KR101525549B1 - Asymmetric synthesis method of unnatural amino acids using ω-transaminase catalysis - Google Patents

Abstract

The present invention relates to a method for synthesizing an enantiomerically pure unnatural amino acid from? -Keto acid using ω-TA catalysis using isopropylamine or the like as an amino donor. According to the present invention, an expensive amino donor is not used By using an inexpensive amino donor such as isopropylamine, non-natural amino acid can be asymmetrically synthesized from the corresponding keto acid with excellent process efficiency by omega transaminase. In addition, the amino acid can be synthesized with a good conversion rate without using an additional enzyme introduction step, which is conventionally used to transfer an unbalanced equilibrium on the basis of alpha-transaminase, or to prevent by-product accumulation. In addition, a variety of unnatural amino acids can be synthesized using an omega-TA mutant capable of accommodating an a-keto acid having a bulky substituent.

Description

[0001] The present invention relates to a method for producing an unnatural amino acid using an omega transaminase,

The present invention relates to a method for preparing an unnatural amino acid by catalytic action of an omega transaminase, wherein isopropylamine or the like is used as an amino donor, and a non-natural amino acid And a method for synthesizing the compound.

Non-proteinaceous amino acids, including L-alpha-amino acids and D-alpha-amino acids with unnatural side chains, are the chiral building blocks of many drugs due to their high enzymatic and pharmacodynamic safety and structural diversity imitated by natural counterparts It is becoming increasingly important. For example, pharmacologically important L-homo-alanines have been used as key intermediates in the production of levetiracetam and brivaracetam (anti-epilepsies) and ethambutol (anti-tuberculosis).

Many chemical catalysts and biocatalytic methods are being developed for cost-effective synthesis of unnatural amino acids. Industrial production of L-homo-alanine has been established by reductive amination of 2-oxobutyric acid with tyrosine transaminase (TTA). To overcome the adverse reaction equilibrium, L-aspartate was used as the amino donor, and the resulting keto acid product (i.e., oxaloacetic acid) underwent spontaneous decarboxylation to pyruvic acid. However, this reaction has a problem that the product contamination by L-alanine occurs because TTA has reactivity to 2-oxobutyric acid as well as skin leucine.

In order to reduce such by-product formation, a technique has been disclosed in which a TTA reaction is coupled with a acetolactate synthase capable of converting acetic acid to acetolactate. In the same strategy, TTA is replaced with D-amino acid transaminase (DATA) It was also applied to the production of D-alanine.

Further, as an alternative to deal with an unfavorable equilibrium, L-glutamic acid is used as the amino donor of the primary transaminase and the reaction of the ornithine transaminase is activated simultaneously to shift the reaction equilibrium, resulting in spontaneous cyclization of the keto acid product I also came.

Catalyzed by? -Transaminase (? _-TA ), unlike the nearly equilibrium equilibrium constant ( K _eq ) in reactions catalyzed by? -Transaminase (? -TA) Transamination between the primary amine and alpha -keto acid is energetically very beneficial. For example, the K _eq of transamination of skin leuco acid and alpha -methylbenzylamine was reported to be 1130. Thus, transamination using primary amines as amino donors allows for asymmetric amination of the keto acids without thermodynamic constraints.

The inventors of the present invention have previously developed and demonstrated the asymmetric synthesis of L-homo-alanine using benzylamine as an amino donor. However, as a significant disadvantage of this method, the resultant demineralized product, the high amino acceptor reactivity of the benzaldehyde, has been shown to result in a sharp decrease in the net reaction rate due to the high reverse reaction rate. For example, ( S ) -selective omega-TA from Paracoccus denitrificans showed 84% reactivity to benzaldehyde compared to skin acid (a typical amino receptor for omega-TA).

Thus, inexpensive amino donors that are not reactive or readily removable from the demineralized product are highly desirable in performing preliminary asymmetric synthesis of unnatural amino acids using < RTI ID = 0.0 > omega-TA. ≪ / RTI >

In the case of isopropylamine, acetamine, which is a deamidation product thereof, is a non-reactive amino acceptor and can be easily removed due to its high volatility, and is considered to be an ideal amino donor of ω-TA .

Despite the fact that isopropylamine can effectively synthesize unnatural amino acids including L-homo-alanine and D-homo-alanine, the inventors of the present invention have found that amino Natural amino acid from? -Keto acid through? -TA catalysis by using isopropylamine or the like as a donor.

In order to solve the above-mentioned problems, the present invention provides a method for preparing a compound of formula (I), which comprises the step of using a ( S ) -selective omega transaminase or ( R ) Asymmetric synthesis of natural amino acids.

