HK1246343A1 - Mutant transaminases as well as methods and uses relating thereto - Google Patents

Description

Mutant transaminases and methods and uses related thereto

Technical Field

The present invention relates to mutant transaminases having increased transaminase activity relative to a wild-type transaminase, fusion proteins comprising the transaminase, polynucleotides encoding the transaminase, host cells comprising the polynucleotides, mutant transaminases and/or fusion proteins, a process for producing amines using the mutant transaminase or fusion protein, and the use of the mutant transaminase or fusion protein for producing amines.

Background

Transaminases (also known as aminotransferases) catalyze transamination reactions, i.e. the transfer of an amino group from an amine donor to an amine acceptor, in particular the amination of ketones and the deamination of amines, in which NH is present on one molecule or domain₂The group is exchanged with an ═ O group on another molecule or domain (see scheme 1).

Amine compounds such as chiral amines are important building blocks for the pharmaceutical, agrochemical or chemical industry. In many of these applications, it is very important to use only one optically pure form. Chemical syntheses have been established for producing such compounds (e.g., asymmetric hydrogenation using transition metals), but these chemical methods are far from perfect in terms of yield, purity, and waste generation. Transaminase-catalyzed amine production is often an advantageous alternative to classical processes, and therefore alternative or improved transaminases are of interest in this field.

The production of chiral amines by enzyme-mediated transamination is advantageous compared to established chemically asymmetric syntheses, as is well demonstrated in the production of the drug sitagliptin (Savile et al, 2010; US 2015/0037869) using an engineered (R) selective transaminase. In this case, the amine aminotransferase (ATA), which was initially inactive towards the desired substrate, was engineered in its entirety by directed evolution. Further mutations of transaminases are described in the art (Steffen-Munsberg et al, 2013; Nobili et al, 2015Deszcz et al, 2015). Other enzymes may also be used to produce optically pure chiral amines and amino acids, such as monoamine oxidases, imine reductases, amino acid dehydrogenases, ammonialyase enzymes or aminomutases. Although transaminases can be used for the kinetic resolution of racemic amines, asymmetric synthesis from keto acids, ketones or aldehydes is of great importance, since they can potentially lead to a conversion of theoretically 100%, while the kinetic resolution mode gives only 50% of the theoretical yield (see scheme 1, schematic synthesis and kinetic resolution).

Scheme 1:

residue R^a、R^b、R^cAnd R^dFor illustrative purposes only, and should represent general organic residues.

However, directed evolution methods do not allow us to gain insight into molecular causes (e.g., acceptance with respect to larger volumes of substrate). The high enantioselectivity of transaminases has driven the theory of large and small binding pockets, however, enlarging small binding pockets to accommodate large volumes of substrate is a goal that is not easily achieved, as evidenced by the following facts: the most successful examples found in the literature to date are transaminases for converting methyl ketones.

It is an object of the present invention to design a mutant transaminase having an increased transaminase activity relative to the wild-type transaminase. Preferably, they should be able to accept a wide range of substrates, especially bulky substrates, especially while maintaining stereoselectivity, and the use of isopropylamine as donor. Thus, increased activity is particularly relevant for bulky substrates (one exemplary option is described in schemes 2 and 3, see below) as well as the amine donor isopropylamine. These stereoselective transaminases are useful for asymmetric synthesis of chiral amines from the corresponding ketones and for kinetic resolution.

Summary of The Invention

Surprisingly, it has been found that a mutant transaminase comprising an amino acid sequence identical to that of SEQ ID NO: 1 (Ruegeriasp) TM1040 transaminase; referred to as 3FCR) is at least 65% identical and has at least two amino acid substitutions relative to the wild-type transaminase, wherein the amino acid sequence corresponds to SEQ ID NO: 1 by Trp or Phe (Trp 59 or Phe59, respectively), and an amino acid at a position corresponding to position 59 of SEQ ID NO: 1 by Ala or Gly (Ala 231 or Gly231, respectively).

As shown in the examples, several amino acid residues in a transaminase have been identified, the substitutions of which increase the transaminase activity of the mutant transaminase relative to the corresponding wild-type transaminase. In particular, it could be shown that the double mutant of 3FCR (i.e., 3FCR (named Y59W/T231A) in which Tyr is substituted with Trp at position 59(Y59W) and Thr is substituted with Ala at position 231(T231A) of SEQ ID NO: 1) has increased activity on the substrate accepted by the wild type (amines 3a and 4a shown in scheme 2), and low activity was also measured for amine 1a(see tables 3 and 4). The results obtained with the transaminase 3FCR mutant can be confirmed by a transaminase (abbreviated as 3GJU) mutant of the intermediate rhizobium kawachii maff303099(Mesorhizobium loti maff303099) (see table 3). Furthermore, in SEQ ID NO: 1-3 FCR mutants with additional Phe substitution of Tyr in position 87 and/or Phe substitution of Tyr in position 152 (named Y87F and Y152F) with two pairsSubstrates with multiple aromatic rings (i.e., amines 1a, 3a and 4a) have increased activity (see Y59W/Y87F/T231A and Y59W/Y87F/Y152F/T231A in Table 4). At the same time, mutation Y152F significantly increased the stability of the mutant (FIG. 1). Further substitution of His for the transaminase of 3FCR SEQ ID NO: 1 Pro at position 423, designated P423H, an even further increase in activity and substrate range of acceptance was obtained (see tables 3 and 4). It can be shown that mutants with substitutions Y59W, Y87F, Y152F, T231A and P423H are particularly active on bulky substrates (e.g. amines 1a, 3a and 4 a). Again, the results obtained with the transaminase 3FCR mutant can be confirmed with the 3GJU mutant (see table 3). In addition, mutant transaminases with mutations Y59W/Y87F/Y152F/T231A, ATA-3, ATA-5, ATA-6, ATA-7, ATA-8 and ATA-9, were demonstrated to have activity on amines 1a, 3a, 4a and 6a (see Table 5). Note that the wild type sequences ATA-3 to ATA-9 have from 65-70% to about 90% sequence identity with wild type 3 FCR. Thus, it can be concluded that the concept of the present invention can be applied to all transaminases having a sequence identity of 65% or more with the sequence of 3FCR (SEQ ID NO: 1). In addition, mutants of 3FCR and 3GJU having an aliphatic hydrophobic amino acid such as Leu or Val at position 87 (Y87L or Y87V, respectively) were found to have increased activity and to be particularly effective for accepting tertiary carbon substituents such as amine 2a (see table 6). Mutants with phenylalanine at position 59 (Y59F) and/or glycine at position 231 (T231G) showed increased activity on bicyclic compounds like amine 5a (see table 7). In addition, 3FCR mutants substituted at position 234 with Phe or Met (referred to as I234F and I234M, respectively) have increased activity on amine 5a (see table 7) and the amine donor isopropylamine (see table 8). The mutants were demonstrated in examples 5 to 11 to maintain stereoselectivity and applicability in asymmetric synthesis and kinetic resolution (see tables 9 to 10).

Accordingly, in a first aspect, the present invention provides a mutant transaminase having increased transaminase activity relative to a wild-type transaminase, wherein the mutant transaminase comprises a sequence identical to SEQ ID NO: 1 (lugger TM1040 transaminase; referred to as 3FCR) has an amino acid sequence that is at least 65% identical, wherein the mutant transaminase has at least two amino acid substitutions relative to the wild-type transaminase, wherein the amino acid sequence corresponds to SEQ ID NO: 1 by Trp or Phe (Trp 59 or Phe59, respectively) and corresponds to SEQ ID NO: 1 by Ala or Gly (Ala 231 or Gly231, respectively).

Disclosure of Invention

The term "transaminase" (classified as EC2.6.1.XX by the Enzyme Commission of the International Union of Biochemistry, of the International Union of Biochemistry; also known as aminotransferase) generally refers to an Enzyme that catalyzes the transfer of an amine group from an amine donor to the carbonyl of an amine acceptor (transamination). The transaminase is a pyridoxal 5' -phosphate-dependent (PLP-dependent) enzyme. As shown in scheme 1, an amine donor donates an amino group to an amine acceptor to synthesize the desired amine and form the corresponding ketone. Since transaminases generally have a high stereoselectivity, transamination reactions can provide the desired amine by asymmetric reduction and/or the remaining amine donor by resolution in the form of an enantiomerically enriched amine by means of oxidative deamination. Omega-transaminases (omega-TA) are of particular interest in the present invention because they are capable of converting ketones without adjacent carboxyl functions to chiral amines, unlike the amino acid transaminases known to synthesize alpha-amino acids from alpha-keto acids. Note that the term amine aminotransferase (ATA) is preferred over ω -TA because these enzymes are named according to their ability to synthesize, for example, L-lysine from the corresponding aldehyde. (S) -Selective ATA has been known for over a decade (Coffen et al, 1994). The transaminase of the invention can be non-selective or selective, such as an (S) -selective or (R) -selective, in particular (S) -selective, transaminase. Suitable examples of these transaminases are also given in table 1.

The term "wild-type transaminase" relates to a transaminase which normally occurs in nature. Examples of naturally occurring wild-type transaminases are given in table 1 below, as shown in SEQ ID NO: 1.3 and 5 to 11 (amino acid sequence). If one or more mutations are introduced, a mutant transaminase is obtained. Thus, the term "mutant transaminase" relates to a transaminase whose amino acid sequence differs from the wild-type sequence. With respect to the mutant transaminases of the invention, it is noted that the mutants are functionally active. This means that the mutant retains its biological function, i.e.retains its transaminase enzymatic activity. According to the invention, the transaminase activity of the mutant is increased relative to the transaminase activity of the wild type. The wild-type transaminase corresponding to the mutant is the wild-type transaminase which has the least number of mutation differences from the mutant. Exemplary wild-type transaminases that can be mutated according to the invention are listed below:

aminotransferase (Lujie's TM1040) (GI: 499859271; abbreviated 3 FCR; SEQ ID NO: 1)

Transaminase (Mesorhizobium baimai maff303099) (GI: 499217058; abbreviated as 3 GJU; SEQ ID NO: 3)

Transaminase (Oceanicola grandis) (GI: 494465841; ATA-3; SEQ ID NO: 5)

Transaminase (Jannaschia Sp) CCS1 (GI: 499773242; ATA-4; SEQ ID NO: 6)

Transaminase (Xanthobacteraceae) (GI: 517199618; ATA-5; SEQ ID NO: 7)

Transaminase (Rhodobacteraceae, RB 2150-13166) (GI: 126705951; ATA-6; SEQ ID NO: 8)

Transaminase (Martelella mediterranean) DSM 17316) (GI: 516720233; ATA-7; SEQ ID NO: 9)

Transaminase (Rugeria pomeroyi) DSS-3 (GI: 56677770; ATA-8; SEQ ID NO: 10)

Transaminase (Sagittula stellata) E-37 (GI: 126711082; ATA-9; SEQ ID NO: 11)

A particularly preferred wild-type transaminase in which mutations described herein can be introduced according to the invention is the one of SEQ id no: 1.3 and 5 to 11, in particular SEQ ID NO: 1 and 3, in particular SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

According to the invention, the mutant transaminase has an increased transaminase activity relative to a corresponding wild-type transaminase without mutation. Enzyme activity is a measure of the activity of an enzyme. The SI unit of the enzyme activity is katal (1katal ═ 1mol s^-1). A more practical and frequently used value is the enzyme unit (U) ═ 1. mu. mol min^-1. 1U corresponds to 16.67 nanokatal. One U is defined as the amount of enzyme catalyzing the conversion of 1 micromole of substrate per minute. The conditions under which the activity is determined are generally standardised: temperatures of 25 ℃ or 30 ℃ are generally employed (as in the examples) and the pH and substrate concentration which give the maximum substrate conversion. The specific activity of the enzyme is the enzyme activity per mg of total protein (in. mu. mol min)^-1mg^-1Representation). Which is the amount of product formed by the enzyme per mg of total protein under given conditions and for a given period of time. Specific activity is equal to the reaction rate multiplied by the reaction volume divided by the total protein amount. SI unit is katal kg^-1But the more practical unit is μmol min^-1mg^-1. Specific activity is a measure of the processivity of an enzyme at a particular (usually saturated) substrate concentration and is usually constant for pure enzymes. If the molecular weight of the enzyme is known, the number of conversions of the active enzyme or. mu. mol product sec can be calculated from the specific activity^-1μmol^-1. The number of conversions can be viewed as the number of times each enzyme molecule has performed its catalytic cycle per second.

