Patent Grant
9376694
This application is a §371 national stage entry of International Application No. PCT/KR2013/006028, filed Jul. 8, 2013, which claims priority to South Korean Patent Application No. 10-2012-0074035 filed Jul. 6, 2012, the entire contents of which are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 8, 2015, is named G1035-00901_SL.txt and is 17,474 bytes in size.
The present disclosure relates to a method for preparing an optically active amino acid using cosubstrate shuttling of transaminase, more particularly to a method for preparing an optically active amino acid with high conversion rate and high purity by coupling transamination by ω-transaminase with high equilibrium constant and a reaction by α-transaminase with low equilibrium constant using an amino acid cosubstrate and its keto acid that shuttle between the two reactions.
Optically active amino acids are important compounds used, for example, as intermediates of various pharmaceuticals. Especially, since unnatural amino acids which are highly valued as optically active intermediates are not metabolites producible by microorganisms, they cannot be produced by the production method of natural amino acids, such as fermentation. Although methods using metal catalysts are developed, they are uneconomical because transition metals are very expensive. Accordingly, an enzyme-based method may be economical and effective for production of optically active amino acids.
In this regard, α-transaminase is an industrially useful enzyme for production of optically active amino acids owing to fairly broad substrate specificity and high optical selectivity. However, its industrial utilization is limited because of low conversion rate due to small equilibrium constant.
Accordingly, for production of optically active amino acids using the enzyme-based method in industrial scale, it is necessary to overcome the low equilibrium constant of α-transaminase.
As existing methods for producing optically active amino acids using enzymes, Korean Patent Publication No. 10-2011-0047789 discloses a method for preparing an amino acid using microorganism-derived chain type transaminase and Korean Patent Publication No. 10-2008-0016287 discloses a method for producing an amino acid using β-transaminase.
The present disclosure is directed to, in order to overcome the low equilibrium constant of α-transaminase, providing a method for preparing an optically active amino acid with high purity by coupling with a ω-transaminase reaction with very large equilibrium constant using an amino acid cosubstrate exhibiting reactivity for the coupled reactions and a keto acid thereof.
In one general aspect, the present disclosure provides a method for preparing an optically active amino acid by a cascade reaction, including coupling a reaction of converting a keto acid to an amino acid by α-transaminase and a reaction of transferring an amino group of an amine substrate by ω-transaminase (TA) using an amino acid cosubstrate.
Specifically, the α-transaminase may be branched-chain transaminase (BCTA),
The amino acid cosubstrate may be any amino acid showing reactivity for both α-transaminase and ω-transaminase. More specifically, it may be selected from alanine, homoalanine, leucine, norvaline and norleucine. Most specifically, it may be alanine or homoalanine.
The keto acid may be any keto acid showing reactivity for α-transaminase. Specific examples include pyruvate, 2-oxobutyrate, 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid, trimethylpyruvate, 3-methyl-2-oxobutyrate, 3-methyl-2oxopentanoic acid, 4-methyl-2-oxopentanoic acid, 2-oxopentanoic acid, 2-oxohexanoic acid, 2-oxooctanoic acid, fluoropyruvate, hydroxypyruvate, mercaptopyruvate, oxaloacetate, ketoglutarate, phenylglyoxylate, phenylpyruvate, 4-hydroxyphenylglyoxylate, 4-dimethyl-2oxopentanoic acid, 3-dimethyl-2-oxopentanoic acid, 3-ethyl-3-methyl-2-oxopentanoic acid, 5-dimethyl-2-oxohexanoic acid, etc.
The amine substrate may be any amine showing reactivity for ω-transaminase. Specific examples include benzylamine, methylbenzylamine, ethylbenzylamine, isopropylamine, 2-butylamine, 1-aminoindane, cyclopropylethylamine, 2-aminopentane, 3-methyl-2-butylamine, 1,3-dimethylbutylamine, 2-aminooctane, 1-methoxy-2-propylamine, 2-aminohexane, p-fluoromethylbenzylamine, mexiletine, 1-methyl-3-phenylpropylamine, etc. Specifically, it may be benzylamine, methylbenzylamine or isopropylamine.
The optically active amino acid produced by the method of the present disclosure may be optically pure amino acid in
In an exemplary embodiment of the present disclosure, in the cascade reaction, the concentration of the amino acid cosubstrate may be 0.1-20% of the concentration of the keto acid substrate.
