KR20140117720A - Method for asymmetric synthesis of d-amino acids using omega-transaminase - Google Patents

Abstract

The present invention relates to a method for synthesis of a pure optical isomer D-amino acid using a (R)-selective omega-transaminase. The (R)-selective omega-transaminase is derived from Aspergillus terreus and Aspergillus fumigatus. The present invention can purely mass produce industrially important D-amino acids optically by an asymmetric synthesis method using a (R)-selective omega-transaminase.

Description

METHOD FOR ASYMMETRIC SYNTHESIS OF D-AMINO ACIDS USING OMEGA-TRANSAMINASE USING OMEGA-TRANSAMINASE BACKGROUND OF THE INVENTION [0001]

The present invention relates to a process for producing pure optically isomeric D-amino acids using (R) -selective omega-transaminase. The (R) -selective omega-transaminase is derived from Aspergillus terreus and Aspergillus fumigatus .

D-amino acids are important chiral building blocks in the synthesis of antibiotics, chiral auxiliaries, pharmaceuticals, food additives and agrochemicals. For example, D-phenylglycine and D- p -hydroxyphenylglycine are conventional structural components of beta-lactam antibiotics, ampicillin and amoxicillin. For example, D-phenylglycine and D- p -hydroxyphenylglycine are essential structural components for various beta-lactam antibiotics, ampicillin and amoxicillin, respectively. D-fluoroalanine is used as an antibiotic by inactivating the bacterial alanine racemase. D-valine is an intermediate used in the synthesis of pyrethroid insecticide fluvulinate. L-amino acids are produced by microbial fermentation, whereas D-amino acids are not produced by fermentation. Because most D-amino acids are not natural metabolites of microorganisms. Thus, the synthesis of D-amino acids can be carried out either by a chemical catalyst preparation method or by the kinetic analysis of racemic amino acid derivatives using biocatalysts such as acylase, amidease, and hydantoinase / carbamoylase resolution has been developed.

The asymmetric synthesis of amino acids by reductive amination of keto acids is an effective method as compared to the kinetic resoultion strategy because keto acids can be converted to the desired enantiomers of amino acids by 100%. The reductive amination method of keto acid can be selected from two natural enzymes. For example, amino acid dehydrogenase (AADH) and transaminase (TA). AADH requires a secondary dehydrogenase to recycle cofactors because it requires NADH as an external cofactor in the case of reductive amination of keto acid by ammonium. A successful example of amino acid synthesis using AADH relates to the industrial production of catalyzed L-tert-leucine by coupling of leucine dihydrogenase with formate dihydrogenase. In contrast, TA catalyzes the reversible transfer of amino groups between the amino donor and acceptor and does not require an external cofactor. However, since the reaction equilibrium of the amino group transfer between amino acid and keto acid is usually close to 1, it is difficult to efficiently produce pure optical isomeric amino acid using TA.

TA is a conventional pyridoxal 5 ' -phosphate (PLP) -dependent enzyme. Amino group transfer reaction consists of two half reactions. Oxidative deamination of an amino donor with conversion of PLP to pyridoxamine 5'-phosphate (PMP); And the reductive amination of an amino acceptor that regenerates from the enzyme PMP (E-PMP) to the enzyme PLP (E-PLP).

TA can be divided into? -Transaminase (? -TA) and? -Transaminase (? -TA) depending on the position of the amino group to be transferred. Unlike α-TA, which accepts only α-amino acids and α-keto acids as substrate pairs, ω-TA exhibits unique properties that deaminate primary amines. Since the ω-TA reaction between the primary amine and α-keto acid (usually pyruvate) is thermodynamically stable, the reaction equilibrium can be shifted to achieve a high conversion rate without removing by-products. In addition to kinetic resolution of racemic primary amines, thermodynamically stable ω-TA reactions have been used for asymmetric synthesis of L-amino acids catalyzed by (S) -selective ω-TA. However, specific studies have not been conducted on D-amino acid asymmetric synthesis using (R) -selective omega transaminase. That is, the first (R) -selective ω-TA can be detected by enrichment screening a long time after the discovery of (S) -selective ω-TA by Arthrobacter sp . It was identified from KNK168. Recently, a variety of (R) -selective ω-TA was identified by the in silico method. However, despite the availability of (R) -selective omega-TA, the application of the omega-TA reaction on the production of D-amino acids has not been reported. There is a continuing need in the pharmaceutical and agrochemical industries for a method for mass-producing D-amino acids inexpensively.