Activity can be determined in an enzyme assay by measuring the consumption of substrate or the production of product over time. There are a number of different methods of measuring substrate and product concentrations, and as known to those skilled in the art, many enzymes can be assayed in several different ways. In the present invention, the transaminase in question is incubated with a suitable amine donor and a suitable amine acceptor for a period of time and under conditions which are conducive to transamination.

Enzyme assays can be divided into two groups according to their sampling method: continuous assays (where the assay gives a continuous reading of activity) and discontinuous assays (where samples are taken, the reaction is stopped, and then the concentration of substrate/product is determined).

In a preferred embodiment, the transaminase of the invention is stereoselective, such as (S) -selective or (R) -selective, in particular (S) -selective. "stereoselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which the formation of one stereoisomer is preferred over the formation of another, or stereoselectivity can be complete, in which only one stereoisomer is formed. When a stereoisomer is an enantiomer, the stereoselectivity is referred to as enantioselectivity. Enantioselectivity is commonly reported in the art as enantiomeric excess (e.e.) (usually as a percentage) calculated according to the formula [ major enantiomer-minor enantiomer ]/[ major enantiomer + minor enantiomer ]. Alternatively, the enantiomeric ratio or er (S: R) can be used to characterize stereoselectivity. Enantiomeric ratio is the ratio of the percentage of one enantiomer (e.g. (S) -enantiomer) to the other enantiomer (e.g. (R) -enantiomer) in a mixture of enantiomers. When stereoisomers are diastereomers, the stereoselectivity is referred to as diastereoselectivity, the fraction of one diastereomer in a mixture of two diastereomers (diastereomers), usually expressed as a percentage, is instead usually reported as diastereomer excess (d.e.). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

Enantiomeric enrichment in this context means that the enantiomeric ratio of the desired chiral amine relative to the undesired chiral amine is greater than 50:50, preferably at least 70:30, even more preferably at least 95:5, most preferably at least 99.5: 0.5, or similarly an enantiomeric excess of greater than 0%, preferably at least 40%, even more preferably at least 90%, most preferably at least 99%.

It has surprisingly been found that a mutation, as defined in the context of the present invention, when introduced into a wild-type transaminase can increase the transaminase activity of the enzyme relative to the wild-type enzyme. As detailed herein, this is of particular interest for the production of amines, in particular for the production of enantiomerically enriched chiral amines, by the following process:

method of producing a composite material

a) Kinetic resolution of racemic amines in the presence of an amine receptor and a mutant transaminase or fusion protein,

or a method

b) Asymmetric transamination of prochiral ketones in the presence of an amine donor and a mutant transaminase or fusion protein,

wherein the mutant transaminase or fusion protein is stereoselective.

In particular, the activity on selected compounds (e.g. bulky substrates, which broadens the substrate range accepted by the transaminase) and/or the amine donor isopropylamine is increased. This is important in the present invention as it allows for a wider industrial applicability, in particular in transaminase-mediated production of amines (e.g. chiral amines).

It has been found that the transaminase of the present invention shows increased activity, in particular on bulky substrates. An illustrative example of a bulky (racemic) amine according to process a) is shown in scheme 2 below.

Scheme 2：

The chemical names are 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a) and 1-phenylethylamine (6 a).

An illustrative example of a bulky prochiral ketone according to process b) is shown in scheme 3 below.

Scheme 3：

The chemical names are (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylpropan-1-one (3b), 2H-acenaphthylene-1-one (4b), 8-benzoyl-8-azabicyclo [3.2.1] oct-3-one (5b) and 1-phenylethanone (6 b).

Various methods for determining enzymatic activity are well known in the art. Including, but not limited to, spectrometry, fluorimetry, calorimetry, chemiluminescence, assays involving light scattering, radiometry, and chromatography. The assay is typically performed under well-controlled conditions including, for example, pH, temperature, salt, buffer, and substrate concentrations. Suitable assays for studying the transaminase activity of an enzyme are well known to those skilled in the art.Suitable assays are described in Analytical Chemistry (2009) et al. This direct photometric assay is based on the significant difference in the absorption spectra of amines and their corresponding ketones. In general, the absorption spectrum of a conjugated aromatic ketone is unique due to the electron delocalization of its benzoyl moiety, which is not present in the corresponding amine. This difference is reflected in the difference in absorption spectra and the production of ketone can be monitored at the wavelength of the highest absorption difference between ketone and amine, as can be readily determined by one skilled in the art. The assay is particularly applicable to substrates 1,2, 3, 4 and 6[ i.e., amines 1a, 2a, 3a, 4a and 6a (see scheme 2) or ketones 1b, 2b, 3b, 4b and 6b (see scheme 3) ]]For substrates that may be unsuitable for the above assays, such as substrates 5a/b or isopropylamine, suitable assays have been developed to screen for amine donors, the principle of which is described in Wei β et al in Analytical Chemistry (2014)Phenol is oxidized to 1, 4-benzoquinone, which spontaneously reacts with 4-aminoantipyrine by a condensation reaction to form a quinonimine dye detectable at visible wavelengths (498 nm). Phenol can be replaced by vanillic acid, which undergoes oxidative decarboxylation and horseradish peroxidase-mediated oxidation to form 2-methoxy-1, 4-benzoquinone. In addition to these two tests, standard chromatographic methods known to those skilled in the art, such as High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Supercritical Fluid Chromatography (SFC) and capillary electrophoresis may also be used.

In addition, exemplary assays are described in the examples (see section B2), particularly suitable substrates are substrates 1 to 5[ e.g., amines 1a to 5a (see scheme 2) or ketones 1B to 5B (see scheme 3) ] or amine donors isopropylamine (2-propylamine).

Furthermore, the person skilled in the art knows statistical procedures, such as Student's t-test or chi-square test, to assess whether one value increases relative to the other. It will be apparent to those skilled in the art that any background signal must be subtracted when analyzing the data. In a specific embodiment, the increase in enzyme activity is at least about 10%. In other embodiments, the increase in enzyme activity is at least 20%, 30%, 40%, 50% or 100%, in particular 150%, 200%, 250% or 300%. The percentage of increase in activity can be determined as [ activity (mutant)/activity (wild-type) -1] × 100.

According to the invention, the mutant transaminase comprises a sequence identical to SEQ ID NO: 1, amino acid sequence at least 65% identical.

The term "SEQ ID NO: 1 "represents SEQ ID NO: 1, which represents the amino acid sequence of a wild-type aminotransferase from lurgeella TM1040 (abbreviated as 3 FCR). In particular, the term "SEQ id no: 1 "refers to the amino acid sequence shown below: (please note that preferred substitution sites according to the present invention are indicated in bold/underline and indicated by position numbers):

the term "at least 65% identical" or "at least 65% sequence identity" as used herein means that the sequence of the mutant transaminase according to the invention has an amino acid sequence which is characterized as follows: in a 100 amino acid chain, at least 65 amino acid residues are identical to the sequence of the corresponding wild-type sequence. The sequence identity according to the invention can be determined, for example, by means of sequence comparison by means of a sequence alignment. Methods of sequence alignment are well known in the art and include various programs and alignment algorithms that have been described, for example, in Pearson and Lipman (1988). In addition, NCBI Basic Local Alignment Search Tool (BLAST) is available from a variety of sources, including the national center for Biotechnology information (NCBI, Bethesda, Md.) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. the percent identity of mutants according to the invention with respect to the amino acid sequence of SEQ ID NO: 1 is typically characterized by NCBIBLAst blastp with a standard set-up or alternatively, the sequence identity can be determined using software GENEious with a standard set-up 70.7% of ATA-4, 69.6% of ATA-5, 71.8% of ATA-6, 77.9% of ATA-7, 90.8% of ATA-8 and 80.3% of ATA-9.

According to the invention, the mutant transaminase has at least two amino acid substitutions relative to the wild-type transaminase, wherein the amino acid substitution in the amino acid sequence corresponding to SEQ ID NO: 1 by Trp or Phe (Trp 59 or Phe59, respectively) and the amino acid at the position corresponding to position 59 of SEQ ID NO: 1 by Ala or Gly (Ala 231 or Gly231, respectively).

In a preferred embodiment, the mutant transaminase of the invention has one or more additional mutations, namely:

-in a sequence corresponding to SEQ ID NO: 1 by a hydrophobic amino acid (HYaa87), in particular wherein the hydrophobic amino acid is Leu (Leu87) or Val (Val87) or Phe (Phe 87); and/or

-in a sequence corresponding to SEQ ID NO: 1 the amino acid in position 152 is substituted by Phe (Phe152), and/or

-in a sequence corresponding to SEQ ID NO: 1 the amino acid in position 234 is replaced by Phe (Phe234) or Met (M234), and/or

-in a sequence corresponding to SEQ ID NO: 1 by His (His 423).

In the present invention, reference is made to SEQ ID NO: 1 (i.e., the amino acid sequence of lurgeella TM1040 transaminase (3FCR for short)), the location of the mutation was identified. For transaminases other than 3FCR, the corresponding mutation site can be identified by performing an amino acid Alignment as described above (e.g.by using BLAST; Basic Local Alignment SearchTool with the Standard settings can be inhttp://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE TYPE ＝BlastSearch&LINK_LOC＝blasthomeObtained) or identified by comparing structures (if any) and identifying the corresponding amino acid. Examples of corresponding positions are given in fig. 2 to 4 and in the following table:

in one embodiment of the invention, the mutant transaminase according to the invention can comprise one or more amino acid deletions, in particular small (e.g. not more than 10 amino acids) N-and/or C-terminal deletions.

In one embodiment, the sequence of a mutant transaminase according to the invention may comprise one or more further amino acid substitutions, in particular one or more conservative amino acid substitutions, in addition to the substitutions specified herein. "conservative amino acid substitution" refers to a residue substituted with a different residue having a similar side chain, and thus generally involves substituting an amino acid in a polypeptide with an amino acid in the same or similar amino acid type as defined. By way of example, and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); the amino acid having a hydroxyl side chain may be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is substituted with another amino acid having a basic side chain (e.g., lysine and arginine); the amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and the hydrophobic or hydrophilic amino acid is replaced by another hydrophobic or hydrophilic amino acid, respectively. Examples of conservative amino acid substitutions include those listed below:

in one embodiment of the invention, the mutant transaminase according to the invention can comprise one or more amino acid additions, in particular small (for example not more than 10 amino acids) internal amino acid additions.

In another embodiment, the sequence of a mutant transaminase according to the invention may comprise, in addition to the substitutions specified herein, a combination of one or more deletions, substitutions or additions as defined above.

However, in another preferred embodiment, the mutant transaminase of the invention has at least the mutations:

trp59 and Ala231, or

-Trp59, Phe87 and Ala 231; or

-Trp59, Leu87 and Ala 231; or

-Trp59, Val87 and Ala 231; or

-Trp59, Phe87, Ala231 and His 423; or

-Trp59, Phe87, Phe152 and Ala 231; or

-Trp59, Leu87, Phe152 and Ala 231; or

-Trp59, Val87, Phe152 and Ala 231; or

-Trp59, Phe87, Phe152, Ala231 and His423, or

-Trp59 and Gly 231; or

-Trp59, Phe87 and Gly 231; or

-Trp59, Leu87 and Gly 231; or

-Trp59, Val87 and Gly 231; or

-Trp59, Phe87, Phe152 and Gly 231; or

-Trp59, Phe87, Phe152, Gly231 and His 423; or

-Phe59, and Ala 231; or

-Phe59, Phe87, and Gly 231; or

-Trp59, Ala231 and Phe 234; or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Phe 234; or

-Trp59, Leu87, Ala231 and Phe 234; or

-Trp59, Val87, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231, Phe234 and His423, or

-Trp59, Gly231 and Phe 234; or

-Trp59, Phe87, Gly231 and Phe 234; or

-Trp59, Leu87, Gly231 and Phe 234; or

-Trp59, Val87, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231, Phe234 and His 423; or

-Phe59, Phe87, Gly231 and Phe234, or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Met 234; or

-Trp59, Leu87, Ala231 and Met 234; or

-Trp59, Val87, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231, Met234 and His423, or

-Trp59, Gly231 and Met 234; or

-Trp59, Phe87, Gly231 and Met 234; or

-Trp59, Leu87, Gly231 and Met 234; or

-Trp59, Val87, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231, Met234 and His 423; or

-Phe59, Phe87, Gly231 and Met 234.