(I) providing an amino acceptor and an amino donor to the ( S ) -selective omega transaminase or ( R ) -selective omega transaminase,

(Ii) transferring an amino group from the amino donor to the amino acceptor through an amino group transfer reaction by the ( S ) -selective omega transaminase or ( R ) -selective omega transaminase to produce an amino acid.

[Reaction Scheme 1]

  2a-2p 3a-3d L-1a-lp 4a-4d

[Reaction Scheme 2]

 2a-2p 3a-3d D-1a-1p 4a-4d

In the above Reaction Schemes 1 to 2, R ₁ and R ₂ And R < ₃ > are specifically defined by the following structural formula.

In addition, the amino acceptor may be represented by the following formula (1) as? -Keto acid.

[Chemical Formula 1]

Wherein R ₁ is selected from a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and a substituted or unsubstituted aryl group having 6 to 18 carbon atoms, and R ₁ is selected from the group consisting of hydrogen, a halogen atom, a hydroxyl group , A thiol group, and an alkyl group having 1 to 10 carbon atoms.

More specifically, the? -Keto acid is at least one selected from the group consisting of 2-oxobutyrate, pyruvate, glyoxylate, fluoropyruvate, hydroxypyrrolate, mercapto pyruvate, bromopyruvate, 3- 2-oxo pentanoate, 2-oxohexanoate, 2-oxooctanoate, a-methyl-2-oxopentanoate, ≪ / RTI > ketoglutarate and phenylglyoxylate.

The amino donor may be an amine compound represented by the following formula (2).

(2)

In the above formula 2, R ₂ And R ₃ are each independently selected from among hydrogen, an alkyl group having 1 to 10 carbon atoms, and an aryl group having 6 to 18 carbon atoms.

More specifically, the amino donor may be selected from [alpha] -methylbenzylamine, benzylamine, isopropylamine and 2-butylamine.

According to a preferred embodiment of the present invention, the ( S ) -selective omega transaminase is selected from the group consisting of P. denitrificans , PDTA), or may be derived from Ochrobactrum anthropi (OATA), and the ( R ) -selective omega transaminase may be derived from Arthrobacter sp., ARTA) or a mutant thereof (AR _mut TA).

The present invention also provides amino acids prepared according to the asymmetric synthesis method of the unnatural amino acids, intermediates containing them, and pharmaceutical products thereof.

According to the present invention, non-natural amino acids can be asymmetrically synthesized from the corresponding keto acids using an omega transaminase with excellent process efficiency by using an inexpensive amino donor such as isopropylamine without using an expensive amino donor. In addition, the amino acid can be synthesized with a good conversion rate without using an additional enzyme introduction step, which is conventionally used to transfer an unbalanced equilibrium on the basis of alpha-transaminase, or to prevent by-product accumulation. In addition, a variety of unnatural amino acids can be synthesized using an omega-TA mutant capable of accommodating an a-keto acid having a large-sized substituent.

1 is a graph showing the time course of reductive amination of 2-oxobutyric acid using benzylamine or isopropylamine as an amino donor in accordance with an embodiment of the present invention.
Figure 2 is a graph confirming the threonine deaminase (TD) and OATA or AR _mut L- or D- homo-homo-alanine-alanine conversion yield of TA is through the coupling reaction according to one embodiment of the invention.

Hereinafter, the present invention will be described in more detail with reference to the drawings, examples, and test examples. It will be apparent to those skilled in the art, however, that these examples and test examples are provided to further illustrate the present invention and that the scope of the present invention is not limited thereby.

Example

(1) Enzyme analysis

A typical enzyme assay was performed at 37 < 0 > C and 50 mM phosphate buffer (pH 7). One unit of ω-TA activity was defined as the amount of enzyme catalyzing the formation of 1 μmole of acetophenone in 20 mM pyruvate and 20 mM ( S ) - or ( R ) -α-methylbenzylamine. The threonine diaminase (TD) 1 unit was defined as the amount of enzyme that produced 1 μmole of 2-oxobutyrate in 1 minute in 50 mM L-threonine.