Disclosure of the Invention The present invention has been made to solve the above-mentioned problems, and provides a method for synthesizing various D-amino acids from (R) -amine and -keto acid using (R) -selective ω-TA. More specifically, it is an object of the present invention to provide a method for mass-synthesizing D-amino acids in a thermodynamically favorable manner.

In order to solve the above problems, the present inventors have investigated the substrate specificity of the two (R) -selective omega transaminases derived from A. terrus and A. fumigatus . We have investigated the substrate specificity of omega transaminases to D-amino acids, alpha -keto acid and primary amines and compared them with previously identified (S) -selective omega transaminases. The present inventors identified 5 reactive? -Keto acids and demonstrated the synthesis of excellent pure optical isomers of 99.7% (ee) or more of the corresponding D-amino acids from? -Keto acid.

In addition, the present invention has found that (R) -selective omega transaminase is similar to (S) -selective omega transaminase as previously identified, and based on this (double recognition in the large pocket and small pocket, ), We could suggest an active site model that plays an important role in recognizing the structural differences of the substrates.

The method for producing D-amino acids using the (R) -selective? -TA of the present invention can be represented by the reaction formula (1).

[Reaction Scheme 1]

Further, the production method of D-amino acid using (R) -selective ω-TA of the present invention is as follows:

(a) providing an amino donor and an amino acceptor to the (R) -selective omega transaminase;

(b) deoxidizing the amino group from the amino donor to the amino acceptor through an amino group transfer reaction by the (R) -selective omega transaminase to produce a D-amino acid.

Alternatively, the amino donor is selected from the group consisting of (R) -α-MBA, benzylamine, (R) -1-ethylbenzylamine, and (R) -1-aminoindan.

Alternatively, the amino acceptor is? -Keto acid.

Alternatively, the? -Keto acid is characterized in that it is selected from the group consisting of pyruvate, fluoropyruvate, hydroxypyruvate, 2-oxobutyrate and 2-oxopentanoate.

Alternatively, the present invention is characterized by using (R) -selective omega transaminase derived from Aspergillus terreus .

Alternatively, the present invention is characterized by the use of (R) -selective omega transaminase derived from Aspergillus fumigatus .

Alternatively, the present invention provides a method for synthesizing D-alanine from (R) -α-MBA and pyruvate using (R) -selective omega transaminase.

Alternatively, the present invention provides a method for synthesizing D-homo-alanine from (R) -α-MBA and 2-oxobutyrate using (R) -selective omega transaminase.

Alternatively, the present invention provides a method for synthesizing D-serine from (R) -α-MBA and hydroxypyruvate using (R) -selective omega transaminase.

Alternatively, the present invention provides a method for synthesizing D-fluoroalanine from (R) -α-MBA and fluoropyruvate using (R) -selective omega transaminase.

Alternatively, the present invention provides a method for synthesizing D-norvaline from (R) -α-MBA and 2-oxopentanoate using (R) -selective omega transaminase.

By virtue of the asymmetric synthesis method using the (R) -selective omega transaminase provided by the present invention, industrially important D-amino acids can be optically purely mass-produced.

Figure 1. Active site model of (R) -selective omega-transaminase. a) Substrate-binding pocket of E-PLP that accommodates (R) -α-MBA or D-alanine. b) Estimated structure of the active site for (R) -α-MBA bound to the internal aluminide and pocket formed between the PLP and the active site lysine.
Figure 2. Effect of acetophenone on enzyme activity. Relative activity was measured in 20 mM (R) -α-MBA, 20 mM pyruvate, and the concentration of acetophenone was varied in 50 mM phosphate buffer.
Figure 3. D-Alanine production in pyruvate was monitored over time using (R) -selective AT-omega-transaminase.

Hereinafter, the present invention will be described in more detail with reference to the drawings, examples, and test examples. It will be apparent, however, to those skilled in the art 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.

