized whole-cell mutant transaminase containing the mutant transaminase of SEQ ID NO: 3. The mixture was heated to 50°C and shaken for a specified time (≧1 d). The formation of the achieved product was determined by HPLC in area percentage (a%). See Table 22 for the results. [Table 22] The reaction mixture contained X (5 mg, 24.4 μmol, 1 equivalent), isopropyl acetate (0.5 ml containing 2.0% H2O), isopropylamine (10 μl, 0.2 mmol, 5 equivalents), and lyophilized whole-cell mutant transaminases (SEQ ID NOs: 1-12). The mixture was heated to 60°C and shaken for a specified time. The formation of the achieved product (XI) was determined by HPLC in area percentage (a%). See Table 23 for the results. [Table 23]

[0117] 3.2 1 ml MARS / 5% [w / w] substrate loading The reaction mixture contained X (50 mg, 48.7 μmol, 1 equivalent), isopropyl acetate (1 ml, 2.0% water-saturated [w / w], as specified in Table 24), isopropylamine (100 μl, 1.2 mmol, 5 equivalents), and lyophilized whole cells containing mutant transaminase (25 mg, s / e 2). The mixture was heated to 60°C (as specified in Table 25) and shaken for the indicated time. The formation of the achieved product (XI) was determined by HPLC in area percentage (a%). [Table 24] [Table 25]

[0118] 3.3 50 mL MARS / 10% [w / w] substrate loading The reaction mixture contained X (5 g, 24.4 mmol, 1 equivalent), isopropyl acetate (30.6 g; 35 ml) containing 2.0% [w / w] water, and lyophilized Escherichia coli (E. coli) cells (2.5 g) containing the mutant transaminase of Sequence ID No. 8. After stirring the mixture for 5 minutes, the reaction was initiated by adding isopropylamine (6.9 g, 10 ml, 4.8 equivalents), and the mixture was heated to 60°C and stirred for 24 hours. The formation of the achieved product (XI) was determined to be 76.4a% by HPLC.

[0119] Subsequently, the biocatalyst (lyophilized whole cells) was removed by filtration and washed with isopropyl acetate (20 ml). The combined filtrate was evaporated to 20 g to remove the remaining isopropylamine, water, and by-product acetone. The concentrated product XI solution was diluted with isopropyl acetate (30 ml), evaporated again to 22 g, and then diluted again with isopropyl acetate (30 ml).

[0120] Product XI solution was heated to 60°C, and the product was precipitated as an HCl salt by adding a 5.8 M HCl solution of isopropyl acetate (3.3 ml, 18.9 mmol, 0.8 equivalents). The solution was aged for a further 16 hours at 60°C with stirring. The crystals were repeatedly isolated by filtration, washed with isopropyl acetate, and digested first in acetone (50 ml) at 50°C for 2 hours, followed by digestion in acetone (50 ml) at 23°C for 16 hours, and finally dried using high vacuum.

[0121] 3.7 g (68.2%) of the HCl salt of the off-white powder product XI.HCl was isolated using HPLC with a purity of 99.6a% XI.HCl (0.4a% substrate X) and NMR with a purity of >95% (≤0.5% [w / w] isopropylamine, containing trace amounts of acetone). The mass was confirmed by LC-MS.

[0122] 3.4 1L scale reaction / 5% [w / w] substrate loading Subsequently, the reaction mixture was prepared by adding X (50 g, 243.7 mmol, 1 equivalent), isopropyl acetate (500 g, 574.7 ml), isopropylamine (72 g, 104.7 ml, 1218.1 mmol, 5 equivalents), and water (21.7 g, 2.6% [w / w]). The mixture was heated to 45°C with stirring before initiating the reaction by adding a suspension of spray-dried Escherichia coli (E. coli) cells (16.6 g) containing the mutant transaminase of Sequence ID No. 8 in isopropyl acetate (225 g, 258.5 ml). The reaction temperature was heated to 60°C and stirred for a further 19 hours. The formation of the achieved product was 84.5a% (determined by HPLC).