(2) Measurement of amino donor and receptor reactivity

Amino donor reactivity was measured with pyruvate (20 mM) and amine (20 mM,? -Methylbenzylamine, benzylamine, isopropylamine) in 50 mM phosphate buffer (pH 7). Enzyme concentration in the reaction mixture is PDTA, OATA and (R) - and ^{1 -} 0.5, 0.7 and 0.2 U mL for selective ω-TA. The reaction was maintained for 10 minutes and the initial rate (i.e., conversion <15%) was determined by analyzing the produced L-alanine (1b) or D-alanine (1b). Amino acceptor reactivity was measured with ( S ) -methylbenzylamine (3a) or ( R ) -methylbenzylamine (3a) (20 mM), a-keto acid (20 mM) and omega-TA (OATA and AR 0.1 and 0.05 U mL ^-1 for _mutant TA, respectively). The initial speed was measured by analyzing the produced acetophenone.

(3) Non-natural  Asymmetric synthesis of α-amino acids

Benzylamine (3b) or isopropylamine (3c) as an amino donor in 100 mM 2 ^- oxobutyrate (2a), 150 mM amine, 0.5 mM PLP and 5 U mL ^-1 OATA in 50 mM phosphate buffer (pH 7) The time course of reductive amination of 2-oxobutyrate (2a) when used was monitored. The reaction volume was 500 占 퐇 and the reaction mixture was incubated at 37 占 폚. Aliquots (10 [mu] l) of the reaction mixture were taken at the defined reaction time and 60 [mu] l acetonitrile was mixed to terminate the reaction. HPLC analysis of 2-oxobutyrate (2a) was performed with the reaction mixture to determine the conversion.

(2-oxobutyrate, pyruvate, fluoropyruvate, hydroxypyruvate, 2-oxooctanoate, 2a, 2b, 2d , 2e and 2n), 150 mM isopropylamine (3c), 0.5 mM PLP and 50 U mL- ¹ ω-TA was performed asymmetric synthesis of the unnatural amino acid (1 mL reaction volume). Keto acids and amino acids were analyzed to determine the conversion and enantiomer excess, respectively.

(4) TD / ω- TA  Coupling reaction

(L-5), 200 mM isopropylamine (3c), 0.5 mM PLP, 5 U mL ^-1 TD and 20 U mL ^-1 ω-TA in 50 mM phosphate buffer (pH 7) Coupling reaction was carried out at 37 ° C to synthesize L-homo-alanine (1a) and D-homo-alanine (1a) (1 mL reaction volume). OATA and AR _mut TA were used for the production of L-homo-alanine (1a) and D-homo-alanine (1a), respectively. The homo-alanine was analyzed to determine conversion yield and enantiomeric excess.

(OATA or AR _mut TA) containing 300 mM L-threonine (L-5), 450 mM isopropylamine (3c), 0.5 mM PLP, 5 U mL ^-1 TD and 10 U mL ^-1 ω- pre-scale synthesis of L-homo-alanine and D-homo-alanine was performed with magnetic stirring at 37 [deg.] C in the reaction mixture. The product was separated from the reaction mixture when the conversion rate exceeded 99%.

(5) Purification and characterization of L-homo-alanine and D-homo-alanine

5 N HCl was added to adjust the pH of the reaction mixture to precipitate the protein. The reaction mixture was filtered through a glass-frit filter funnel to remove protein precipitate. The filtrate was loaded onto a glass column packed with Dowex 50WX8 cation-exchange resin (40 g), then washed with 0.1 N HCl (120 mL) and water (120 mL) and then eluted with 145 mL of 10% ammonia solution . The eluent fractions were pooled and evaporated at 30 &lt; 0 &gt; C and 0.1 bar. The resulting solid was washed with EtOH (50 mL) and then oven-dried overnight. Purified L-homo-alanine and D-homo-alanine were structurally characterized by ¹ H NMR, ¹³ C NMR, IR, elemental analysis and LC / MS to confirm the recovery of the desired pure product.

Experimental Example  One.

Although isopropylamine is an ideal amino donor group, most omega-TA's do not exhibit substantial reactivity to isopropylamine. For example, two typical ω-TA, ie Vibrio flat ruby Alice (Vibrio fluvialis) JS17 derived from (.. JS Shin and BG Kim , J. Org Chem, 2002, 67, 2848-2853) (S) - Selective ω-TA and Arthrobacter ( R ) -selected omega-TA derived from E. coli, sp., ARTA) was found not to be reactive with isopropylamine.

Thus, while showing reactivity to isopropylamine, optimal ω-TA was found which can utilize isopropylamine as an effective amino donor.