Amino receptor specificity

To perform asymmetric synthesis of D-amino acids using (R) -selective omega transaminase, the inventors first examined keto acids which can effectively act as amino receptors. The range of D-amino acids that can be produced by the two (R) -selective omega transaminases derived from A. terrus and A. fumigatus (abbreviated as AT ω-TA and AF ω-TA, respectively) To do this, we tested 17 alpha -keto acids. The results are shown in Table 1. Two kinds of ω-TA showed similar amino receptor specificity for α-keto acid. Among 17 alpha -keto acids, pyruvate showed the highest reactivity. The present inventors have found that the reactivity of amino acceptors is dramatically reduced in [alpha] -keto acid having a substituent larger than that of the ethyl group. Other? -Keto acids with aliphatic or aromatic substituents greater than ethyl groups have proved to be inert.

In Table 1, "relative reactivity" indicates an initial velocity normalized by pyruvate. The reaction conditions are 20 mM (R) -α-MBA, 20 mM α-keto acid, 0.1 U / mL ω-TA and 50 mM phosphate buffer (pH 7), AT ω -TA and AF ω-TA The initial rates for pyruvate used were 0.12 and 0.11 mM / min, respectively. In Table 1, n.r. means non-reactivity and indicates that the relative reactivity is 1% or less.

Two kinds of ω-TA showed little reactivity to α-ketoglutarate in a similar manner to (S) -selective ω-TA. Of the four pyruvate analogues, bromopyrate and mercapto pyruvate were inactive. This is due to spatial limitations due to large substituents. The spatial impairment in the binding pocket of E-PMP appears to overwhelm the electrical effects of negatively charged substituents that increase the electrophilic properties of carbonyl carbon. The low reactivity of fluoropyruvate and hydroxypyruvate to pyruvate suggests that the spatial impairment overwhelms the electrical effect. The glyoxylate having a substituent smaller than the methyl group is less reactive than the pyruvate, indicating that the binding pocket accepting the substituent is suitable for the methyl group.

Comparing the relative reactivity of the four reactive α-keto acids with substituents larger than the methyl group, despite the similar amino receptor specificity of the two ω-TAs, the binding pocket of AT ω-TA has AF ω-TA It can be seen that it is more flexible. Thus, based on the reactivity of? -Keto acid, AT? -TA is more suitable for the asymmetric amination of? -Keto acid to produce D-amino acid.

Amino donor specificity

To investigate whether the spatial impairment of E-PMP, which defines the amino-receptor specificity, is present in E-PLP, we measured the relativity of the 13 amino acids to (R) -α-MBA, Respectively.

In Table 2, "relative reactivity" indicates an initial velocity normalized by (R) -α-MBA. The reaction conditions were 20 mM racemic amino donor (except 10 mM glycine), 10 mM pyruvate, 0.3 U / mL of? -TA and 50 mM phosphate buffer (pH 7), AT? -TA and AF? -TA The initial velocities for (R) -α-MBA were 0.15 and 0.14 mM / min, respectively. In Table 2, n.r. means non-reactivity and indicates that the relative reactivity is 1% or less. In Table 2 above, 2-oxobutyrate (10 mM) was used as the amino acceptor. Relative reactivity was based on the reactivity of (R) -α-MBA using 2-oxobutyrate as the amino acceptor.

No more reactive D-amino acids than (R) -α-MBA were found. Compared to amino-receptor specificity, the two ω-TA showed similar amino donor specificities for amino acids. Comparison of amino acceptor specificity and amino donor specificity showed that the spatial impairment of E-PMP is exactly the same as the binding pocket of E-PLP. All amino acids with larger side chains than ethyl group except D-norvaline were not reactive. Consistent with little reactivity of? -ketoglutarate, the two amino acids with carboxylate on the side chain, such as D-aspartate and D-glutarate, were inactive.

Consistent with the amino acceptor reactivity of α-keto acid, relative reactivity higher than 1% was observed in D-alanine, D-serine, D-homoalanine and D-norvaline among the 13 amino acids tested. It is interesting that glycine is inactive, although the corresponding keto acid (i.e., glyoxylate) of glycine shows substantial amino acceptor reactivity.