More preferably, the mutant transaminase of the invention differs from the corresponding wild-type transaminase only in the following mutations:

trp59 and Ala231, or

-Trp59, Phe87 and Ala 231; or

-Trp59, Leu87 and Ala 231; or

-Trp59, Val87 and Ala 231; or

-Trp59, Phe87, Ala231 and His 423; or

-Trp59, Phe87, Phe152 and Ala 231; or

-Trp59, Leu87, Phe152 and Ala 231; or

-Trp59, Val87, Phe152 and Ala 231; or

-Trp59, Phe87, Phe152, Ala231 and His423, or

-Trp59 and Gly 231; or

-Trp59, Phe87 and Gly 231; or

-Trp59, Leu87 and Gly 231; or

-Trp59, Val87 and Gly 231; or

-Trp59, Phe87, Phe152 and Gly 231; or

-Trp59, Phe87, Phe152, Gly231 and His 423; or

-Phe59, and Ala 231; or

-Phe59, Phe87, and Gly 231; or

-Trp59, Ala231 and Phe 234; or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Phe 234; or

-Trp59, Leu87, Ala231 and Phe 234; or

-Trp59, Val87, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231, Phe234 and His423, or

-Trp59, Gly231 and Phe 234; or

-Trp59, Phe87, Gly231 and Phe 234; or

-Trp59, Leu87, Gly231 and Phe 234; or

-Trp59, Val87, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231, Phe234 and His 423; or

-Phe59, Phe87, Gly231 and Phe234, or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Met 234; or

-Trp59, Leu87, Ala231 and Met 234; or

-Trp59, Val87, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231, Met234 and His423, or

-Trp59, Gly231 and Met 234; or

-Trp59, Phe87, Gly231 and Met 234; or

-Trp59, Leu87, Gly231 and Met 234; or

-Trp59, Val87, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231, Met234 and His 423; or

-Phe59, Phe87, Gly231 and Met 234.

In an even more preferred embodiment, the mutant transaminase of the invention has at least a mutation

-Trp59, Phe87, Phe152 and Ala231, or

-Trp59, Phe87, Phe152, Ala231 and Met234, or

-Trp59, Phe87, Phe152, Ala231 and H423, or

-Phe59, Phe87 and Gly 231.

More preferably, the mutant transaminase of the invention differs from the corresponding wild-type transaminase only in the following mutations

-Trp59, Phe87, Phe152 and Ala231, or

-Trp59, Phe87, Phe152, Ala231 and Met234, or

-Trp59, Phe87, Phe152, Ala231 and H423, or

-Phe59, Phe87 and Gly 231.

A preferred and specific amino acid sequence of the mutant transaminase comprises SEQ ID NO: 30 to 38, 43 to 51, 53, 55 and 58-64 (see figures 3, 4 and 5) and 57 (see the "sequences" section below) or those amino acid sequences consisting of the sequences defined in any of them.

As described above, the mutant transaminase of the present invention comprises a sequence identical to SEQ ID NO: 1, amino acid sequence at least 65% identical. In a preferred embodiment, the mutant transaminase comprises a sequence identical to SEQ ID NO: 1, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of seq id no. Sequence identity was determined as above. In a preferred embodiment of the invention, the mutant transaminase comprises a sequence identical to a sequence selected from SEQ ID NO: 1.3 and 5 to 11, an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98% or 99% identical.

According to the invention, the mutant transaminase has an increased transaminase activity relative to the corresponding wild-type enzyme. As mentioned above, this is of particular interest for bulky substrates as well as the amine donor isopropylamine. Thus, in the context of the present invention, increased activity on bulky substrates/isopropylamine is preferred. Suitable large-volume test substrates for determining whether activity on these substances is increased are substrates 1 to 5, including those shown in scheme 2, i.e., 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), and 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a), as well as those shown in scheme 3, (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylprop-1-one (3b), 2H-acenaphthylene-1-one (4b) and 8-benzoyl-8-azabicyclo [3.2.1] oct-3-one (5 b). It will be understood by those skilled in the art that if the activity for at least one substrate or isopropylamine (in particular at least one of substrates 1 to 5 or isopropylamine) is increased, there is already an increase in activity.

Thus, in a preferred embodiment of the invention, the mutant transaminase has an increased transaminase activity for the transamination of at least one compound selected from the group consisting of: 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), and 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a), 3 (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylpropan-1-one (3b), 2H-acenaphthylene-1-one (4b), and 8-benzoyl-8-azabicyclo [3.2.1] octan-3 -ketone (5b) and isopropylamine.

In a preferred embodiment, the mutant transaminase has at least a 2-fold increase in transaminase activity, preferably at least a 2.5-fold increase in transaminase activity, preferably at least a 3-fold increase in transaminase activity, more preferably at least a 3.5-fold increase in transaminase activity, and most preferably at least a 4-fold increase in transaminase activity, relative to the corresponding wild-type enzyme, in particular for at least one compound selected from the group consisting of: 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), and 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a), 3 (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylprop-1-one (3b), 2H-acenaphthylene-1-one (4b), 8-benzoyl-8-azabicyclo [3.2.1] oct-3-one (5b) and isopropylamine.

In another aspect, the invention relates to a fusion protein comprising the transaminase of the invention.

Fusion proteins are proteins produced by linking two or more otherwise separate proteins or peptides. This process produces polypeptides with functional properties derived from each of the original proteins. Thus, depending on the intended use of the transaminase, it can be combined with additional peptides or proteins into a fusion protein. Proteins may be fused via a linker or spacer, which increases the likelihood that the proteins fold independently and function as intended. Particularly where the linker enables protein purification, the linker in the protein or peptide fusion is sometimes engineered to have a cleavage site for a protease or chemical agent, thereby enabling the release of the two separate proteins. Dimeric or multimeric fusion proteins can be prepared by genetic engineering, by fusing the original protein to a peptide domain (e.g., streptavidin or leucine zipper) that induces dimerization or multimerization of the artificial protein. Fusion proteins to which toxins or antibodies are attached can also be prepared. Other fusions include the addition of signal sequences such as lipidation signal sequences, secretion signal sequences, glycosylation signal sequences, translocation signal peptides, and the like.

Preferably, the fusion protein of the invention comprises a tag. The attachment of the tag to the protein may be used for various purposes, e.g. for simplifying purification, for assisting correct folding of the protein, for preventing precipitation of the protein, for changing chromatographic properties, for modifying the protein or for labelling or marking the protein.

A number of (affinity) tags or (affinity) labels are currently known. They are generally classified into 3 categories according to their size: the small tag has a maximum of 12 amino acids, the medium tag has a maximum of 60 amino acids, and the large tag has more than 60 amino acids. Small tags include Arg-tag, His-tag, Strep-tag, Flag-tag, T7-tag, V5-peptide-tag, and c-Myc-tag, while medium-sized tags include S-tag, HAT-tag, calmodulin-binding peptide, chitin-binding peptide, and some cellulose-binding domains. The latter may contain up to 189 amino acids and thus may be considered as large affinity tags, such as GST-and MBP-tags. In order to produce particularly pure proteins, so-called ditags or tandem tags have been developed. In this case, the protein is purified in two separate chromatographic steps, using the affinity of the first tag and subsequently the affinity of the second tag, respectively. Examples of such dual or tandem tags are the GST-His-tag (glutathione-S-transferase fused to a polyhistidine tag), the 6 xHis-Strep-tag (6 histidine residues fused to a Strep-tag (see below)), the 6 xHis-tag 100-tag (6 histidine residues fused to the 12 amino acid protein of mammalian MAP-kinase 2), the 8 xHis-HA-tag (8 histidine residues fused to a hemagglutinin epitope tag), the His-MBP (His-tag fused to maltose-binding protein), the FLAG-HA-tag (FLAG-tag fused to a hemagglutinin epitope tag), and the FLAG-Strep-tag. Most of these tags were specifically developed for purification of proteins produced by prokaryotic cells. Suitable labels are given in the specification, including SEQ ID NO: 56 (SHHHHHH).

In another aspect, the invention relates to a nucleic acid encoding a transaminase of the invention.

The term "nucleic acid" as used herein generally relates to any nucleotide molecule encoding a mutant transaminase of the present invention, which may be of variable length. Examples of nucleic acids of the invention include, but are not limited to, plasmids, vectors, or any kind of DNA and/or RNA fragments that can be isolated by standard molecular biology procedures, including, for example, ion exchange chromatography. The nucleic acids of the invention may be used to transfect or transduce a particular cell or organism.

The nucleic acid molecules of the invention may be in the form of RNA (such as mRNA or cRNA) or may be in the form of DNA (including, for example, cDNA and genomic DNA), for example, produced by cloning or by chemical synthesis techniques or a combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded. The single-stranded DNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand. Herein, a nucleic acid molecule may also refer to, for example, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA (which may be single-stranded or more typically double-stranded or triple-stranded, or a mixture of single-and double-stranded regions). Further, herein, a nucleic acid molecule may refer to a triple-stranded region comprising RNA or DNA or both RNA and DNA.

In addition, the nucleic acid may comprise one or more modified bases. Such nucleic acids may also comprise modifications, for example in the ribose-phosphate backbone, to increase the stability and half-life of such molecules in physiological environments. Thus, a DNA or RNA that modifies the backbone for stability or for other reasons is a "nucleic acid molecule" as this property is desired herein. Furthermore, DNA or RNA comprising a rare base (such as inosine) or a modified base (such as tritylated base) are also nucleic acid molecules in the context of the present invention, to name just two examples. It is understood that various modifications of DNA and RNA are known to those skilled in the art for many useful purposes. The term nucleic acid molecule as used herein includes chemically, enzymatically or metabolically modified forms of nucleic acid molecules, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.

In addition, a nucleic acid molecule encoding a mutant transaminase of the present invention can be functionally linked to any desired sequence (e.g., a regulatory sequence, a leader sequence, a heterologous marker sequence, or a heterologous coding sequence) using standard techniques (e.g., standard cloning techniques) to produce a fusion protein.

The nucleic acids of the invention can be initially formed in vitro or in cultured cells, typically by manipulating the nucleic acids by endonucleases and/or exonucleases and/or polymerases and/or ligases and/or recombinases or other methods known to the skilled person.

The nucleic acids of the invention may be contained in an expression vector in which the nucleic acid is operably linked to a promoter sequence capable of promoting expression of the nucleic acid in a host cell.

As used herein, the term "expression vector" generally refers to any kind of nucleic acid molecule that can be used to express a protein of interest in a cell (see also details above for nucleic acids of the invention). In particular, the expression vector of the present invention may be any plasmid or vector known to those skilled in the art to be suitable for expressing a protein in a particular host cell, including but not limited to mammalian cells, bacterial cells, and yeast cells. The expression construct of the invention may also be a nucleic acid encoding the transaminase of the invention, which is subsequently used for cloning into the corresponding vector to ensure expression. Plasmids and vectors for protein expression are well known in the art and are commercially available from a variety of suppliers, such as Promega (Madison, Wis., USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, Calif., USA), or MoBiTec (Germany). Methods for protein expression are well known to those skilled in the art and are described, for example, in Sambrook et al, 2000(Molecular Cloning: A laboratory, third edition).

The vector may additionally comprise nucleic acid sequences which allow it to replicate in the host cell, such as an origin of replication, one or more therapeutic genes and/or selectable marker genes, and other genetic elements known in the art, such as regulatory elements which direct the transcription, translation and/or secretion of the encoded protein. The vector may be used to transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins that are not native to the cell. The vector optionally includes materials that aid in achieving entry of the nucleic acid into the cell, such as viral particles, liposomes, protein capsids, and the like. Many types of suitable expression vectors are known in the art for protein expression by standard molecular biology techniques. Such vectors may be selected from conventional vector types, including insect (e.g., baculovirus expression systems), or yeast, fungal, bacterial or viral expression systems. Many types of other suitable expression vectors known in the art may also be used for this purpose. Methods for obtaining such expression vectors are well known (see, e.g., Sambrook et al, supra).

As described above, the nucleic acid encoding the mutant transaminase of the present invention is operably linked to sequences suitable for driving protein expression in a host cell to ensure expression of the protein. However, the following are also included in the present invention: the claimed expression construct may be an intermediate product which is subsequently cloned into a suitable expression vector to ensure expression of the protein. The expression vectors of the present invention may further comprise all kinds of nucleic acid sequences including, but not limited to, polyadenylation signals, splice donor and splice acceptor signals, intervening sequences, transcriptional enhancer sequences, translational enhancer sequences, drug resistance genes, and the like. Optionally, the drug resistance gene may be operably linked to an Internal Ribosome Entry Site (IRES), which may be cell cycle specific or cell cycle independent.