[0123] Subsequently, the biocatalyst (spray-dried whole cells) was removed by filtration and washed with isopropyl acetate (200 ml, 45°C). The combined filtrate was evaporated to 334 g to remove the remaining isopropylamine, water, and by-product acetone. The concentrated product XI solution was diluted with isopropyl acetate (800 ml).

[0124] The product XI solution was heated to 60°C, and the product was precipitated as an HCl salt by adding a 5.8 M HCl solution of isopropyl acetate (42.0 ml, 243.7 mmol, approximately 1.2 equivalents). The solution was aged at 60°C for a further 16 hours with stirring. The crystals were repeatedly isolated by filtration, washed with isopropyl acetate (400 ml) and acetone (250 ml), and then dried using high vacuum.

[0125] 50.4 g (83.8%) of the HCl salt of the off-white powder product XI.HCl was isolated using HPLC with a purity of 98.3a% (1.0a% substrate X) and NMR with a purity of >95% (≤1.0% [w / w] isopropylamine, containing trace amounts of acetone). The mass was confirmed by LC-MS.

[0126] 3.5 2L scale reaction / 5% [w / w] substrate loading / s / e3 Subsequently, the reaction mixture was prepared by adding X (90.0 g, 192.5 mmol, 1 equivalent), isopropyl acetate (875 g, 1006 ml), isopropylamine (129.6 g, 5 equivalents), and water (31.1 g, 2.0% [w / w]). The mixture was stirred at 22°C before initiating the reaction by adding a suspension of spray-dried Escherichia coli (E. coli) cells (30.0 g) and PLP (0.6 g) containing the mutant transaminase of SEQ ID NO: 8 (02219422AN0826) in isopropyl acetate (386 g, 444 ml). The reaction temperature was heated to 60°C and stirred for a further 8 hours. The formation of the achieved product was approximately 80a% (determined by HPLC). The reaction volume was halved by distillation off the by-product acetone. The reaction mixture was supplemented with isopropyl acetate (652 g, 750 ml), isopropylamine (129.6 g, 5 equivalents), and water (30 g), and the reaction was completed for another 16 hours, achieving 91.5% product formation.

[0127] Next, 15g of Dicalite was added and stirred for 45 minutes, after which the biocatalyst was filtered (spray-dried whole cells). The biocatalyst was washed with isopropyl acetate (345ml) at 45°C. The combined filtrate was concentrated to 180ml by evaporation (2V, removal of remaining isopropylamine, water, and acetone).

[0128] The concentrated product XI solution was diluted with isopropyl acetate (450 ml, 5V) and water (450 ml, 5V) and heated to 50°C. The pH of the mixture was adjusted to less than 1 by adding aqueous HCl (47.5 g, 0.95 equivalents). The mixture was heated to 65°C to separate the phases. The organic phase was extracted again with water (180 ml, 2V). The aqueous solutions were combined, and the solvent was changed to isopropanol by azeotropic removal of water while maintaining a constant volume (450 ml, 5V). The temperature was reduced to 0°C to completely crystallize product XI. After filtering out product XI, it was washed with isopropanol (180 ml, 2V) at 0°C and then dried using high vacuum.

[0129] 94.7 g (89.0%) of the HCl salt was isolated as the off-white crystalline product XI.HCl by HPLC purity of 99.9a% XI.HCl (0.05a% substrate X, ≤0.1% isopropanol).

[0130] 3.6 0.8L scale reaction / 5% [w / w] substrate loading / s / e4 Subsequently, the reaction mixture was prepared by adding X (39.5 g, 192.5 mmol, 1 equivalent), isopropyl acetate (385 g, 442 ml), isopropylamine (56.7 g, 5 equivalents), and water (13.6 g, 2.0% [w / w]). The mixture was stirred at 22°C before initiating the reaction by adding a suspension of spray-dried Escherichia coli (E. coli) cells (10.0 g) containing the mutant transaminase of SEQ ID NO: 8 in isopropyl acetate (170 g, 195 ml). The reaction temperature was heated to 60°C and stirred for a further 8 hours. The formation of the achieved product was approximately 80a% (determined by HPLC). The reaction volume was halved by distillation off the by-product acetone. The reaction mixture was supplemented with isopropyl acetate (278 g, 320 ml), isopropylamine (56.7 g, 5 equivalents), and water (13.1 g), and the reaction was completed for another 16 hours, achieving 91.5% product formation.