To this end, P. denitrificans , PDTA) and the spectrum not trophy foil oak (Ochrobactrum anthropi, OATA) with two (S) cloned from - optionally ω-TA (respectively SEQ ID NO: 1, SEQ ID NO: 2) and sub-reducing the ketone by isopropylamine folk the genetically modified to (R) - selective ARTA (SEQ ID NO: 3) and variants thereof (AR TA _mut, SEQ ID NO: 4) four primary amine (α- methylbenzylamine, benzylamine, diisopropylamine for 2 -Butylamine) was tested for amino donor reactivity, and the results are shown in Table 1 below.

Amine Relative reactivity (%) PDTA OATA ARTA AR _mut TA 3a alpha -methylbenzylamine 100 100 100 100 3b Benzylamine 128 259 3 28 3c Isopropylamine 7 43 2 8 3d 2-butylamine n.r. 23 2 14

Table 1 above shows the amino donor reactivity of primary amines to omega-TA, where the relative reactivity (%) is normalized to ( S ) -? - methylbenzylamine or ( R ) Represents the initial reaction rate (i.e., < 15% conversion).

The reaction conditions and the amine (20 mM) and f Brewer acid (20 mM), a pure enantiomer (S) - and (R) -α- methylbenzylamine (20 mM) is (S) - and (R) - selective ω -TA respectively. In addition, Rac -2-butylamine (40 mM) was used for the reactivity measurement, and nr indicates that the relative reactivity is less than 1%, indicating no reactivity.

Although two ( S ) -selective ω-TA showed higher reactivity than ( S ) -α-methylbenzylamine in benzylamine, the two ( R ) Lt; RTI ID = 0.0 &gt; methylbenzylamine. &Lt; / RTI &gt; PDTA and ARTA showed normal reactivity with alkylamines (i.e., isopropylamine, 2-butylamine). On the other hand, OATA exhibited substantial reactivity to alkylamines and the reactivity of isopropylamine was 43% relative to ( S ) -? - methylbenzylamine. In addition, AR _mut TA showed higher reactivity to isopropylamine than ARTA.

Thus, the ( S ) -selective ω-TA (OATA, SEQ ID NO: 2) cloned from Ochrobactrum anthropi (OATA) and the Arthrobacter In an optional variant of ω-TA (TA _mut AR, SEQ ID NO: 4) is an asymmetric synthesis of D- and L- amino acids, respectively, using isopropyl amine as an effective amino donor - sp, ARTA) of (R) derived from .

Experimental Example  2.

In order to confirm that isopropylamine has a better amino donor than benzylamine, the progress of the amination reaction of 2-oxobutyric acid catalyzed by OATA using the two amines as amino donor was compared, Respectively.

1 shows the result of monitoring the time course of the reductive amination of 2-oxobutyric acid using benzylamine or isopropylamine as an amino donor. The reaction conditions were 2-oxobutyric acid (100 mM), amine (150 mM) and OATA (5 U mL ^-1 ).

Although the reactivity of benzylamine was 6 times higher than isopropylamine, both reactions showed similar conversion rates until 30 minutes, after which the reaction with benzylamine was much slower than with isopropylamine. Also, after 4 hours reaction, the conversion rate reached> 99% for isopropylamine and 86% for benzylamine. This clearly shows that the product inhibition caused by the high amino acceptor reactivity of benzaldehyde (35% relative to hyaluronic acid) is very detrimental to the effective amination of keto acid.

Experimental Example  3.

It is known that most ω-TA have a severe steric constraint on bonding, making it impossible to enter a substituent larger than an ethyl group. Was α- Kane experiment the substrate specificity of the ω-TA for the acid to determine the extent of amino acid that can be produced by the OATA and AR TA _mut derived as optimum ω-TA in accordance with the present invention, and the results [Table 2].

α-keto acid Relative reactivity (%) OATA AR _mut TA 2a 2-oxobutyrate 13 33 2b Pyruvate 100 100 2c Glyoxylate 135 36 2d Fluoropyruvate 43 5 2e Hydroxypyruvate 14 6 2f Mercaptopurylate n.r. n.r. 2g Bromopyruvate n.r. n.r. 2h 3-methyl-2-oxobutyrate n.r. n.r. 2i Trimethylpyruvate n.r. n.r. 2j 2-oxopentanoate n.r. 2 2k 3-methyl-2-oxopentanoate n.r. n.r. 2l 4-methyl-2-oxopentanoate n.r. n.r. 2m 2-oxohexanoate n.r. 2 2n 2-oxooctanoate n.r. 8 2o a-ketoglutarate n.r. n.r. 2p Phenylglyoxylate n.r. n.r.

The relative reactivity (%) in Table 2 represents the initial reaction rate normalized to that of pyruvic acid. The reaction conditions are α-keto acid (20 mM) and ( S ) -α-methylbenzylamine or ( R ) -α-methylbenzylamine (20 mM), and nr indicates no reactivity.