The term "operably linked" as used herein generally means that the genetic elements are arranged so that they function in concert for their intended purposes, e.g., wherein transcription is initiated by a promoter and proceeds through a DNA sequence encoding a protein of the invention. That is, the sequence encoding the fusion protein is transcribed into mRNA by RNA polymerase, which is then spliced and translated into protein.

The term "promoter sequence" as used in the context of the present invention generally refers to any kind of regulatory DNA sequence operably linked to a downstream coding sequence, wherein said promoter is capable of binding RNA polymerase and initiating transcription of the encoded open reading frame in a cell, thereby driving expression of said downstream coding sequence. The promoter sequence of the present invention may be any kind of promoter sequence known to those skilled in the art, including but not limited to constitutive promoters, inducible promoters, cell cycle specific promoters, and cell type specific promoters.

Another aspect of the invention relates to a host cell comprising a transaminase of the invention, a fusion protein of the invention or a polynucleotide or an expression vector of the invention. In one embodiment of the invention, host cells having transaminase activity are used in the context of the present invention. In particular, cells that are optionally lysed or otherwise permeabilized are used for transamination, e.g., in the methods and uses of the invention. The "host cell" of the present invention may be any kind of organism suitable for use in recombinant DNA technology, including but not limited to a wide variety of bacterial and yeast strains suitable for expression of one or more recombinant proteins. Examples of host cells include, for example, various strains of Bacillus subtilis or Escherichia coli. Various E.coli bacterial host cells are known to those skilled in the art, including but not limited to strains such as DH 5-alpha, HB101, MV1190, JM109, JM101 or XL-1blue, which may be purchased from various suppliers, including, for example, Stratagene (CA, USA), Promega (WI, USA) or Qiagen (Hilden, Germany). A particularly suitable host cell, i.e.E.coli BL21(DE3) cell, is also described in the examples. Bacillus subtilis strains that can be used as host cells include, for example, 1012 wild-type: leuA8metB5trpC2hsdRM1 and 168 Marburg: trpC2(Trp-), which is available, for example, from MoBiTec (Germany).

The cultivation of the host cells according to the invention is a routine procedure known to the person skilled in the art. That is, a nucleic acid encoding the mutant transaminase of the present invention can be introduced into a suitable host cell to produce the corresponding protein by recombinant methods. These host cells may be any kind of suitable cells that can be grown in culture, preferably bacterial cells such as E.coli. In a first step, the method may comprise cloning the corresponding gene into a suitable plasmid vector. Plasmid vectors are widely used for gene cloning and can be easily introduced, i.e., transfected into bacterial cells that have been prepared to be transiently permeable to DNA. After the protein is expressed in the host cell, the cells can be harvested and used as starting material for preparing a cell extract containing the protein of interest. Cell extracts containing the protein of interest are obtained by lysing the cells. Methods for preparing cell extracts by means of chemical or mechanical cell lysis are well known to those skilled in the art and include, but are not limited to, for example, hypotonic salt treatment, homogenization, or sonication.

In another aspect, the present invention provides a method of preparing an amine, the method comprising reacting an amine acceptor with a mutant transaminase of the present invention or a fusion protein of the present invention in the presence of an amine donor.

The reaction of the present invention follows in principle the scheme given in scheme 1: (See above). This scheme illustrates the transfer of an amino group from an amine donor to an amine acceptor. The amine donor may be any molecule comprising a transferable amino group that is acceptable for use by the mutant transaminase or fusion protein of the invention. The amine acceptor may be any molecule to which an amino group may be transferred by the mutant transaminase or fusion protein of the invention. Typically, amino (NH)₂) Is transferred from a primary amine (amine donor) to a carbonyl group (C ═ O) (amine acceptor) by a transaminase. The terms "amine acceptor" and "amino acceptor" are used interchangeably with "amine donor" and "amino donor" on the one hand and on the other hand.

In a preferred embodiment, enantiomerically enriched chiral amines are produced by

a) Kinetic resolution of racemic amines in the presence of an amine receptor and a mutant transaminase or fusion protein, or

b) Asymmetric transamination of prochiral ketones in the presence of an amine donor and a mutant transaminase or fusion protein,

wherein the mutant transaminase or fusion protein is stereoselective.

It is to be understood that the mutant transaminase or fusion protein can be used as a solution, lyophilisate, immobilized or whole-cell catalyst.

Method a)

In this process, known as kinetic resolution, the enantiomer of the racemic amine is reacted with a stereoselective transaminase or fusion protein (optionally in a host cell) at different reaction rates to give an enantiomerically enriched composition of the less reactive enantiomer. Thus, the racemic mixture of the amine is enantiomerically enriched for the desired enantiomer.

The racemic amine may have the formula

Wherein the content of the first and second substances,

R¹or R²Independently of one another, represents an optionally substituted alkyl, aryl, carbocyclyl or heterocyclyl group; or

R¹And R²Together with the carbon atoms to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring.

In a preferred embodiment

R¹Is optionally substituted C_1-12-an alkyl or aryl group;

R²is an optionally substituted aryl or heterocyclyl group; or

R¹And R²Together with the carbon atom to which they are attached form an optionally substituted mono-or polycyclic carbocyclic or heterocyclic ring, wherein the optional substituents are selected from C_1-12Alkyl radical, C_1-12Alkoxy, aryl, aryloxy, halogen, hydroxy or cyano.

In another preferred embodiment

R¹Is optionally substituted C_1-7-alkyl or phenyl;

R²is optionally substituted phenyl; or

R¹And R²Together with the carbon atom to which they are attached form an optionally substituted mono-or polycyclic carbocyclic or heterocyclic ring, wherein the optional substituents are selected from C_1-7Alkyl radical, C_1-7-alkoxy, phenyl, phenoxy, chloro, hydroxy or cyano.

Illustrative examples of amines are shown in scheme 2 (above).

Suitable amine acceptors for the kinetic resolution in process a) may be selected from ketones and ketocarboxylic acids. It is preferred to use ketocarboxylic acids such as 2-ketocarboxylic acids (e.g., 2-ketoglutaric acid, glyoxylic acid, pyruvic acid, oxaloacetic acid, etc.) and suitable salts thereof. The most commonly used and therefore most preferred are the conjugate bases of 2-ketocarboxylic acids, such as 2-ketoglutarate, pyruvate, glyoxylate or oxalate.

The kinetic resolution of the racemic amine with the mutant transaminase or fusion protein can be conveniently carried out in an aqueous medium, suitably containing a physiological buffer such as CHES buffer, at a pH in the range 5 to 11, especially 7.5 to 10, and at a temperature in the range 10 ℃ to 50 ℃, preferably 20 ℃ to 40 ℃.

When 2-ketocarboxylic acids are used as amine acceptors, the molar ratio of racemic amine to amine acceptor is usually chosen to be in the range from 1.1 to 10, in particular from 1.1 to 2.5. This molar ratio can be significantly increased when ketones are used as amine acceptors.

Method b)

Method b entails asymmetric transamination of a prochiral ketone in the presence of an amine donor and a stereoselective mutant transaminase or fusion protein, optionally in a host cell.

The prochiral ketone may have the formula

R³Or R⁴Independently of one another, represents an optionally substituted alkyl, aryl, carbocyclyl or heterocyclyl group; or

R³And R⁴Together with the carbon atoms to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring.

In a preferred embodiment

R³Is optionally substituted C_1-12-an alkyl or aryl group;

R⁴is an optionally substituted aryl or heterocyclyl group; or

R³And R⁴Together with the carbon atom to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring, wherein optionallyIs selected from C_1-12Alkyl radical, C_1-12Alkoxy, aryl, aryloxy, halogen, hydroxy or cyano.

In another preferred embodiment

R³Is optionally substituted C_1-7-alkyl or phenyl;

R⁴is optionally substituted phenyl; or

R³And R⁴Together with the carbon atom to which they are attached form an optionally substituted mono-or polycyclic carbocyclic or heterocyclic ring, wherein the optional substituents are selected from C_1-7Alkyl radical, C_1-7-alkoxy, phenyl, phenoxy, chloro, hydroxy or cyano.

Scheme 3 (above) shows illustrative examples of prochiral ketones.

Suitable amine donors may generally be selected from achiral or chiral amines or amino acids. Typical amines are primary aliphatic amines such as isopropylamine, 1-phenylethylamine, 2-amino-4-phenylbutane, o-xylylenediamine, or amino acids such as alpha amino acids, glycine, glutamic acid or alanine. Preferred amine donors are isopropylamine and L-alanine.

In a preferred embodiment of the invention, the amine acceptor and amine donor are reacted with the mutant transaminase or fusion protein using a system that favors the shift of the equilibrium towards the desired enantiomerically enriched chiral amine.

Various biocatalytic strategies are employed, either to remove the formed by-product (e.g. pyruvate) by a second enzyme (e.g. lactate dehydrogenase, pyruvate decarboxylase) or to recycle/remove by alanine dehydrogenase, to shift the equilibrium of the asymmetric transamination reaction.

When a multi-enzyme system is used, the reaction conditions need to meet the requirements of all enzymes involved. The reaction may conveniently be carried out in an aqueous medium, suitably containing a physiological buffer such as CHES buffer, at a pH in the range 5 to 11, especially 7.5 to 10, and a temperature in the range 10 ℃ to 50 ℃, preferably 20 ℃ to 40 ℃.

The molar ratio of prochiral ketone to amine donor depends on the second enzyme used and the respective reaction conditions. In general, the molar ratio of prochiral ketone and amine donor can be chosen in the range from 1:100 to 1:5, in particular from 1:50 to 1: 10.

The use of a large excess of isopropylamine as the preferred amine donor, especially in combination with the in situ removal of the acetone formed (its corresponding ketone), can shift the equilibrium towards the formation of the target amine in asymmetric transamination. Addition of organic co-solvents (water miscible and immiscible) or other specific additives can improve the reaction.

The terms used herein in the racemic amine of formula I and the prochiral ketone of formula II have the following meanings.

The term "halogen" denotes fluorine, chlorine, bromine or iodine, especially chlorine.

The term "alkyl" denotes a monovalent straight or branched chain saturated hydrocarbon group of 1 to 12 carbon atoms. In particular embodiments, the alkyl group has 1 to 7 carbon atoms, and in more particular embodiments 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

The term "alkoxy" refers to an alkyl group as defined above attached to an oxy group. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

The term "aryl" denotes a monovalent aromatic carbocyclic mono-or bicyclic ring system comprising 6 to 10 carbon ring atoms. Examples of aryl moieties include phenyl and naphthyl.

The term "aryloxy" denotes an aryl group as defined above attached to an oxy group. Suitable examples are phenoxy or naphthoxy.

The term "monocyclic or polycyclic" refers to compounds characterized by one or more closed atom(s) (primarily carbon) rings. These ring substructures include cycloalkanes, arenes, and other rings. Although "poly" is literally "many," it also includes bicyclic, tricyclic, tetracyclic, and the like.

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

The term "heterocyclyl" refers to a saturated, partially unsaturated or unsaturated 5-to 6-membered monocyclic or 8-to 10-membered bicyclic ring, which may contain 1,2 or 3 heteroatoms selected from nitrogen, oxygen and/or sulfur. The ring system may be aromatic or non-aromatic. Typical heterocyclic residues are pyridyl, piperidyl, pyrrolidinyl, pyrimidinyl, furanyl, pyranyl, benzimidazolyl, quinolyl or isoquinolyl.

Optional substituents for the racemic amines of formula I and the prochiral ketones of formula II herein may be selected from alkyl, alkoxy, aryl, aryloxy, halogen, hydroxy or cyano groups. The preferences and examples described above also apply to these substituents.

In another aspect, the invention relates to the use of a mutant transaminase of the invention or a fusion protein of the invention for transamination of a ketone, in particular for the synthesis of a chiral amine having an asymmetric carbon atom bound to the transferred amino group.

With respect to the use of the present invention, the terms, examples, and specific embodiments used in other aspects of the disclosure also apply to that aspect. In particular, the mutant transaminases or fusion proteins according to the invention can be used as described in detail for the process of the invention.

Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Definition of commonly used terms in molecular biology can be found in Benjamin Lewis, Genes V (ISBN 0-19-854287-9), published by Oxford university Press 1994; the Encyclopedia of molecular biology (ISBN0-632-02182-9) by Kendrew et al (eds) published by Blackwell science Ltd 1994; and Robert A.Meyers (eds.), Molecular Biology and Biotechnology, published by VCH Publishers, Inc.1995, a Comprehensive Desk Reference (ISBN 1-56081-.

The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but the preferred methods and materials are described herein. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "comprising," and "include" are to be construed as inclusive and not exclusive. Likewise, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" means two or more.