[0131] Next, 5g of Dicalite was added and stirred for 45 minutes, after which the biocatalyst was filtered (spray-dried whole cells). The biocatalyst was washed with isopropyl acetate (150 ml, 45°C). The combined filtrate was concentrated to 80 ml by evaporation (2V, removal of remaining isopropylamine, water, and acetone).

[0132] The concentrated product XI solution was diluted with isopropyl acetate (200 ml, 5V) and water (200 ml, 5V) and heated to 50°C. The pH of the mixture was adjusted to less than 1 by adding aqueous HCl (23 g, 1.05 equivalents). The mixture was heated to 65°C to separate the phases. The organic phase was extracted again with water (80 ml, 2V). The aqueous solutions were combined, and the solvent was changed to isopropanol by azeotropic removal of water while maintaining a constant volume (200 ml, 5V). The temperature was reduced to 0°C to completely crystallize product XI. After filtering out product XI, it was washed with isopropanol (80 ml) at 0°C and then dried under high vacuum.

[0133] 38.3 g (82.0%) of the HCl salt was isolated as the off-white crystalline product XI.HCl by HPLC purity of 99.8a% XI.HCl (0.1a% substrate X, ≤0.1% [w / w] isopropylamine, water and / or isopropanol).

[0134] Example 4: Aqueous and organic reactions with other substrates 4.1 Selection of commercially available ketones TIFF2026528779000033.tif150170

[0135] 4.2 Small-scale general enzymatic amine synthesis with 1% substrate loading The reaction mixture contained ketones (5 mg, 1 equivalent, Ia-I-n and X), isopropyl acetate (0.5 ml containing 2.0% H2O), isopropylamine (10 μl, 0.2 mmol, 5 equivalents), and lyophilized mutant transaminase (SEQ ID NO: 8) (1.67 mg; s / e3). The mixture was heated to 60°C and shaken for 24 hours. The formation of the achieved product II and its enantiomer excess were determined in area percentage (a%) and percentage (%) by corresponding chromatographic methods. [Table 26]

[0136] array Sequence ID 1 Length: 466 Type: Protein Organism: Ruegeria pomeroyi Other information: Wild type MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDAMAGLWCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVICGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMRHVGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0137] Sequence ID 2 Length: 466 Type: Protein Biological: Artificial Other information: E00023 Mutation: Leu61_Met65_Val266_Pro419 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPHVGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0138] Sequence ID 3 Length: 466 Type: Protein Biological: Artificial Other information: E00026 Mutation: Leu61_Met65_Val266_Val419_Val426 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMVHVGDRMVISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0139] Sequence ID 4 Length: 466 Type: Protein Biological: Artificial Other information: E00040 Mutation: Leu61_Ser62_Met65_Phe171_Val266_Lys318_Pro419_Asn420_Ser421 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0140] Sequence ID 5 Length: 466 Type: Protein Biological: Artificial Other information: E00041 Mutation: Leu61_Ser62_Met65_Phe171_Val266_Lys318_Met320_Pro419_Asn420_Ser421 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFMHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0141] Sequence ID 6 Length: 466 Type: Protein Biological: Artificial Other information: E00046 Mutations: Leu61_Met65_Phe171_Val266_Cys320_Val419_Asn420_Ser421_Val426 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFCHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMVNSGDRMVISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0142] Sequence ID 7 Length: 466 Type: Protein Biological: Artificial Other information: E00058 Mutation: Tyr9_Leu61_Ser62_Met65_Phe171_Val266_Lys318_Pro419_Asn420_Ser421 MSLATITNYMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0143] Sequence ID 8 Length: 466 Type: Protein Biological: Artificial Other information: E00059 Mutation: Tyr9_Leu61_Ser62_Met65_Phe171_Val266_Lys318_Met320_Pro419_Asn420_Ser421 MSLATITNYMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFMHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0144] Sequence ID 9 Length: 466 Type: Protein Biological: Artificial Other information: E00060 Mutation: Tyr9_Leu61_Met65_Phe171_Val266_Cys320_Val419_Asn420_Ser421_Val426 MSLATITNYMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFCHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMVNSGDRMVISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSAA

[0145] Sequence ID 10 Length: 466 Type: Protein Biological: Artificial Other information: E00061 Mutation: Leu61_Ser62_Met65_Phe171_Asp198_Val266_Lys318_Pro419_Asn420_Ser421_Met464 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGLMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFNHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKMAA

[0146] Sequence ID 11 Length: 466 Type: Protein Biological: Artificial Other information: E00062 Mutations: Leu61_Ser62_Met65_Phe171_Asp198_Val266_Lys318_Met320_Pro419_Asn420_Ser421_Met464 MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALSGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDL NHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHINQPNWWAEGGLMDPEEFGLARARELEEAILELGENRVAAFIAEPVQ GAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDKFMHGYTYSGHPVAAAVALENLRILEEENILD HVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMPNSGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKMAA

[0147] Sequence ID 12 Length: 466 Type: Protein Biological: Artificial Other information: E00063 Mutation: Leu61_Met65_Phe171_Asp198_Val266_Cys320_Val419_Asn420_Ser421_Val426_Met464 MSLATITNHMPTAEALQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDALAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTHVPAIALAQKLAELAPGDLNHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGFAGMHAQSGLIPDVHHINQPNWWAEGGLMDPEEFGLARARELEEAILELGENRVAAFIAEPVQGAGGVIVAPDSYWPEIQRICDKYDILLIADEVVCGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFCHGYTYSGHPVAAVALENLRILEEENILDHVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMVNSGDRMVISPPLVITPAEIDEMFVRIRKSLDEAQAEIEEKQGLMKMAA

Claims

1. A mutant transaminase in which asymmetric reductive amination activity is increased compared to wild-type transaminase, The mutant transaminase contains an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 1 (transaminase derived from Ruegeria pomeroyi), The mutant transaminase has at least four amino acid substitutions at positions 61, 65, 266, and 419 of the amino acid sequence of SEQ ID NO: 1, Of these substitutions, at least three substitutions are - The amino acid at position 61 of sequence number 1 is substituted with Leu (Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met (Met65). - The amino acid at position 266 of SEQ ID NO: 1 is substituted with Val(Val266), and - The amino acid at position 419 of SEQ ID NO: 1 is substituted with Pro (Pro419) or Val (Val419). A mutant transaminase selected from the group consisting of the following.

2. - The amino acid at position 61 of sequence number 1 is substituted with Leu (Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met (Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), - The amino acid at position 419 of sequence number 1 is substituted with Pro (Pro419) or Val (Val419). The mutant transaminase according to claim 1.

3. - The amino acid at position 426 of the sequence number is substituted with Val (Val426). The mutant transaminase according to claim 1 or 2.

4. - The amino acid at position 61 of sequence number 1 is substituted with Leu (Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met (Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), - The amino acid at position 419 of SEQ ID NO: 1 is substituted with Pro(Pro419), or - The amino acid at position 61 of sequence number 1 is substituted with Leu (Leu61). - The amino acid at position 65 of sequence number 1 is substituted with Met (Met65). - The amino acid at position 266 of sequence number 1 is substituted with Val(Val266), - The amino acid at position 419 of sequence number 1 is substituted with Val (Val419). - The amino acid at position 426 of sequence number 1 is substituted with Val(Val426). A mutant transaminase according to any one of claims 1 to 3.