(R) -elective ω- TA Active site model

The present inventors have been able to propose an active site model of (R) -selective ω-TA by substrate specificity for α-keto acid (Table 1) and D-amino acid (Table 2). In a previous report, we used Vibrio < RTI ID = 0.0 > An active site model of (R) -selective ω-TA isolated from fluvialis JS17 (VF -TA) was constructed. Despite the opposite optical stereospecificity, the substrate specificity of the two (R) -selective ω-TA suggested the active site of (R) -selective ω-TA based on the similarity of VF ω-TA. The active site model consists of two different sized pockets, such as large (L) and small (S) pockets (Fig. 1A). The high reactivity of (R) -α-MBA and D-alanine suggests that the L pocket recognizes a hydrophobic substituent (eg, a phenyl group of (R) -α-MBA) as well as a carboxylate (eg D-alanine) do. This dual recognition mode is reminiscent of aspartate aminotransferase and aromatic amino acid aminotransferase. It is also observed in (S) -selective omega-TA. The spatial size limitation in the S pocket for substituents larger than ethyl groups is the main determinant of substrate specificity for amino donors as well as for amino acceptors.

In order to estimate the directionality of the binding pocket, it should be located near the C4 'of the internal aldimine formed between the PLP and the active site lysine for continuous reaction of the amino group of (R) -α-MBA (FIG. However, when (S) -α-MBA is bound to a binding pocket with a phenyl group and a methyl group docked in the L and S pockets, the amino group will be far away from the internal aldimines and will not perform a nucleophilic attack on C4 ' . This may account for the severe optical stereospecificity of (R) -selective omega-TA.

Since the proposal for the active site model of (R) -selective ω-TA was based on (S) -selective ω-TA, the opposite optical stereospecificity of (R) - and (S) The binding site model should be explained. The present inventors speculate that the two binding pockets of (R) - and (S) -selective ω-TA are inversely related to the relative positions of the internal aldimines. Thus, the optical stereospecificity can be determined by the relative accessibility of the internal amino groups and the aldimines of each optical isomer of the substrate attached to the binding pocket.

Selection of amino donors for asymmetric synthesis of D-amino acids

Reaction equilibrium of amino group transfer between α-keto acid and amino acid occurs naturally between substrate and product. Thus, instead of D-amino acids, primary amines should be used as amino donors for the thermodynamically favorable amination of α-keto acids. For this purpose, the inventors measured the reactivity of five primary amines [Table 3]. Benzylamine and (R) -a-ethylbenzylamine showed lower reactivity than (R) -α-MBA in two kinds of ω-TA, and S pocket of (R) . The 1-aminoindan showed high reactivity and was also observed with (S) -selective ω-TA. Recently, isopropylamine is very attractive as an inexpensive amino donor of? -TA. However, the present inventors have found that isopropylamine is inactive for two types of omega-TA. Based on the reactivity of amines, (R) -α-MBA proved to be the best amino donor in the asymmetric synthesis of D-amino acids.

In Table 3, "relative reactivity" refers to the initial velocity normalized by (R) -α-MBA. The reaction conditions are 20 mM racemic amino donor (except 10 mM benzylamine and isopropylamine), 10 mM pyruvate, 0.3 U / mL of ω-TA and 50 mM phosphate buffer (pH 7). In Table 3, nr means non-reactivity and indicates that the relative reactivity is 1% or less.

Production inhibition by acetophenone

Enzyme inhibition by ketone products produced by the depolymerization of amine substrates was generally observed in (S) -selective ω-TA. This product inhibition has a deleterious effect on the efficient amination of? -Keto acid when amines such as? -MBA are used as amino donors. We investigated whether the two (R) -selective ω-TA is susceptible to this product inhibition. Interestingly, product inhibition by acetophenone was not found up to 40 mM, and removal of acetophenone during the amine transfer between? -MBA and? -Keto acid was not required.

Asymmetric synthesis of D-amino acids

Since enantiopure (R) -α-MBA is expensive, rac-α-MBA was used as an amino donor to make D-amino acids. Asymmetric amination of 60 mM rac-α-MBA and 20 mM pyruvate proceeded efficiently using AT ω-TA and 99.3% conversion from pyruvate to D-alanine over 0.5 hour (FIG. 3). In addition to D-alanine, the present inventors have also found that D-homoalanine, D-serine, D-fluoroalanine and D-serine can be deduced from 2-oxobutyrate, hydroxypyrrobait, fluoropyruvate and 2-oxopentanoate, D-norvaline was synthesized asymmetrically, which is shown in Table 4. Due to the low reactivity of 2-oxopentanoate, conversion rates of over 99% were achieved within 10 hours, except for D-norvaline. The enantiomeric purity of the resulting D-amino acid was more than 99.7%.