The following figures and examples are intended to illustrate various embodiments of the present invention. Therefore, the specific modifications discussed 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 may be made without departing from the scope of the invention, and it is therefore to be understood that such equivalent embodiments are to be included in the present invention.

Brief Description of Drawings

FIG. 1: thermostability of the 3FCR variant in HEPES buffer (50mM) pH7.5 at 25 ℃; 3FCR Y59W/T231A (open circle), 3FCR Y59W/Y87F/T231A (closed circle), 3FCR Y59W/Y87F/Y152F/T231A (triangle), 3FCRY59W/Y87F/Y152F/T231A/P423H (square). The activity in the kinetic resolution mode was assessed using 6a as the amine donor, as described in paragraph B2, with the activity before incubation defined as 100%.

FIG. 2: preferred wild type transaminases are 3FCR, 3GJU, ATA-3, ATA-4, ATA5, ATA-6, ATA-7, ATA-8 and ATA-9. The sites of the mutations Trp59, Phe87, Phe152, Ala231, Met234 and His423 according to the invention are underlined in each transaminase.

FIG. 3: preferred specific mutant transaminases are 3FCR, 3GJU, ATA-3, ATA-4, ATA5, ATA-6, ATA-7, ATA-8 and ATA-9 (each having 4 amino acids substituted as indicated). The mutations Trp59(Y59W), Phe87(Y87F), Phe152(Y152F) and Ala231(T231A) of the invention are underlined in each transaminase.

FIG. 4: preferred specific mutant transaminases are 3FCR, 3GJU, ATA-3, ATA-4, ATA5, ATA-6, ATA-7, ATA-8 and ATA-9 (each having 5 amino acids substituted as indicated). The mutations Trp59(Y59W), Phe87(Y87F), Phe152(Y152F), Ala231(T231A) and His423(P423H) according to the invention are underlined in each transaminase.

FIG. 5: preferred specific mutant transaminases are 3FCR, 3GJU, ATA-3, ATA-4, ATA5, ATA-6, ATA-7, ATA-8 and ATA-9 (each having 5 amino acids substituted as indicated). The mutations Trp59(Y59W), Phe87(Y87F), Phe152(Y152F), Ala231(T231A) and Met234(I234M) according to the invention are underlined in each transaminase.

Examples

General procedure

A biocatalyst production

A1 transaminase gene acquisition and construction of expression vector

Codon optimization algorithm OptimumGene based on the reported amino acid sequence of the transaminase (NCBI GI code provided in table 1) and GenScript (Piscataway, u.s.a.)^TMIn all cases, a C-terminal histidine tag (with a stop codon at the end) was added to facilitate purification of the expressed TA by metal affinity chromatography (see corresponding paragraphs). restriction sites were added to the nucleotide sequence for subsequent cloning in the vector of interest pET22 b. the 5 'end was NdeI-restricted and the 3' end was BamHI-restricted.the initiation codon (ATG) was incorporated into the restriction site of NdeI, thereby encoding only one methionine.the vector contains an ampicillin resistant bla coding sequence (β -lactamase). expression was controlled by the lac promoter according to the cloning strategy.the resulting plasmid was transformed into E.coli BL21(DE 3). the codon optimized gene sequence and the encoded polypeptide sequence are provided in the "sequence" section.

With modified versionsThe PCR method prepared in the invention of the use of mutants. Primers were designed with mismatches that provided the desired mutation. For each reaction, we used Pfu buffer, 0.2mM dNTP, 0.2 ng/. mu.L parental plasmid, 0.2. mu.M of each primer, 2% (w/w) DMSO and 0.2. mu.L Pfu Plus! A polymerase. Amplification was performed as follows:

a) 5 minutes at 95 ℃;

b)20 cycles: 95 ℃ for 45 seconds; 60 ℃ for 45 seconds; 72 ℃ for 7 minutes;

c)72 ℃ for 14 minutes

The PCR product was digested with DpnI (20. mu.L/mL) at 37 ℃ for 2 hours, and then the restriction enzyme was inactivated by heating at 80 ℃ for 20 minutes. The digested PCR product was transformed into E.coli TOP-10 cells using standard methods. Plasmids were isolated from clones transformed with PCR products and the correct sequence was confirmed by DNA sequencing. The isolated plasmid with the correct mutation was transformed into E.coli BL21(DE3) cells using standard methods.

Table 1: the transaminase used in the present invention is also the NCBI gene identification code (GI) of the wild-type sequence.

Production of A2 transaminase

A single colony of the microorganism of Escherichia coli BL21(DE3) was inoculated in 3mL of Luria Bertani medium containing 100. mu.g/mL of ampicillin in a test tube and cultured overnight (about 18h) at 30 ℃ with shaking at 180 rpm. In a 300mL shake flask, 30mL Terrific broth containing 100. mu.g/mL ampicillin was inoculated with 1% (v/v) of an overnight culture (0.3mL) and cultured with shaking at 180rpm at 37 ℃ to an Optical Density (OD) of 600nm₆₀₀) 0.5 to 0.7. the culture was cooled to 20 ℃ and isopropyl β -D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce expression of TA. shaking at 20 ℃ was continued overnight incubation for more than about 16 hours. cells were harvested by centrifugation (6000g, 10 min, 4 ℃) and the supernatant discarded. cell pellet was resuspended in 6mL of precooled (4 ℃) HEPES buffer (50mM, pH7.5) containing 0.1mM pyridoxal-5-phosphate (PLP) and sonicated (Bandelin Sonopuls HD 2070; 5 min 2 times, 5 min intervals, 50% pulses, 60% power, always kept on ice) to lyse the cells. cell debris was removed by centrifugation (6000g, 30 min, 4 ℃). the lysate supernatant was filtered with a 0.2 μ M filter (Millipore) and 30% glycerol was added and stored at +4 ℃ or-20 ℃ followed by a purification step of NaCl containing 0.3M lysis buffer.

Purification of A3 transaminase

Protein purification was performed using metal affinity chromatography using the C-terminal HisTag in all expressed TAs and standard procedures for those skilled in the art. Crude lysate prepared as described in paragraph A2 was loaded into equilibration with lysis buffer (HEPES buffer, 50mM, 0.3M NaCl, 0.1mM PLP)The latter HisTrap Fastflow 5mL column (this column was assembled inOn a protein chromatography system). Once TA was loaded onto the column, non-specifically bound protein was washed off using 2 column volumes of 10mM imidazole. TA was eluted from the column by applying 2 column volumes of 0.3M imidazole. The purified TA fractions were combined and then purified using a Sephadex G60 column equilibrated with HEPES (50mM, pH7.5) buffer containing 0.1mM PLP, using standard procedures known to those skilled in the art, byAnd (5) desalting the system. 30% glycerol was added and the purified enzyme solution was stored at +4 ℃ or-20 ℃.

B biocatalysis

B1 preparation of stock solutions of the Compound of interest

Due to the hydrophobicity of the substrate of interest (see schemes 2 and 3), stock solutions are prepared in water-soluble organic solvents such as 2-propanol or dimethyl sulfoxide. In the standard procedure of the kinetic resolution experiment, stock solutions of 20 to 40mM of the ketone and amine of interest were prepared in 2-propanol. When amines 1 and 4 were prepared as hydrochlorides, the solution was sonicated at room temperature for 30 seconds (bandaging Sonorex RK 512H) to facilitate dispersion. After dispersion, the homogeneous solution was stable and no precipitation of the amine or amine salt was observed.

Activity measurement in B2 kinetic resolution mode

Using a microtiter plate reader, in TECANIn a 200 PRO reader, activity measurements were performed. For theA direct photometric assay described in Analytical Chemistry (2009; see also above), the enzyme reaction is performed in CHES buffer (50mM, pH) at 30 ℃9.0) in a total volume of 200. mu.L. 1mM of racemic amine of interest (rac-amine) and 2mM of amine acceptor (preferably pyruvate or glyoxylate) are added to the buffer containing the TA of interest (final concentration). Since the amine stock solution was prepared in an organic solvent, the final concentration of the organic solvent (2-propanol or DMSO) was 5% (v/v). The reaction was initiated by the addition of a second substrate (pyruvate). The concentration of enzyme varies from 2 to 800. mu.g/mL final concentration, depending on the specific activity on the substrate tested. The production of ketones is monitored at the optimum wavelength for each compound, as determined by methods known to those skilled in the art. The wavelengths used for each substrate, as determined by the exact reaction conditions, and the apparent extinction coefficients used to determine the specific activity of TA are listed in table 2.

Table 2: optimum wavelength (nm) and apparent extinction coefficient (M) for the ketone of interest^-1cm^-1). Ketone 5a was not included as it could not be monitored by direct photometry. The calculation of the apparent coefficient simulates the reaction conditions, since some amines have a small, but not disadvantageous, absorbance at the wavelength monitored.

Substrate	Optimum wavelength	Apparent extinction coefficient
			1a	265nm	16562M-1cm-1
2a	245nm	7953M-1cm-1
			3a	245nm	9646M-1cm-1
4a	340nm	5714M-1cm-1
			6a	245nm	6115M-1cm-1

For compounds that cannot be applied with direct photometry, such as substrates 5a/b and isopropylamine, the assay described in Analytical Chemistry (2014) by Wei β et al is used to prepare a master mixture in CHES buffer (pH9.0,50mM) containing 2mM amine 5a or 50mM isopropylamine (amine donor), 2.4mM glyoxylate (amine acceptor), 0.12mg/mL glycine oxidase from Bacillus thermophilus (Geobacillus kaustophilus), 5U/mL horseradish peroxidase, 3.9mM vanillic acid and 1.2mM 4-aminoantipyrin for use in this assay, the reaction is initiated by adding 20. mu.L of an enzyme solution standardized to a concentration of 1mg/mL to 130. mu.L of the master mixture and carried out at 37 ℃ for 1 hour, the absorbance at 498nm is increased by the addition of quinone 4654M (4654M) due to the formation of a quinone imine dye^-1cm^-1) And measuring the enzyme activity. Since the assay relies on the activity of three enzymes, it is not possible to calculate the specific activity. Thus, the activity of TA is always expressed relative to the reference variant [ activity (mutant)/activity (reference) × 100)]。

Example 1: improvement of TA specific Activity for kinetic resolution of racemic amines 1a, 3a and 4a by incorporation of mutations

The specific activity of the proposed mutants in the kinetic resolution of racemic amines 1a, 3a, 4a and 6a was determined using purified TA according to the assay described in section B2. These positions were identified in 3FCR scaffolds and then transferred to 3GJU scaffolds to confirm their importance for acceptance of the amine of interest. SEQ ID NO: 1 and SEQ ID NO: identity of 3 was 71.3%. Table 3: specific activity (U/mg) of 3FCR and 3GJU mutants in kinetic resolution mode. n.a. no activity; n.d. not determined.

The advantages of the double mutant Y59W/T231A compared to the wild type and the single mutant in 3FCR, and compared to the enzyme with only 71.3% sequence identity (3GJU) were demonstrated. In addition, the cumulative beneficial effects at other locations are exemplified by the mutants Y59W/Y87F/Y152F/T231A/P423H.

More specifically, the most significant intermediate mutants generated on the scaffold of 3FCR had the specific activities shown in table 4.

Table 4: specific activity (U/mg) of the 3FCR mutant in the kinetic resolution mode.

In the table, it is exemplified that the activities on the substrates 1a, 3a and 4a gradually increase with the addition of each mutation. The increased activity on these bulky substrates is not necessarily accompanied by an increased activity on the standard substrate 6 a.

The most interesting mutants were evaluated for operational stability at 25 ℃. The purified enzyme was incubated in storage buffer (HEPES buffer, 50mM, pH7.5, 0.1mM PLP added) at the specified temperature without shaking. At specific time intervals, aliquots of the incubated enzyme were taken and the residual activity was monitored using the direct test described in paragraph B2 of the general procedure, using racemic amine 6a as the amine donor. The activity measured before incubation for each mutant was defined as 100%. The results are shown in FIG. 1.

Mutations Y59W, Y87F, Y152F and T231A were incorporated into several putative transaminases identified by BLAST searches at NCBI. The amino acid sequence of 3FCR Y59W/Y87F/Y152F/T231A was used for queries in non-redundant protein sequence databases. None of the four mutations were found in the first 100 results; all sequences are conserved in these positions, i.e., with Tyr 59, Tyr 87, Tyr 152, and Thr 231. Sequences with identities as low as 69.6% were selected (SEQ ID NOS: 5-11) and synthetic genes with four mutations in the corresponding positions were customized (amino acid sequences SEQ ID NOS: 32 and 34-38, respectively). All TA's were soluble expressed except for ATA-4. The specific activities of these transaminases for kinetic resolution of amines 1a, 3a, 4a and 6a were determined according to the test described in paragraph B2 of the general procedure, using purified TA.