5. - The amino acid at the position corresponding to position 9 of sequence number 1 is substituted with Tyr (Tyr9), and / or - The amino acid at position 62 of sequence number 1 is substituted with Ser (Ser62), and / or - The amino acid at position 65 of sequence number 1 is substituted with Phe(Phe65), Gly(Gly65), or Met(Met65), and / or, - The amino acid at position 171 of sequence number 1 is substituted with Phe (Phe171) or Trp (Trp171), and / or - The amino acid at position 198 of Sequence ID No. 1 is substituted with Leu(Leu198) or Met(Met198), particularly Leu(Leu198), and / or - The amino acid at position 318 of sequence number 1 is substituted with Lys(Lys318), and / or - The amino acid at position 320 of Sequence ID No. 1 is substituted with Cys (Cys320), Leu (Leu318), Lys (Lys320), or Met (Met320), and / or, - The amino acid at position 419 of SEQ ID NO: 1 is substituted with Ala (Ala419), Cys (Cys419), Gly (Gly419), Pro (Pro419), or Val (Val419), and / or, - The amino acid at position 420 of Sequence ID No. 1 is substituted with Asn (Asn420), Asp (Asp420), Cys (Cys420), or Ser (Ser420), and / or - The amino acid at position 421 of sequence number 1 is substituted with Ser (Ser421), and / or - The amino acid at position 464 of the sequence number is substituted with Met (Met464). A mutant transaminase according to any one of claims 1 to 4.

6. - The mutant transaminase has substitutions Leu61, Met65, Val266 and Pro419, or - The mutant transaminase has substitutions Leu61, Met65, Val266, Val419 and Val426, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - The mutant transaminase has the substituted Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions of Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions of Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426 and Met464, A mutant transaminase according to any one of claims 1 to 5.

7. - The mutant transaminase has substitutions Leu61, Met65, Val266 and Pro419, and the amino acids at positions 9, 62, 171, 198, 318, 320, 420, 421, 426 and 464 of SEQ ID NO: 1 are unsubstituted. - The mutant transaminase has substitutions Leu61, Met65, Val266, Val419 and Val426, and the amino acids at positions 9, 62, 171, 198, 318, 320, 420, 421 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase, It has substituted Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, and the amino acids at positions 9, 198, 320, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, and the amino acids at positions 9, 198, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, and the amino acids at positions 9, 62, 198, 318 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substituted Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, and the amino acids at positions 198, 320, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substituted Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, and the amino acids at positions 198, 426 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions of Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, and the amino acids at positions 62, 198, 318 and 464 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, and the amino acids at positions 9, 320 and 426 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, and the amino acids at positions 9 and 426 of SEQ ID NO: 1 are unsubstituted, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426 and Met464, and the amino acids at positions 9, 62 and 318 of SEQ ID NO: 1 are unsubstituted. A mutant transaminase according to any one of claims 1 to 6.

8. - The mutant transaminase has substitutions Leu61, Met65, Val266 and Pro419, or - The mutant transaminase has substitutions Leu61, Met65, Val266, Val419 and Val426, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - The mutant transaminase has the substituted Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions of Tyr9, Leu61, Ser62, Met65, Phe171, Val266, Lys318, Met320, Pro419, Asn420 and Ser421, or - The mutant transaminase has substitutions of Tyr9, Leu61, Met65, Phe171, Val266, Cys320, Val419, Asn420, Ser421 and Val426, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Pro419, Asn420, Ser421 and Met464, or - The mutant transaminase has substitutions Leu61, Ser62, Met65, Phe171, Asp198, Val266, Lys318, Met320, Pro419, Asn420, Ser421 and Met464, or - The mutant transaminase has substitutions Leu61, Met65, Phe171, Asp198, Val266, Cys320, Val419, Asn420, Ser421, Val426 and Met464, The substitution is the only substitution in the mutant transaminase relative to the amino acid sequence of SEQ ID NO:

1. A mutant transaminase according to any one of claims 1 to 7.

9. A mutant transaminase according to any one of claims 1 to 8, comprising or containing an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, particularly 100%, identical to any of the amino acid sequences of SEQ ID NOs: 2 to 12.