The reaction conditions are 37 to 60 mM rac-α-MBA, 20 mM α-keto acid, 3 U / mL AT ω-TA, 50 mM phosphate buffer (pH 7) and 0.1 mM PLP.

Example  1. Genes Cloning

Two (R) -selective ω-TA genes derived from A. terrus and A. fumigatus were synthesized (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) and cloned into pGEM-T vector. The ω-TA gene was amplified using a PCR primer to subclone the gene into pET28a (+) expression vector (WI, Novagen). The primers for A. terreus are shown in Table 5 below, and the primers for A. fumigatus are shown in Table 6 below.

division Baseball Forward primer (5'-AGAAGGAGATATA CCATGG CCTCCATGGACAAAGTCT-3 ') Reverse primer (5'-GGTGGTGGTGGTG CTCGAG GTTCCTCTCGTTATAATC-3 ')

division Base sequence Forward primer (5'-AGAAGGAGATATA CCATGG CCTCTATGGACAAAGTCT-3 ') Reverse primer (5'-GGTGGTGGTGGTG CTCGAG GCCACTGCCATAGTCAAC-3 ')

The forward primer has an NcoI site and the reverse primer has an XhoI site. The underlined portion of the primer in [Table 5] and [Table 6] represents the restriction enzyme site in the nucleotide. The gene fragment amplified by PCR was digested with restriction enzymes and ligated with the NcoI / XhoI fragment of pET28a (+) using Infusion HD cloning kit (CA, Clontech). The cloning products were identified by DNA sequencing.

Example  2. Cell culture and enzyme purification

(R) - Escherichia with the pET28a (+) expression vector carrying the optional ω-TA gene coli BL21 (DE3) cells were cultured at 37 占 폚 in LB medium containing 50 占 퐂 / ml kanamycin. Protein expression was induced by IPTG at about 0.6 OD ₆₀₀ (final concentration = 1 mM) and cells were incubated for 6 hours. The cell pellet was resuspended in 20 mL resuspension buffer (50 mM Tris-HCl, 50 mM sodium chloride, 1 mM EDTA, 1 mM [beta] -mercaptoethanol, 0.1 mM PMSF, 0.5 mM PLP, pH 7.0). The cell suspension was sonicated, centrifuged at 17,000 x g for 30 minutes at 4 DEG C to remove cells, and the supernatant was obtained as crude extract.

The crude extract was loaded on a HisTrap HP column (GE Healthcare) and the His ₆ -tagged omega-TA was purified and elution buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 mM PLP, pH 7.4). The active enzyme fractions were loaded on a HiTrap desolating column (GE Healthcare) for buffer exchange using elution buffer (50 mM sodium phosphate, 0.15 M sodium chloride, 0.5 mM PLP, pH 7).

Example  3. Analysis of purified enzyme

Analysis of the enzyme was performed at 37 ° C, pH 7 (50 mM phosphate buffer). Normally, the reaction volume was 50 占 퐇, and the reaction was stopped after 10 minutes by adding 300 占 퐇 of acetonitrile. One unit of (R) -selective omega transaminase means that 1 μmole of acetophenone is produced in 1 minute in the presence of 20 mM (R) -methylbenzylamine and 20 mM pyruvate. Acetophenone was analyzed by HPLC to determine the initial reaction rate.

Example  4. (R) - Selective ω- TA Of amino acid receptors

The initial reaction rate (ie, conversion rate <20%) was measured to investigate the substrate specificity of (R) -selective ω-TA. To investigate amino-receptor specificity, the reactivity of 17 alpha -keto acids was compared (see Table 1). The concentration of both the amino donor and acceptor in the reaction mixture was 20 mM. (R) -α-MBA was used as the amino donor. The initial reaction rate was determined by measuring the amount of acetophenone produced by HPLC.