Table 5: the putative transaminases identified in the BLAST search had specific activities (U/mg) in the kinetic resolution mode following incorporation of the mutations Y59W/Y87F/Y152F/T231A.

The motif found in 3FCR and tested in 3GJU can be transferred to enzymes with at least 65% sequence identity and give rise to meaningful activity on substrates 1a, 3a and 4 a.

Example 2: improvement of TA specific Activity for kinetic resolution of racemic amines 1a, 2a, 3a, 4a and 6a by incorporation of mutations

The specific activity of the proposed mutants on the kinetic resolution of racemic amines 1a, 2a, 3a, 4a and 6a was determined according to the assay described in general procedure paragraph B2, using purified TA. These positions were identified in 3FCR scaffolds and then transferred to 3GJU scaffolds to confirm their importance in accepting the amine of interest. SEQ ID NO: 1 and SEQ ID NO: identity of 3 was 71.3%. Table 6: specific activity (U/mg) of 3FCR and 3GJU mutants in kinetic resolution mode. n.a. no activity n.d. not determined.

Position 87 is important for receiving substrate 2 a/b. Aliphatic hydrophobic residues such as Leu or Val are necessary at this position to aid acceptance. This effect can be transferred to homologous proteins (e.g.3 GJU, sequence identity: 71.3%).

Example 3: improvement of the specific activity of TA on the kinetic resolution of racemic amine 5a by introduction of mutations

The specific activity of the proposed mutants on the kinetic resolution of racemic amine 5a was determined according to the assay described in general procedure paragraph B2, using purified TA. These positions were identified in the 3FCR scaffold.

Table 7: relative activity of the 3FCR mutant in kinetic resolution of racemic amine 5 a. The activity of 3FCR Y59W/T231A was used as reference (100%). For comparison, the specific activity (U/mg) for substrate 6a is provided.

It was demonstrated that Y59F and T231G substitutions in the 3FCR scaffold had a beneficial effect on the activity of substrate 5 a. The combination of these substitutions in variant Y59F/Y152F/T231G produced the best variant of substrate 5a relative to substrate 6a to date. In addition, the beneficial effect of a substitution at position 234 to Phe or Met is shown.

Example 4: improving specific activity of TA in Using isopropylamine as an amine Donor

The specific activity of the proposed mutants on the use of isopropylamine as amine donor was determined according to the assay described in general procedure paragraph B2, using purified TA. These positions were identified in the 3FCR scaffold.

Table 8: relative activity of the 3FCR mutant using isopropylamine as the amine donor. The activity of 3FCR Y59W/T231A was used as a reference (100%). For comparison, the specific activity (U/mg) for substrate 6a is provided.

It can be concluded that the substitution at position 234 has a beneficial effect on the activity on isopropylamine.

Example 5: asymmetric synthesis of chiral amines 1a, 3a and 4a using L-alanine as amine donor

A2 mL glass vial containing 0.5mL of a reaction mixture (HEPES buffer, 50mM, pH8.0) of 8mM ketone (1, 3 or 4), 200mM L-alanine, 20% DMSO, 25mM glucose, 5mM NADH, 0.5mg/mL glucose dehydrogenase GDH-105(Codexis), 5. mu.L/mL L-Lactate Dehydrogenase (LDH) from bovine heart (L2625-50KU, Sigma), 1mM PLP and TA of interest was incubated at 30 ℃ on a heated shaker (Eppendorf) at a shaking speed of 600 rpm. After 20 hours, the reaction was terminated by heat denaturation of the enzyme at 90 ℃ for 10 minutes. The reaction was cooled to room temperature and an equal amount of acetonitrile containing 0.1% diethylamine was added to the reaction mixture. The mixture was centrifuged at 17000g for 1 min to remove precipitated protein and the supernatant was filtered into the inlet for HPLC analysis (Chiralpak OD-RH column, 150 x 4.6mm,5 μm particles, 30 ℃; using 50% acetonitrile containing 0.1% diethylamine and 50% H₂Isocratic method of O, flow rate 0.5 mL/min). Transformation is defined as [ total amine, mM/(total amine, mM + ketone, mM). 100]。

Table 9: conversion (%) and enantiomeric ratio (er,%) in the asymmetric synthesis of chiral amines using L-alanine as amine donor. The enzyme concentration is 1.8 mg/mL; n.d. not determining

Using L-alanine as amine donor, asymmetric synthesis can be achieved in less than one day (20 hours) on a 8mM ketone scale (for the production of chiral amines 1a, 3a and 4a) with the desired conversion and enantioselectivity. The various variants effectively catalyze the reaction.

Example 6: asymmetric synthesis of amine 5a using alanine as amine donor

A2 mL glass vial containing 0.5mL of a reaction mixture (HEPES buffer, 50mM, pH8.0) of 8mM ketone 5, 200mM L-alanine, 20% DMSO, 25mM glucose, 5mM NADH, 0.5mg/mL glucose dehydrogenase GDH-105(Codexis), 5. mu.L/mL L-Lactate Dehydrogenase (LDH) from bovine heart (L2625-50KU, Sigma), 1mM PLP and TA of interest was incubated at 30 ℃ on a heated shaker (Eppendorf) with a shaking speed of 600 rpm. After 20 hours, the reaction was terminated by heat denaturation of the enzyme at 90 ℃ for 10 minutes. The reaction was cooled to room temperature and an equal amount of acetonitrile containing 0.1% diethylamine was added to the reaction mixture. The mixture was centrifuged at 17000g for 1 min to remove precipitated protein and the supernatant was filtered into the inlet for HPLC analysis (Chiralpak OD-RH column, 150 x 4.6mm,5 μm particles, 30 ℃; using 30% acetonitrile containing 0.1% diethylamine and 70% H₂Isocratic method of O, flow rate 0.5 mL/min). Transformation is defined as [ total amine, mM/(total amine, mM + ketone, mM). 100]。

Table 10: conversion (%) of asymmetric synthesis of amine 5a using L-alanine as an amine donor.

Using L-alanine as amine donor, asymmetric synthesis can be achieved in less than one day (20 hours), on a 8mM ketone scale for substrate 5a/b, with the desired conversion and enantioselectivity. Various TA mutants efficiently catalyzed the reaction.

Example 7: asymmetric synthesis of chiral amine 1a, (R) - (4-chlorophenyl) -benzylamine using alanine as amine donor

In 53.5ml of reaction buffer (HEPES 100mM pH 8.0; 4mM PLP; 400mM D, L-alanine), 4.5g of pyruvate reductase mixture (5.4mg of GDH-102[ Codexis ]]；21.6mg LDH-101[Codexis]；126mgNAD[Roche](ii) a 4.32g D-glucose). The purified mutant transaminase 3FCR Y59W/Y87F/Y152F/T231A (22ml, protein 2.6 mg/ml; see A3) was then added as a solution and stirred for 5 minutes at 30 ℃. The reaction was started by adding 100mg of (4-chlorophenyl) (phenyl) methanone dissolved in 20ml of DMSO. The reaction pH was manually adjusted to 8.0(2N NaOH) and kept constant by automatic addition of 0.1N NaOH. Initially, the yellow reaction solution was slightly turbid. After 24 hours and 98 area% conversion (IPC-HPLC), the reaction was acidified to ph2.0 to precipitate the enzyme. 1.125g of filter aid (Dicalite) were added and the mixture was stirred for 20 minutes. Subsequently, the reaction mixture was filtered through a bed of 20g of filter aid (Dicalite) and the filter cake was washed with 40ml of 0.1N HCl. The pH of the combined, slightly yellow, clear aqueous solution was adjusted to pH10 and the solution changed color to orange, and two 50ml TBME extractions were performed. The combined organic phases were washed with MgSO₄Drying, filtration and evaporation in vacuo at 40 ℃ gave 122mg (122%) of a yellowish oil as the crude product of the title compound 1 a.

Hydrochloride formation: the crude product was dissolved in 5ml of TBME and 350. mu.l of 2MHCl were added with stirring using a syringe. The resulting suspension was stirred at room temperature for 2 hours and then filtered (paper filter). The isolated hydrochloride salt was washed with TBME and dried under high vacuum at room temperature for 3 hours, yielding 95mg (82%) of a white powder.

Chemical purity HPLC: 99.7 area% [210 nm; X-Bridge C8; 50 x 4.6mm,2.5 μm, 2ml flow, 45 ℃ A: H₂O/ACN (95/5), 50 → 10% in 3 minutes, hold for 0.5 minutes, B: ACN, 40 → 80% in 3 minutes, hold for 0.5 minutes, C: H in H₂100mmol ammonium acetate in O/ACN (95/5), 10% isocratic](ii) a Chiral HPLC 97.1% (R), 2.9% (S) [235 nm; chrowpack CR-I (+); 150 x 3mm,5 μm, 0.4ml flow, 40 ℃, A: 55% in H₂3.6g perchloric acid in O, B45% ACN]；¹H NMR(600MHz,CH₃OD)ppm7.20-7.49(m,9H),5.57(s,1H)；GC-MS:217(M)⁺。

Example 8: kinetic resolution of amine 2a for the preparation of (S) -2, 2-dimethyl-1-phenylpropan-1-amine using pyruvate as amine acceptor

To 89.75ml of reaction buffer (TRIS 50mM pH 8.5; 1mM PLP) were subsequently added 129mg of sodium pyruvate and a solution of the purified mutant transaminase 3FCR Y59W/Y87L/T231A (18.5ml, protein 2.47 mg/ml; see A3) and stirred for 5 minutes at 30 ℃. The reaction was started by adding 100mg of 2, 2-dimethyl-1-phenylpropan-1-amine dissolved in 1ml of DMSO. After 48 hours and about 55 area percent conversion (IPC-HPLC), the reaction was acidified to pH2.0 to precipitate the enzyme and stirred for 20 minutes. Subsequently, the reaction mixture was filtered through a bed of 25g filter aid (Dicalite) and the filter cake was subsequently washed with deionized water and 50ml TBME. After phase separation, the aqueous phase was extracted with 50ml of TBME to remove 2, 2-dimethyl-1-phenyl-propan-1-one 2 b. The combined organic phases were washed with MgSO₄Dried, filtered and evaporated in vacuo at 40 ℃ to yield 25mg (26.5%; chiral HPLC: 99 area%) 2, 2-dimethyl-1-phenyl-propan-1-one 2b as a yellow viscous oil. The pH of the aqueous phase was adjusted to 12 using 2N NaOH and then extracted twice with 50ml TBME. The combined organic phases were washed with MgSO₄Dried, filtered and evaporated in vacuo at 40 ℃ to yield 38mg (40%) (S) -2, 2-dimethyl-1-phenyl-propan-1-amine 2a as a yellow oil.

Chiral HPLC 98.1% (S), 1.9% (R) [220 nm; chiracel OD-3R; 150 x 4.6mm,3 μm, flow 1.0ml,at 25 ℃, A is 50% ACN, B is 50% H in 950ml₂6.3g ammonium formate in O50 ml ACN]；¹H NMR(600MHz,CDCl3)ppm 7.27-7.31(m,4H),7.24(dt,J＝6.0,2.6Hz,1H),3.71(s,1H),0.91(s,9H)；GC-MS:162(M)。

Example 9: asymmetric synthesis of chiral amines 3a, (S) -1, 3-diphenylpropylamines using alanine as amine donor

In 68ml of reaction buffer (HEPES 100mM pH 8.0; 4mM PLP; 400mM D, L-alanine) were dissolved 4.5g of pyruvate reductase mixture (5.4mg of GDH-102[ Codexis)])；21.6mg LDH-101[Codexis]；126mgNAD[Roche](ii) a 4.32g D-glucose). The purified mutant transaminase 3FCR Y59W/Y87F/Y152F/T231A (6.8ml, protein 2.6 mg/ml; see A3) was then added as a solution and stirred for 5 minutes at 30 ℃. The reaction was started by adding 100mg of 1, 3-diphenylpropan-1-one dissolved in 20ml DMSO. The reaction pH was kept constant by automatic addition of 0.1N NaOH. Initially, the yellow reaction solution was slightly turbid. After 24 hours and 98 area% conversion (IPC-HPLC), the reaction was acidified to pH2.0 to precipitate the enzyme. 1.125g of filter aid (Dicalite) were added and the mixture was stirred for 20 minutes. Subsequently, the reaction mixture was filtered through a bed of 20g of filter aid (Dicalite) and the filter cake was washed with 40ml of 0.1N HCl. The pH of the combined, slightly yellow, clear aqueous solution was adjusted to pH10 to change its color to orange and the amine extraction was performed using two 50ml TBME. The combined organic phases were washed with MgSO₄Drying, filtration and evaporation in vacuo at 40 ℃ gave 95mg (95%) of a yellowish oil as the crude product of the title compound 3 a.