As shown in the above Table 1, OATA exhibited substantial reactivity only to the α-keto acids having substituents smaller than the ethyl group, namely 5 α-keto acids (2a-2e), out of the 16 tested. Further, though α- cake of solid pharmaceutical ARTA (parent enzyme) for the acid is also a multi-mutation was introduced to accommodate butyl substituent of the aryl alkyl ketone, and was found to be still preserved in AR TA _mut.

However, the extended binding pocket of AR _mut TA has improved reactivity to alpha-keto acids with a linear alkyl substituent (i.e., 2j, 2m-2n). In particular, it is noteworthy that 2n exhibited much higher reactivity than 2j and 2m.

Experimental Example  4.

For asymmetric synthesis of unnatural amino acids, in the example according to the present invention, OATA proceeds with α-keto acids which exhibit a relative reactivity of 5% or more, ie 2a and 2d in the following Table 3, and AR _mut TA is 2a -2b, 2d-2e and 2n.

temperament ω-TA Reaction time
(h) Conversion Rate
(%) product
(% ee ) 2a 2-oxobutyrate OATA 0.5 99 L-homo alanine
(&Gt; 99.9) 2d Fluoropyruvate OATA 10 79 L-fluoroalanine
(&Gt; 99.9) 2a 2-oxobutyrate AR _mut TA 0.5 99 D-homo-alanine
(&Gt; 99.9) 2b Pyruvate AR _mut TA 0.2 98 D-alanine
(&Gt; 99.9) 2d Fluoropyruvate AR _mut TA 10 86 D-fluoroalanine
(&Gt; 99.9) 2e Hydroxypyruvate AR _mut TA 2 99 D-serine
(&Gt; 99.9) 2n 2-oxooctanoate AR _mut TA 0.5 99 D-norleucine
(&Gt; 99.9)

Reductive amination of α-keto acid (100 mM) with isopropylamine (1.5 molar equivalents) resulted in a conversion of> 98% and an excellent enantiomeric purity (> 99.9%) within 2 hours, except for the fluoropyruvate (2d) Lt; RTI ID = 0.0 &gt; amino acids. &Lt; / RTI &gt; The amination of fluoropyruvate (2d) in comparison to other α-keto acids showed unexpectedly slow reaction progress in both ω-Ta. Since the formation of enzyme aggregates in the amination reaction was observed, it can be concluded that fluoro pyruvate (2d) or its aminated product, fluoroalanine, induced irreversible enzyme inactivation. It was also found that enzyme precipitation was not observed in the incubation solution containing fluoropyruvate (2d) or isopropylamine (3c), leading to enzyme inactivation. Therefore, for effective amination of fluorpyruvate (2d), a method of enhancing enzyme stability such as enzyme fixation needs to be utilized.

The unnatural amino acids listed in Table 3 above are very important industrially. L-homo-alanine is very useful in the pharmaceutical industry. In addition, D-alanine is a building block of Abarelix (antineoplastic agent), D-serine is a precursor of D-cycloserine to be. D-Fluoroalanine is also being used as an antibiotic due to its ability to inactivate bacterial alanine racemization enzymes.

Experimental Example  5.

In the α-keto acids used in the above Table 3, 2-oxobutyrate was already confirmed to be easily obtainable from an inexpensive precursor, ie, L-threonine (L-5) using threonine diaminase (TD) L-threonine is one of the inexpensive natural amino acids produced by microbial fermentation in large quantities.

Thus, in an embodiment of the present invention, both enantiomers of homoalanine (1a) are coupled to the L-threonine (TD) by coupling with the L-threonine deaminase (TD) -Threonine (L-5) and isopropylamine (3c).

[Reaction Scheme 2]

The resulting carbon skeletons and amino groups of the L-homoalanine (1a) and D-homoalanine (1a) are derived from L-threonine (L-5) and isopropylamine (3c), respectively. The reaction using TD / OATA-coupled L-5 (100 mM) and 3c (2 equivalents) was completed at 60 minutes and the conversion yield was 99% as shown in FIG. 2, and the produced L-homoalanine 1a) was> 99.9% ee .

The OATA to produce D- homo-alanine (1a) under the same conditions, the substrate is substituted by AR _mut TA, TD / TA _mut AR reaction D- homo-alanine as the conversion yield and> 99.9% ee of 98% to 90 bunjjae ( 1a). The longer reaction time required to complete the TD / AR _mut TA reaction was due to the relatively lower reactivity of isopropylamine (3c) to AR _mut TA than to OATA.