10. - Having increased reductive amination activity against wild-type transaminase in the presence of organic solvents and amine donors, and / or - It has increased stability in aqueous organic media, particularly in aqueous media containing 4.0% v / v to 40.0% v / v organic solvents, and / or - Has increased stability in the presence of organic solvents and residual water, and / or - Reductive amination from ketones to primary amines, particularly from prochiral ketones to chiral primary amines, is possible, and / or It exhibits reductive amination activity at temperatures from -40°C to 70°C, particularly from 50°C to 70°C. A mutant transaminase according to any one of claims 1 to 9.

11. Reductive amination from ketones to primary amines is possible, and the ketone is of formula I (In the formula, R １ and R ２ These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R １ and R ２ These have (which, together with the carbon atoms to which they are bonded, form a monocyclic or polycyclic carbocyclic or heterocyclic carbocyclic ring, which may be substituted), The mutant transaminase according to any one of claims 1 to 10, wherein the optionally substituted substituent is selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano.

12. Convert the ketone of formula I to the primary amine of formula II. (In the formula, R １ and R ２ These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R １ and R ２ They have the ability to be converted into monocyclic or polycyclic carbocyclic or heterocyclic rings (which may be substituted) together with the carbon atoms to which they are bonded. The mutant transaminase according to any one of claims 1 to 11, wherein the optionally substituted substituent is selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano.

13. A nucleic acid encoding a mutant transaminase according to any one of claims 1 to 12, optionally included in the vector.

14. A cell comprising a mutant transaminase according to any one of claims 1 to 12 and / or the nucleic acid according to claim 13.

15. A method for enzymatic reductive transamination of a ketone and formation of a primary amine in the presence of a mutant transaminase according to any one of claims 1 to 12.

16. The method according to claim 15, wherein the ketone is a prochiral ketone and the primary amine is a chiral primary amine.

17. The aforementioned ketone, Formula I (In the formula, R １ and R ２ each independently represents an optionally substituted alkyl, aryl, carbocyclic or heterocyclic group, or R １ and R ２ These have (which, together with the carbon atoms to which they are bonded, form a monocyclic or polycyclic carbocyclic or heterocyclic carbocyclic ring, which may be substituted), The substituents that may be substituted are selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano. The resulting primary amine is, Formula II (In the formula, R １ and R ２ These independently represent an alkyl, aryl, carbocykryl, or heterocycline which may be substituted, or R １ and R ２ These have (which, together with the carbon atoms to which they are bonded, form a monocyclic or polycyclic carbocyclic or heterocyclic carbocyclic ring, which may be substituted), The method according to claim 15 or 16, wherein the optionally substituted substituent is selected from alkyl, alkoxy, aryl, heteroaryl, aryloxy, halogen, hydroxyl, or cyano.

18. The method according to any one of claims 15 to 17, wherein the enzymatic reductive amino group transfer is carried out in the presence of an organic solvent containing 1% to 5% by weight of water.

19. The method according to claim 18, wherein the organic solvent is a lower alkyl ester of acetic acid or propionic acid.

20. The method according to any one of claims 15 to 19, wherein the enzymatic reductive amino group transfer is carried out in the presence of an amine donor.

21. The method according to claim 20, wherein the amine donor is a primary aliphatic amine.

22. The method according to claim 20 or 21, wherein the amine donor is used in an amount of 2 to 20 equivalents.

23. The method according to any one of claims 15 to 22, wherein the mutant transaminase is used as an enzyme-immobilized product or as all Escherichia coli (E. coli) cells.

24. The method according to any one of claims 15 to 23, wherein the enzymatic reductive amino group transfer is carried out at 40°C to 70°C.

25. The method according to any one of claims 15 to 24, wherein the ketone of formula I is loaded in an amount of 1% to 15% by weight.

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