Hydrochloride formation: the crude product was dissolved in 5ml of TBME and 360. mu.l of 2MHCl were added with stirring using a syringe. The resulting suspension was stirred at room temperature for 2 hours and then filtered (paper filter). The isolated hydrochloride salt was washed with TBME and dried at room temperature under high vacuum for 3 hours to yield 84mg (71%) of a white powder as the hydrochloride salt of the title compound 3 a.

Chemical purity HPLC: 99.0 area% [210 nm; X-Bridge C8; 50 x 4.6mm of the total length of the steel,2.5 μm, flow 2ml,45 ℃, A: H2O/ACN (95/5), 60 → 10% in 3 minutes, hold 0.5 minutes, B: ACN, 30 → 80% in 3 minutes, hold 0.5 minutes, C: H₂100mmol ammonium acetate in O/ACN (95/5), 10% isocratic](ii) a Chiral HPLC 100% (S) [222 nm; chiralpak IF 3; 150X 4.6mm, flow 1ml,40 ℃, A90% heptane with 0.1% diethylamine, B10% ethanol]；¹H NMR(600MHz,CH₃OD)ppm 7.44-7.59(m,5H),7.28-7.33(m,2H),7.12-7.24(m,3H),4.24(dd,J＝9.6,5.6Hz,1H),2.55(br t,J＝7.9Hz,2H),2.22-2.42(m,2H)；LC-MS:212(M+H)⁺。

Example 10: asymmetric synthesis of chiral amine 4a, (S) -acenaphthen-1-ylamine using alanine as amine donor

In 60.5ml of a reaction buffer (HEPES 100mM pH 8.0; 4mM PLP; 400mM D, L-alanine), 4.5g of a pyruvate reductase mixture (5.4mg of GDH-102[ Codexis)])；21.6mg LDH-101[Codexis]；126mgNAD[Roche](ii) a 4.32g D-glucose). The purified amino transaminase 3FCR Y59W/Y87F/Y152F/T231A (15ml, protein 2.6 mg/ml; see A3) was then added as a solution and stirred for 5 minutes at 30 ℃. The reaction was started by adding 100mg of 2H-acenaphthylene-1-one dissolved in 20ml DMSO. The reaction pH was manually adjusted to 8.0(2N NaOH) and kept constant by automatic addition of 0.1N NaOH. Initially, the yellow reaction solution was slightly turbid. To prevent potential oxidative aromatization at N₂The reaction was carried out under an atmosphere and in the dark. After 46 hours and 98 area% conversion (IPC-HPLC), the reaction was acidified to pH2.0 to precipitate the enzyme. 5g of filter aid (Dicalite) were added and the mixture was stirred for 20 minutes. Subsequently, the reaction mixture was filtered through a bed of 20g filter aid (Dicalite) and the filter cake was washed with 40ml 0.1N HCl and 50ml TBME. The pH of the combined, slightly yellow, clear aqueous solution was adjusted to pH10 and then amine extracted using 50ml TBME. The combined organic phases were washed with MgSO₄Drying, filtration and evaporation in vacuo at 40 ℃ gave 112mg (112%) of an orange-brown oil as the crude product of the title compound 4 a.

Hydrochloride formation: the crude product was dissolved in 5ml of TBME and 445. mu.l of 2MHCl were added with stirring using a syringe. The resulting very fine suspension was stirred at room temperature for 1 hour and then centrifuged (5 min; 4000 rpm). After decanting the TBME, the remaining solid was resuspended in 10ml TBME, vigorously mixed, centrifuged (5 min; 4000rpm) and decanted again. The moist hydrochloride salt was evaporated in vacuo at 40 ℃ and dried under high vacuum at room temperature for 3h to yield 84mg (69%) of the hydrochloride salt of the title compound 4a as a beige powder.

Chemical and chiral purity HPLC: 81.5% (S) 18.5% (R) [232 nm; chiracel OJ-3; 150 x 4.6mm,3 μm; flow 2.5ml,30 ℃, A: 96% heptane with 0.1% diethylamine, B: 4% ethanol]；¹H NMR(600MHz,CH₃OD)ppm:7.90(d,J＝8.1Hz,1H),7.77(d,J＝8.3Hz,1H),7.54-7.70(m,3H),7.46(d,J＝6.9Hz,1H),5.32(dd,J＝8.1,2.7Hz,1H),3.99(dd,J＝17.8,8.1Hz,1H),3.41(dt,J＝17.8,1.3Hz,1H)；LC-MS:171.1(M+H)⁺。

Example 11: asymmetric synthesis of amine 5a, exo-3-amino-8-aza-bicyclo [3.2.1] oct-8-yl) -phenyl-methanones using 2-propylamine as amine donor

Purified mutant transaminase 3FCR Y59W/Y87F/Y152F/T231A/I234M solution (78 ml total, protein 1.1 mg/ml; see A3) and 100mg 8-benzoyl-8-aza-bicyclo [3.2.1] dissolved in 20ml DMSO]Oct-3-one 5b was added to 82ml of reaction buffer (HEPES 50 mM; 2mM PLP; 0.2M 2-propylamine hydrochloride; pH7.5) with stirring at 30 ℃. The reaction was carried out for 9 days, resulting in a conversion of 75 area% (IPC-HPLC). After 9d, the reaction was acidified to ph2.0, the enzyme was precipitated and stirred for 15 min. Subsequently, the reaction mixture was filtered through a bed of 25g filter aid (Dicalite) and extracted twice with 50ml dichloromethane to remove 8-benzoyl-8-aza-bicyclo [3.2.1]]Oct-3-one 5 b. The combined organic phases were washed with MgSO₄Dried, filtered and evaporated in vacuo at 40 ℃ to yield 37mg (HPLC: 97 area%) of 8-benzoyl-8-aza-bicyclo [3.2.1]Oct-3-one 5b, as a micro-butter. The pH of the aqueous phase was adjusted to 12 using 2N NaOH and then extracted four times with 50ml of dichloromethane. The combined organic matterPhase MgSO₄Drying, filtration and evaporation in vacuo at 40 ℃ and high vacuum drying at 60 ℃ for 36 h gave 60mg (60%) of the title compound 5a as a yellow oil.

Chemical purity HPLC: 98.9 area% [210 nm; X-Bridge C8; 50 x 2.1mm,2.5 μm, flow ml,40 ℃, A: 90% in H₂O/ACN (95/5) 10mmol ammonium acetate, B ACN, 10%](ii) a Chiral SFC 100% exo [210 nm; chiralpak AD-3; 150 x 4.6mm,5 μm; 3ml of flow; left 40 ℃; the right 42 ℃; a82% CO2, B18% methanol with 0.2% 2-propylamine]；¹H NMR(600MHz,DMSO-d₆,120℃)ppm 7.44(s,5H),4.29(br s,2H),3.16(dt,J＝11.1,5.5Hz,1H),1.86-2.00(m,3H),1.79(ddd,J＝13.2，5.1，3.0Hz，3H)，1.67-1.75(m3H)，1.34-1.47(m，2H)；LC-MS：231(M+H)+。

Sequence of

Wild type sequence (tagging):

3FCR wild type (tagged): amino acids (SEQ ID NO: 1+ SEQ ID NO: 56)

3FCR wild type tagging: nucleic acid (SEQ ID NO: 2)

ATGCTGAAAAACGACCAACTGGACCAATGGGACCGTGATAACTTCTTCCACCCGTCAACGCACCTGGCGCAACATGCCCGTGGCGAATCAGCTAACCGTGTGATCAAAACCGCGTCGGGCGTTTTTATTGAAGATCGCGACGGTACGAAACTGCTGGATGCTTTCGCGGGCCTGTATTGCGTTAATGTCGGCTACGGTCGTCAGGAAATTGCCGAAGCAATCGCTGATCAAGCGCGCGAACTGGCCTATTACCATAGCTATGTGGGCCACGGTACCGAAGCTTCTATCACGCTGGCGAAAATGATTCTGGATCGTGCCCCGAAAAACATGAGTAAAGTTTACTTTGGTCTGGGCGGTTCCGACGCAAACGAAACCAATGTCAAACTGATCTGGTATTACAACAATATTCTGGGCCGCCCGGAGAAAAAGAAAATTATCAGTCGTTGGCGCGGTTATCATGGCAGTGGTCTGGTTACCGGCTCCCTGACGGGTCTGGAACTGTTTCATAAAAAATTCGATCTGCCGGTGGAACAGGTTATTCACACCGAAGCCCCGTATTACTTTCGTCGCGAAGACCTGAACCAGACGGAAGAACAATTCGTCGCACACTGTGTGGCTGAACTGGAAGCGCTGATCGAACGTGAAGGCGCGGATACCATTGCGGCCTTCATCGGCGAACCGATTCTGGGTACGGGCGGTATTGTGCCGCCGCCGGCCGGTTATTGGGAAGCAATCCAGACCGTCCTGAATAAACATGATATTCTGCTGGTTGCGGACGAAGTGGTTACCGGCTTTGGTCGCCTGGGCACGATGTTCGGTTCTGATCACTATGGCCTGGAACCGGACATTATCACCATCGCGAAAGGTCTGACGTCAGCGTACGCCCCGCTGAGCGGTTCTATTGTGTCGGATAAAGTCTGGAAAGTGCTGGAACAGGGCACCGACGAAAACGGTCCGATCGGCCATGGTTGGACGTATAGCGCACACCCGATTGGTGCAGCTGCAGGTGTTGCAAATCTGAAACTGCTGGATGAACTGAACCTGGTTAGCAATGCCGGCGAAGTCGGTGCCTACCTGAACGCAACCATGGCAGAAGCTCTGTCCCAACATGCTAATGTTGGCGATGTCCGTGGCGAAGGTCTGCTGTGCGCGGTGGAATTTGTTAAAGATCGTGACAGCCGCACGTTTTTCGATGCCGCAGACAAAATCGGTCCGCAGATTTCTGCGAAACTGCTGGAACAAGATAAAATTATCGCGCGTGCCATGCCGCAGGGCGACATTCTGGGTTTTGCCCCGCCGTTCTGTCTGACCCGCGCAGAAGCTGATCAAGTCGTGGAAGGTACGCTGCGCGCTGTCAAAGCCGTTCTGGGTTCACATCACCATCACCACCACTAA

3GJU wild type (tagged): amino acids (SEQ ID NO: 3+ SEQ ID NO: 56)

3GJU wild-type tagging: nucleic acid (SEQ ID NO: 4)

ATA-3: the amino acids illustrated in FIG. 2 (SEQ ID NO: 5)

ATA-4: the amino acids illustrated in FIG. 2 (SEQ ID NO: 6)

ATA-5: the amino acids illustrated in FIG. 2 (SEQ ID NO: 7)

ATA-6: the amino acids illustrated in FIG. 2 (SEQ ID NO: 8)

ATA-7: the amino acids illustrated in FIG. 2 (SEQ ID NO: 9)

ATA-8: the amino acids illustrated in FIG. 2 (SEQ ID NO: 10)

ATA-9: the amino acids illustrated in FIG. 2 (SEQ ID NO: 11)

Sequence of the double mutant:

3FCR Y59W/T231A: amino acid (SEQ ID NO: 12)

3FCR Y59W/T231G: amino acid (SEQ ID NO: 13)

3FCR Y59F/T231A: amino acid (SEQ ID NO: 14)

3GJU Y59W/T231A: amino acid (SEQ ID NO: 15)

3GJU Y59W/T231G: amino acid (SEQ ID NO: 16)

3GJU Y59F/T231A: amino acid (SEQ ID NO: 17)

Sequences of the three mutants:

3FCR Y59W/Y87F/T231A: amino acid (SEQ ID NO: 18)

3FCR Y59W/Y87L/T231A: amino acid (SEQ ID NO: 19)

3FCR Y59W/Y87V/T231A: amino acid (SEQ ID NO: 20)

3FCR Y59W/Y87F/T231G: amino acid (SEQ ID NO: 21)

3FCR Y59F/Y87F/T231G: amino acid (SEQ ID NO: 57)

3FCR Y59W/T231A/I234F: amino acid (SEQ ID NO: 22)