From the above results, it can be seen that two enantiomers of pharmacologically important homo alanine (1a) can be easily prepared from inexpensive substrates.

Experimental Example  6.

Using the TD / omega-TA method of Experimental Example 5 in a 50 mL reaction mixture charged with L-threonine (L-5) (1.79 g, 15 mmol) and isopropylamine (3c) (1.94 mL, 22.5 mmol) The synthesis of L-homo-alanine (1a) and D-homo-alanine (1a) was carried out.

As a result, conversion rates exceeding 99% in TD / OATA and TD / AR _mut TA reactions were obtained within 12 hours and 24 hours, respectively. Purification and structural characterization of the desired amino acid products were performed to obtain pure L- (1.20 g, 77.6% yield,> 99.9% ee ) and D-homoalanine 1a (1.21 g, 78.2% yield,> 99.9% ee ).

As described above, according to the present invention,

1. Isopropylamine can be used to asymmetrically synthesize unnatural amino acids from the corresponding keto acids by an effective biocatalyst.

2. Moreover, unlike the? -Ta reaction, the method using? -TA according to the present invention using an inexpensive achiral amino donor is thermodynamically very advantageous. This shows that the process efficiency is very high because it can eliminate the need for additional enzymes to shift the unfavorable equilibrium that is absolutely necessary on the basis of α-TA or to prevent by-product accumulation.

3. Also, with respect to the synthesis of D-amino acids, a significant disadvantage of the method using α-TA is that it requires expensive D-amino acids as amino donors, such as the use of D-aspartic acid in DATA reactions, According to the method described above, this disadvantage can be solved.