To investigate the amino donor specificity, five primary amines and 13 amino acids were tested in the presence of pyruvate (10 mM) used as the amino acceptor (see Table 2). A racemic form of the amino donor (20 mM) was used except for the acylamino donor (10 mM). In the case of alanine, 2-oxobutyrate was used as the amino donor. The initial reaction rate was determined by analyzing D-alanine or D-homo-alanine GITC derivative by HPLC.

Example  5. Product inhibition

(0-40 mM) of acetophenone under the reaction conditions of 20 mM (R) -α-MBA, 20 mM pyruvate, 0.1 U / mL enzyme and 50 mM potassium phosphate buffer (pH 7) Product inhibition was investigated. The initial reaction rate was determined by analyzing D-alanine.

Example  6. Asymmetric synthesis of D-amino acids

The reaction conditions for the asymmetric synthesis of D-amino acids were 20 mM α-keto acid, 60 mM rac-α-MBA, 50 mM potassium phosphate buffer (pH 7), 0.1 mM PLP and 3 U / mL purified A. terreus The result is -TA. Conversion rates and enantiomeric purity were determined by analyzing the residual? -Keto acid and the resulting amino acid.

Analysis method

Acetophenone analysis was performed on a Waters HPLC system (Milford, MA) using a Symmetry column (Waters, USA) with 60% methanol (A) / 40% water (B) containing 0.1% trifluoroacetic acid at 1 mL / ). &Lt; / RTI &gt; The analysis was carried out with a UV detector adjusted to 254 nm. For the aciral analysis of amino acids, 2,3,4,6-tetra-O-acetyl -? - D-glucopyranosyl isothiocyanate (GITC) was used for amino acid derivatization. 20 μl of triethylamine stock (100 mM dispersed in acetonitrile) and 90 μl of TDW were diluted with acetonitrile to a final total concentration of 0.3 molar equivalent or less of GITC in a conventional GITC derivatization method. . The resulting solution was allowed to stand at room temperature for 90 minutes. Analysis of the amino acid derivatives was carried out on a Symmetry column using isocratic elution at 1 mL / min of 40% A / 60% B. UV analysis was performed at 254 nm. The retention times of the L / D enantiomers of alanine, homo-alanine and fluoroalanine were 11.5 / 16.3, 26.9 / 28.7 and 11.1 / 14.8 min, respectively. The stasis time of the L / D enantiomer of serine was 212.6 / 228.3 min at 20% A / 80% B. The stagnation time of the L / D enantiomer of norvaline was 4.0 / 5.1 min at 60% A / 40% B.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> method for asymmetric synthesis of D-amino acids using          omega-transaminase <130> HPC3950 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 978 <212> DNA <213> Aspergillus terreus <400> 1 atggcctcca tggacaaagt ctttgccggc tacgccgccc gccaagcgat cctcgaatca 60 accgagacca ccaacccctt tgcgaagggt atcgcctggg tagaaggcga gctggtgccc 120 ctggcagagg cacgcattcc actgctcgac cagggcttca tgcacagcga tctcacctac 180 gacgtgccct ccgtctggga cggccgcttc ttccggctag acgaccacat cacgcggctc 240 gaagccagct gcaccaagct ccggctgcga ctgccactcc cgcgcgacca ggtcaagcag 300 attctcgtcg agatggtggc caagagcggc atccgcgacg cctttgtcga gctgatcgtg 360 acgcgcgggc tgaagggcgt gcgggggaca cgccccgagg acatcgtcaa caatctgtac 420 atgtttgtgc agccgtacgt gtgggtgatg gagccggata tgcagcgtgt cggcggcagc 480 gcggtcgtcg cccgcaccgt gcgccgggtg cccccgggtg ccatcgaccc aaccgtcaag 540 aacctgcaat ggggcgatct cgtgcgcggc atgttcgagg ctgcggatcg cggtgcaact 600 tatccgttct tgacggacgg agatgcccat ctcaccgaag gctctgggtt caatattgtg 660 ctcgtcaagg acggcgtgct gtacacacca gaccgtggtg tgctgcaggg cgtgacacga 720 aagagtgtta tcaatgcggc ggaagccttc gggattgaag tccgcgttga gtttgtgccg 780 gttgagctgg cgtaccgttg tgatgagatc tttatgtgta ccaccgctgg cggcatcatg 840 cctatcacta cgctggatgg gatgcccgtg aatggaggac agatcggtcc tattacgaag 900 aagatttggg atggatattg ggctatgcat tatgatgcgg cttacagctt cgagattgat 960 tataacgaga ggaactga 978 <210> 2 <211> 972 <212> DNA <213> Aspergillus fumigatus <400> 2 atggcctcta tggacaaagt cttttcggga tattatgcgc gccagaagct gcttgaacgg 60 agcgacaatc ctttctctaa gggcattgct tatgtggaag gaaagctcgt cttacctagt 120 gatgctagaa taccgctact cgacgaaggt ttcatgcaca gtgacctaac ctatgatgtt 180 atatcggttt gggatggtcg cttctttcga ttggacgatc atttgcaacg gattttggaa 240 agctgcgata agatgcggct caagttccca cttgcactga gctcagtgaa aaatattctg 300 gctgagatgg tcgccaagag tggtatccgg gatgcgtttg tggaagttat tgtgacacgt 360 ggtctgacag gtgtacgtgg ttcgaagcct gaggatctgt ataataacaa catatacctg 420 cttgttcttc catacatttg ggttatggcg cctgagaacc agctccatgg tggcgaggct 480 atcattacaa ggacagtgcg acgaacaccc ccaggtgcat ttgatcctac tatcaaaaat 540 ctacagtggg gtgatttaac aaagggactt tttgaggcaa tggaccgtgg cgccacatac 600 ccatttctca ctgatggaga caccaacctt actgaaggat ctggtttcaa cattgttttg 660 gtgaagacg gtattatcta tacccctgat cgaggtgtct tgcgagggat cacacgtaaa 720 agtgtgattg acgttgcccg agccaacagc atcgacatcc gccttgaggt cgtaccagtg 780 gagcaggctt atcactctga tgagatcttc atgtgcacaa ctgccggcgg cattatgcct 840 ataacattgc ttgatggtca acctgttaat gacggccagg ttggcccaat cacaaagaag 900 atatgggatg gctattggga gatgcactac aatccggcgt atagttttcc tgttgactat 960 ggcagtggct aa 972