3FCR Y59W/T231A/I234M: amino acid (SEQ ID NO: 23)

3GJU Y59W/Y87F/T231A: amino acid (SEQ ID NO: 24)

3GJU Y59W/Y87L/T231A: amino acid (SEQ ID NO: 25)

3GJU Y59W/Y87V/T231A: amino acid (SEQ ID NO: 26)

3GJU Y59W/Y87F/T231G: amino acid (SEQ ID NO: 27)

3GJU Y59W/T231A/I234F: amino acid (SEQ ID NO: 28)

3GJU Y59W/T231A/I234M: amino acid (SEQ ID NO: 29)

Sequence of the 4-fold mutant:

3FCR Y59W/Y87F/Y152F/1231A: the amino acids shown in FIG. 3 (SEQ ID NO: 30)

3GJU Y59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 31)

ATA-3Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 32)

ATA-4Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 33)

ATA-5Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 34)

ATA-6Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 35)

ATA-7Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 36)

ATA-8Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 37)

ATA-9Y 59W/Y87F/Y152F/T231A: the amino acids shown in FIG. 3 (SEQ ID NO: 38)

3FCR Y59W/Y87L/Y152F/T231A: amino acid (SEQ ID NO: 39)

3FCR Y59W/Y87F/T231A/P423H: amino acid (SEQ ID NO: 40)

3GJU Y59W/Y87L/Y152F/T231A: amino acid (SEQ ID NO: 41)

3GJU Y59W/Y87F/T231A/P423H: amino acid (SEQ ID NO: 42)

Sequence of 5-fold mutant:

3FCR Y59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 43)

3GJU Y59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 44)

ATA-3Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 45)

ATA-4Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 46)

ATA-5Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 47)

ATA-6Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 48)

ATA-7Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 49)

ATA-8Y 59W/Y87F/Y152F/T231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 50)

ATA-9Y 59W/Y87F/Y152F/7231A/P423H: the amino acids shown in FIG. 4 (SEQ ID NO: 51)

3FCR Y59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 53)

3GJU Y59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 55)

ATA-3Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 58)

ATA-4Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 59)

ATA-5Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 60)

ATA-6Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 61)

ATA-7Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 62)

ATA-8Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 63)

ATA-9Y 59W/Y87F/Y152F/T231A/I234M: the amino acids shown in FIG. 5 (SEQ ID NO: 64)

3FCR Y59W/Y87F/Y152F/T231A/234F: amino acid (SEQ ID NO: 52)

GJU Y59W/Y87F/Y152F/T231A/I234F: amino acid (SEQ ID NO: 54)

Reference to the literature

Coffen D.L.,Okabe M.,Sun R.C.,Lee S.&Matcham G.W.J.(1994)Aminotransferase Catalysis Applied to the Synthesis of a PAFAntagonist.Bioorg.Med.Chem.2,411–413.

Deszcz D.,Affacati P.,Ladkau N.,Gegel A.,Ward J.M.,Hailes H.C.,DalbyP.A.(2015)Single active-site mutants are sufficient to enhance serine:pyruvateα-transaminase activity in anω-transaminase.FEBS J DOI:10.1111/febs.13293

Nobili,A.,Steffen-Munsberg,F.,Kohls,H.,Trentin,I.,Schulzke,C.,M., Bornschele, U.T. (2015) Engineering the active site of the amine-transferase from video fluorescence for the enzyme synthesis of aryl-alkyl amides and aminocohols,ChemCatChem,7,757-760.

Pearson W.R.,Lipman D.J.(1988)Improved tools for biological sequencecomparison.Proc Natl Acad Sci U S A,85,2444-2448.

Savile C.K.,Janey J.M.,Mundorff E.C.,Moore J.C.,Tam S.,Jarvis W.R.,Colbeck J.C.,Krebber A.,Fleitz F.J.,Brands J.,Devine P.N.,Huisman G.W.&HughesG.J.(2010)Biocatalytic Asymmetric Synthesis of Chiral Amines from KetonesApplied to Sitagliptin Manufacture.Science 329,305–309.

S.,M., Redstad, E., Robins, K., Bornscheuer U.T (2009), Arapid and reactive kinetic assay for chromatography of omega-transaminases, anal. chem,81,8244-

Steffen-Munsberg F.,Vickers C.,Thontowi A.,S.,Meinhardt T.,Svedendahl Humble M.,Land H.,Berglund P.,Bornscheuer U.T.,M.(2013)Revealing the Structural Basis of Promiscuous Amine TransaminaseActivity.ChemCatChem,5,154–157.US 2015/0037869 A1

Weiβ,M.S.,Pavlidis,I.V.,Vickers,C.,M., Bornscheuer, U.T, (2014), agarose oxidase based high-through high-phase-assay for substretching and direct evolution of (R) -and (S) -selective amine transaminases, anal. chem,86,11847-11853.

Claims (18)

1. A mutant transaminase having increased transaminase activity relative to a wild-type transaminase, wherein the mutant transaminase comprises an amino acid sequence that is identical to SEQ ID NO: 1 (lugger TM1040 transaminase; referred to as 3FCR) has an amino acid sequence that is at least 65% identical, wherein the mutant transaminase has at least two amino acid substitutions relative to the wild-type transaminase, wherein the amino acid sequence corresponds to SEQ ID NO: 1 by Trp or Phe (Trp 59 or Phe59, respectively) and corresponds to SEQ ID NO: 1 by Ala or Gly (Ala 231 or Gly231, respectively).

2. The mutant transaminase of claim 1,

-wherein in a sequence corresponding to SEQ ID NO: 1 by a hydrophobic amino acid (HYaa87), in particular wherein the hydrophobic amino acid is Leu (Leu87) or Val (Val87) or Phe (Phe 87); and/or

-wherein in a sequence corresponding to SEQ ID NO: 1 the amino acid in position 152 is substituted by Phe (Phe152), and/or

-wherein in a sequence corresponding to SEQ ID NO: 1 the amino acid in position 234 is replaced by Phe (Phe234) or Met (M234), and/or

-wherein in a sequence corresponding to SEQ ID NO: 1 by His (His 423).

3. The mutant transaminase of claim 1 or 2, wherein the transaminase has at least a mutation

Trp59 and Ala231, or

-Trp59, Phe87 and Ala 231; or

-Trp59, Leu87 and Ala 231; or

-Trp59, Val87 and Ala 231; or

-Trp59, Phe87, Ala231 and His 423; or

-Trp59, Phe87, Phe152 and Ala 231; or

-Trp59, Leu87, Phe152 and Ala 231; or

-Trp59, Val87, Phe152 and Ala 231; or

-Trp59, Phe87, Phe152, Ala231 and His423, or

-Trp59 and Gly 231; or

-Trp59, Phe87 and Gly 231; or

-Trp59, Leu87 and Gly 231; or

-Trp59, Val87 and Gly 231; or

-Trp59, Phe87, Phe152 and Gly 231; or

-Trp59, Phe87, Phe152, Gly231 and His 423; or

-Phe59 and Ala 231; or

-Phe59, Phe87 and Gly 231; or

-Trp59, Ala231 and Phe 234; or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Phe 234; or

-Trp59, Leu87, Ala231 and Phe 234; or

-Trp59, Val87, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231 and Phe 234; or

-Trp59, Phe87, Phe152, Ala231, Phe234 and His423, or

-Trp59, Gly231 and Phe 234; or

-Trp59, Phe87, Gly231 and Phe 234; or

-Trp59, Leu87, Gly231 and Phe 234; or

-Trp59, Val87, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231 and Phe 234; or

-Trp59, Phe87, Phe152, Gly231, Phe234 and His 423; or

-Phe59, Phe87, Gly231 and Phe234, or

-Trp59, Ala231 and Met 234; or

-Trp59, Phe87, Ala231 and Met 234; or

-Trp59, Leu87, Ala231 and Met 234; or

-Trp59, Val87, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231 and Met 234; or

-Trp59, Phe87, Phe152, Ala231, Met234 and His423, or

-Trp59, Gly231 and Met 234; or

-Trp59, Phe87, Gly231 and Met 234; or

-Trp59, Leu87, Gly231 and Met 234; or

-Trp59, Val87, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231 and Met 234; or

-Trp59, Phe87, Phe152, Gly231, Met234 and His 423; or

-Phe59, Phe87, Gly231 and Met 234.

4. The mutant transaminase enzyme of any one of claims 1 to 3 in which the transaminase enzyme has at least a mutation

-Trp59, Phe87, Phe152 and Ala231, or

-Trp59, Phe87, Phe152, Ala231 and Met234, or

-Trp59, Phe87, Phe152, Ala231 and H423, or

-Phe59, Phe87 and Gly 231.

5. The mutant transaminase enzyme of any one of claims 1 to 4 in which the transaminase enzyme comprises or consists of a sequence selected from SEQ ID NO: 30 to 38, 43 to 51, 53, 55 and 57-64.

6. The mutant transaminase enzyme of any one of claims 1 to 5 in which the mutant transaminase enzyme comprises a sequence identical to that of SEQ ID NO: 1, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.3 or 5 to 11, or an amino acid sequence which is at least 90%, 95%, 96%, 97%, 98% or 99% identical.

7. The mutant transaminase enzyme of any one of claims 1 to 6 in which the mutant transaminase enzyme has increased transaminase activity for the transamination of at least one compound selected from: 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), and 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a), 3 (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylpropan-1-one (3b), 2H-acenaphthylene-1-one (4b), and 8-benzoyl-8-azabicyclo [3.2.1] octan-3 -ketone (5b) and isopropylamine.

8. The mutant transaminase of any one of claims 1 to 7, wherein the mutant transaminase has at least a 2-fold increase in transaminase activity, preferably at least a 2.5-fold increase in transaminase activity, preferably at least a 3-fold increase in transaminase activity, more preferably at least a 3.5-fold increase in transaminase activity, and most preferably at least a 4-fold increase in transaminase activity, relative to the corresponding wild-type enzyme, in particular for at least one compound selected from: 1- (4-chlorophenyl) -1-phenyl-1-aminomethane (1a), 2-dimethyl-1-phenyl-1-aminopropane (2a), 1, 3-diphenyl-1-aminopropane (3a), 1, 2-dihydroacenaphthylene-1-amine (4a), and 3-amino-8-benzoyl-8-azabicyclo [3.2.1] octane (5a), (4-chlorophenyl) -phenyl-methanone (1b), 2-dimethyl-1-phenyl-propan-1-one (2b), 1, 3-diphenylprop-1-one (3b), 2H-acenaphthylene-1-one (4b), 8-benzoyl-8-azabicyclo [3.2.1] oct-3-one (5b) and isopropylamine.

9. A fusion protein comprising the transaminase enzyme of any one of claims 1 to 8.

10. A nucleic acid encoding the transaminase of any one of claims 1 to 8 or the fusion protein of claim 9.

11. A host cell comprising the transaminase of any one of claims 1 to 8, the fusion protein of claim 9, or the polynucleotide of claim 10.

12. A method of making an amine, the method comprising reacting an amine acceptor with the mutant transaminase of any one of claims 1 to 8 or the fusion protein of claim 9 in the presence of an amine donor.

13. The method of claim 12, wherein an enantiomerically enriched chiral amine is produced by

a) Kinetic resolution of racemic amines in the presence of an amine receptor and a mutant transaminase or fusion protein, or

b) Asymmetric transamination of prochiral ketones in the presence of an amine donor and a mutant transaminase or fusion protein,

wherein the mutant transaminase or fusion protein is stereoselective.

14. The process of claim 13, wherein in process a) the racemic amine has the formula

Wherein the content of the first and second substances,

R¹or R²Independently of one another, represents an optionally substituted alkyl, aryl, carbocyclyl or heterocyclyl group; or

R¹And R²Together with the carbon atoms to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring.

15. The process according to any one of claims 13 to 14, wherein in process a) the amine acceptor is selected from ketones and ketocarboxylic acids.

16. The process of claim 13 wherein in process b) the prochiral ketone has formula (la)

Wherein

R³Or R⁴Independently of one another, represents an optionally substituted alkyl, aryl, carbocyclyl or heterocyclyl group; or

R³And R⁴Together with the carbon atoms to which they are attached form an optionally substituted monocyclic or polycyclic carbocyclic or heterocyclic ring.

17. The process according to claim 13 or 16, wherein in process b) the amine donor is an achiral or chiral amine or amino acid.

18. Use of a mutant transaminase according to any one of claims 1 to 8 or a fusion protein of claim 9 for producing amines, in particular for producing enantiomerically enriched chiral amines by kinetic resolution or by asymmetric transamination.