4. In addition, to apply the method according to the present invention to the asymmetric synthesis of a variety of unnatural amino acids structurally, the steric constraints of the substrate binding pocket of? -TAs must be resolved. At present more than 20 ω-TA have been reported, but naturally occurring enzymes capable of accommodating α-keto acids with bulky substituents than ethyl groups have not been identified. The present invention solves this problem by designing an omega-TA mutant capable of accommodating a large a-keto acid, thereby producing an omega-TA mutant capable of accommodating various bulky substituents in the substrate binding pocket.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Asymmetric synthesis method of unnatural amino acids using omega          가사 <130> HPC-4357 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1362 <212> DNA <213> Paracoccus denitrificans <400> 1 atgaaccaac cgcaaagctg ggaagcccgg gccgagacct attcgctcta cggtttcacc 60 gacatgccct cggtccatca gcggggcacg gtcgtcgtga cccatggcga ggggccctat 120 atcgtcgatg tccatggccg ccgctatctg gatgccaatt cgggcctgtg gaacatggtc 180 gcgggcttcg accacaaggg cctgatcgag gccgccaagg cgcaatacga ccgctttccc 240 ggctatcacg cctttttcgg ccgcatgtcc gaccagaccg tgatgctgtc ggaaaagctg 300 gtcgaggtct cgccattcga caacggccgg gtcttctata ccaattccgg ctccgaggcg 360 aacgacacca tggtcaagat gctgtggttc ctgcatgccg ccgagggcaa gccgcaaaag 420 cgcaagatcc tgacgcgctg gaacgcctat cacggcgtga ccgcggtttc ggcctcgatg 480 accggcaagc cctacaactc ggtcttcggc ctgccgctgc ccggcttcat ccacctgacc 540 tgcccgcatt actggcgcta tggcgaggaa ggcgagaccg aggcgcaatt cgtcgcccgc 600 ctggcacgcg agcttgagga taccatcacc cgcgagggcg ccgacaccat cgccggcttc 660 ttcgccgagc cggtgatggg cgcggggggg gtgatcccgc cggcgaaggg ttatttccag 720 gccatcctgc cgatcttgcg caagtatgac atcccgatga tctcggacga ggtgatctgc 780 ggcttcgggc gcaccggcaa cacctggggc tgcctgacct acgacttcat gcccgatgcg 840 atcatctcgt ccaagaacct gactgcgggc ttcttcccga tgggcgccgt catcctcggg 900 cccgacctcg ccaagcgggt cgaggccgcg gtcgaggcga tcgaggagtt cccgcacggc 960 ttcaccgcct cgggccatcc ggtcggctgc gccatcgcgc tgaaggccat cgacgtggtg 1020 atgaacgagg ggctggccga gaatgtccgc cgcctcgcac cccgcttcga ggcggggctg 1080 aagcgcatcg ccgaccgccc gaacatcggc gaataccgcg gcatcggctt catgtgggcg 1140 ctggaggcgg tcaaggacaa gccgaccaag acccccttcg acgccaatct ttcggtcagc 1200 gagcgcatcg ccaatacctg caccgatctg gggctgatct gccggccgct gggccagtcc 1260 atcgtgctgt gcccgccctt catcctgacc gaggcgcaga tggacgagat gttcgaaaag 1320 ctggaaaagg cgctcgacaa ggtctttgcc gaggtggcct ga 1362 <210> 2 <211> 1371 <212> DNA <213> Ochrobactrum anthropi <400> 2 atgactgctc agccaaactc tcttgaagct cgcgatatcc gttatcatct ccattcttat 60 accgatgctg tccgcctcga agcggaaggt ccgctcgtca tcgagcgtgg cgatggcatt 120 tacgtcgaag atgtatcggg caagcgctat atcgaagcga tgtcaggact gtggagtgtt 180 ggcgtgggct tttccgaacc gcgtctggcc gaagcagctg cacgacagat gaagaagctg 240 cctttctacc atacattctc ctaccgttcg catggtcctg tcattgatct ggcagaaaag 300 cttgtctcaa tggctcctgt tccgatgagc aaggcctact tcaccaattc aggttccgaa 360 gccaacgata cggtcgtcaa gttgatctgg tatcgctcca atgcgctggg tgaaccggag 420 cgcaagaaaa tcatctcacg caagcgcggc tatcacggtg tgacgattgc ctctgccagc 480 ctgaccggct tgcccaacaa tcaccgttct ttcgatctgc cgatcgatcg tatcctgcat 540 acgggctgcc cgcattttta tcgcgaagga caggctggcg agagtgagga acaattcgca 600 acgcggctgg cggatgagct ggaacagttg atcatcgcgg aaggtcctca caccatcgct 660 gctttcattg gcgagccggt gatgggggct ggcggcgtag tcgtgccgcc caaaacctat 720 tgggaaaaag tgcaggctgt tctcaagcgc tacgatattc tgctgatcgc cgacgaggtt 780 atttgcggct tcggacggac aggcaatctg ttcggcagcc agactttcga tatgaaaccg 840 gacattctgg tgatgtcgaa gcagctttcg tcatcctatc tgccgatttc ggccttcctc 900 atcaacgagc gtgtgtacgc gccaattgcc gaagaaagcc acaagatcgg cacgcttggc 960 acgggcttca cggcatctgg ccatccggtg gcggcagcgg tagcgctgga aaacctcgcc 1020 attattgaag agcgtgatct ggtcgccaat gcgcgcgacc gcggcaccta tatgcagaag 1080 cgcctgcgtg agttgcagga tcatcctctg gtcggcgaag tgcgtggcgt tggtctcata 1140 gccggtgtcg agcttgtcac cgacaagcag gccaagacgg gccttgaacc aaccggcgct 1200 ctgggcgcaa aggcaaacgc cgttcttcag gagcgcggcg tcatttcccg cgcaatgggc 1260 gatacgcttg ccttctgccc gccgctcatc atcaacgatc agcaggttga tacgatggtg 1320 tccgcgctcg aggcgacgct gaacgatgtt caggcaagcc tcaccaggta a 1371 <210> 3 <211> 993 <212> DNA <213> Arthrobacter sp. <400> 3 atggcattca gcgccgatac cagcgaaatt gtttataccc atgataccgg tctggattat 60 atcacctata gcgattatga actggatccg gcaaatccgc tggcaggcgg tgcagcatgg 120 attgaaggtg catttgttcc gcctagcgaa gcacgtatta gcatttttga tcagggctat 180 ctgcatagtg atgttaccta taccgtgttt catgtgtgga atggtaatgc atttcgcctg 240 gatgatcata ttgaacgtct gtttagcaat gcagaaagca tgcgtattat tccgcctctg 300 acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360 gcatttgtta gcgttagcat tacccgtggt tatagcagca caccgggtga acgtgatatt 420 accaaacatc gtccgcaggt ttatatgtat gcagttccgt atcagtggat tgttccgttt 480 gatcgtattc gtgatggtgt tcatgcaatg gttgcacaga gcgttcgtcg tacaccgcgt 540 agcagcattg atccgcaggt taaaaatttt cagtggggtg atctgattcg tgcagttcaa 600 gaaacccatg atcgtggttt tgaagcaccg ctgctgctgg atggtgatgg tctgctggcc 660 gaaggtagcg gttttaatgt tgttgtgatt aaagatggtg tggttcgtag tccgggtcgt 720 gcagcactgc ctggtattac ccgtaaaacc gttctggaaa ttgcagaaag cctgggtcat 780 gaagcaattc tggcagatat taccctggca gaactgctgg atgcagatga agttctgggt 840 tgtaccaccg caggcggtgt ttggccgttt gttagcgtgg atggtaatcc gatttcagat 900 ggtgttccgg gtccgattac ccagagcatt attcgtcgtt attgggaact gaatgttgaa 960 agcagcagcc tgctgacacc ggttcagtat tga 993 <210> 4 <211> 993 <212> DNA <213> Arthrobacter sp. <400> 4 atggcattta gcgcagatac accggaaatt gtttataccc atgataccgg tctggattat 60 attacctata gcgattatga actggatccg gcaaatccgc tggcaggcgg tgcagcatgg 120 attgaaggtg catttgttcc tccgagcgaa gcacgtatta gcatttttga tcagggcttt 180 tataccagtg atgcaaccta taccaccttt catgtgtgga atggtaatgc atttcgtctg 240 ggtgatcata ttgaacgtct gtttagcaat gccgaaagca ttcgtctgat tcctccgctg 300 acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360 gcaatggtta ccgttaccat tacccgtggt tatagcagca ccccgtttga acgtgatatt 420 accaaacatc gtccgcaggt ttatatgagc gcatgtccgt atcagtggat tgttccgttt 480 gatcgtattc gtgatggtgt tcatctgatg gttgcacaga gcgttcgtcg tacaccgcgt 540 agcagcattg atccgcaggt taaaaatttt cagtggggtg atctgattcg tgcaattcag 600 gaaacccacg atcgcggttt tgaactgccg ctgctgctgg attgtgataa tctgctggcc 660 gaaggtccgg gttttaatgt tgttgttatt aaagatggcg tggttcgtag tccgggtcgt 720 gcagcactgc ctggtattac ccgtaaaacc gttctggaaa ttgcagaaag cctgggtcat 780 gaagcaattc tggcagatat tacaccggca gaactgtatg atgcagatga agttctgggt 840 tgtagcaccg gtggtggtgt ttggccgttt gttagcgttg atggtaatag cattagtgat 900 ggcgttccgg gtccgattac ccagagcatt attcgtcgtt attgggaact gaatgttgaa 960 ccgagcagcc tgctgacacc ggttcagtat tga 993