Claims (12)

A method for producing a D-amino acid from a specific substrate using (R) -selective omega transaminase,
(a) providing an amino donor and an amino acceptor to the (R) -selective omega transaminase;
(b) transferring an amino group from the amino donor to the amino acceptor through an amino group transfer reaction by the (R) -selective omega transaminase to produce a D-amino acid
Amino acid. &Lt; / RTI &gt; The method of claim 1, wherein the (R) -selective omega transaminase is derived from Aspergillus terreus . 3. The method according to claim 2, wherein said (R) -selective omega transaminase is transcribed after being transcribed from the nucleotide sequence of SEQ ID NO: 1. The method of claim 1, wherein the (R) -selective omega transaminase is derived from Aspergillus fumigatus . 5. The method according to claim 4, wherein said (R) -selective omega transaminase is transcribed after being transcribed from the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1, wherein the amino donor is selected from the group consisting of (R) -α-MBA, benzylamine, (R) -1-ethylbenzylamine and - A method for producing an amino acid. A method for producing a D-amino acid according to claim 1, wherein the amino acid is a-keto acid 8. The composition according to claim 7, wherein the alpha -keto acid is selected from the group consisting of pyruvate, fluoropyruvate, hydroxypyrrhizate, 2-oxobutyrate and 2-oxopentanoate. / RTI &gt; The method according to claim 1, wherein (D) -alpha.-MBA is provided as said amino donor and pyruvate is provided as said amino acceptor to synthesize D-alanine. 2. The method according to claim 1, wherein the D-amino acid is produced by providing (R) -a-MBA with the amino donor and providing 2-oxobutyrate with the amino acceptor to synthesize D-homoalanine Way. 2. The method according to claim 1, wherein D-serine is synthesized by providing (R) -a-MBA with the amino donor and providing hydroxypyruvate with the amino acceptor Way. The method according to claim 1, wherein the D-amino acid is produced by providing (R) -a-MBA with the amino donor and providing fluoropyruvate with the amino acceptor to synthesize D-fluoroalanine How to.

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