Claims (12)

A method of asymmetric synthesis of unnatural amino acids using ( S ) -selective omega transaminase or ( R ) -selective omega transaminase,
(i) providing? -keto acid and isopropylamine to the ( S ) -selective omega transaminase or ( R ) -selective omega transaminase; And
(ii) transferring an amino group from the isopropylamine to the? -keto acid through an amino group transfer reaction by the ( S ) -selective omega transaminase or ( R ) -selective omega transaminase to produce an amino acid Lt; / RTI &gt;
A method for asymmetric synthesis of an unnatural amino acid, wherein the? -Keto acid is represented by the following formula 1:
[Chemical Formula 1]

In the above formula (1)
R ₁ is selected from a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and a substituted or unsubstituted aryl group having 6 to 18 carbon atoms,
The R ₁ may further be substituted with at least one member selected from the group consisting of hydrogen, a halogen atom, a hydroxyl group, a thiol group, and an alkyl group having 1 to 10 carbon atoms. delete The method according to claim 1,
The? -Keto acid may be at least one selected from the group consisting of 2-oxobutyrate, pyruvate, glyoxylate, fluoropyruvate, hydroxypyrrolate, mercapto pyruvate, bromopyruvate, 3- 2-oxopentanoate, 2-oxohexanoate, 2-oxoctanoate, a-ketoglutarate, 2-oxoheptanoate, Lt; RTI ID = 0.0 &gt; aminocarboxylic &lt; / RTI &gt; acid and a phenyl glyoxylate. delete delete The method according to claim 1,
The ( S ) -selective omega transaminase may be selected from the group consisting of P. denitrificans , PDTA), or from Ochrobactrum anthropi (OATA). &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 6,
Characterized in that said ( S ) -selective omega transaminase is transcribed after being transcribed from the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1,
The ( R ) -selective omega transaminase may be selected from the group consisting of Arthrobacter sp., ARTA) or a mutant thereof (AR _mut TA). 9. The method of claim 8,
Characterized in that said ( R ) -selective omega transaminase is transcribed after being transcribed from the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. delete delete delete

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