In an exemplary embodiment of the present disclosure, the cascade reaction may be conducted by further adding an organic solvent such as hexane if the reactivity of the ω-transaminase is inhibited by a ketone or an aldehyde.
In an exemplary embodiment of the present disclosure, the ratio of the α-transaminase (U/mL) and the ω-transaminase (U/mL) added for the cascade reaction may be 1:1-10.
In an exemplary embodiment of the present disclosure, the α-transaminase may be branched-chain transaminase isolated from E. coli and may have a DNA sequence of SEQ ID NO 7 and an amino acid sequence of SEQ ID NO 8.
In an exemplary embodiment of the present disclosure, the ω-transaminase may be one isolated from Paracoccus denitrificans and may have a DNA sequence of SEQ ID NO 9 and an amino acid sequence of SEQ ID NO 10.
In an exemplary embodiment of the present disclosure, the ω-transaminase may be one isolated from Ochrobactrum anthropi and may have a DNA sequence of SEQ ID NO 11 and an amino acid sequence of SEQ ID NO 12.
The present disclosure allows production of various optically active amino acids with high purity by solving the low equilibrium constant problem of transaminase and is applicable to production of various optically active amino acids in industrial scale. Since the present disclosure allows easy production of various unnatural amino acids having high reactivity and stability, which are used as pharmaceutical precursors, it can be usefully employed in preparation of pharmaceuticals, food additives and various animal feeds.
Hereinafter, the present disclosure is described in more detail.
Transaminase is a very important enzyme in amino acid metabolism. It catalyzes the transfer of an amino group between an amino acid and a keto acid with pyridoxal phosphate (PLP) as a coenzyme. Unlike other transaminases, ω-transaminase transfers an amino group of an amino acid or an amine compound having no carboxyl group, having the amino group at α-position, to an amino acceptor such as a keto acid or a ketone. Accordingly, ω-transaminase is a very useful enzyme in the production of optically active amine compounds.
The present disclosure relates to a method for preparing various optically active amino acids with high efficiency by coupling a reaction by α-transaminase such as branched-chain transaminase,
In an exemplary embodiment of the present disclosure, to provide a method for preparing various optically active amino acids with high purity and high efficiency by solving the low equilibrium constant problem of the conversion of a keto acid to an amino acid by α-transaminase, a reaction of converting a keto acid to an
In another exemplary embodiment of the present disclosure, a reaction of converting a keto acid to a
The amino acid cosubstrate that shuttles between the two reactions may be any amino acid which shows reactivity for both reactions and can be converted to its keto acid. Specifically, alanine, homoalanine, leucine, valine, norleucine, etc. may be used. More specifically, alanine or homoalanine having 1 or 2 carbon atoms may be used.
The keto acid may be any keto acid for which α-transaminase shows reactivity. Specific examples include pyruvate, 2-oxobutyrate, 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid, trimethylpyruvate, 3-methyl-2-oxobutyrate, 3-methyl-2oxopentanoic acid, 4-methyl-2-oxopentanoic acid, 2-oxopentanoic acid, 2-oxohexanoic acid, 2-oxooctanoic acid, fluoropyruvate, hydroxypyruvate, mercaptopyruvate, oxaloacetate, ketoglutarate, phenylglyoxylate, phenylpyruvate, 4-hydroxyphenylglyoxylate, 4-dimethyl-2oxopentanoic acid, 3-dimethyl-2-oxopentanoic acid, 3-ethyl-3-methyl-2-oxopentanoic acid, 5-dimethyl-2-oxohexanoic acid, etc.
The amine substrate may be any amine for which ω-transaminase shows reactivity. Specific examples include benzylamine, methylbenzylamine, ethylbenzylamine, isopropylamine, 2-butylamine, 1-aminoindane, cyclopropylethylamine, 2-aminopentane, 3-methyl-2-butylamine, 1,3-dimethylbutylamine, 2-aminooctane, 1-methoxy-2-propylamine, 2-aminohexane, p-fluoromethylbenzylamine, mexiletine, 1-methyl-3-phenylpropylamine, etc. More specifically, benzylamine, methylbenzylamine or isopropylamine may be used.
The optically active amino acid produced by the method of the present disclosure may be an optically pure amino acid in
In the cascade reaction according to the present disclosure, the concentration of the amino acid cosubstrate may be 0.1-20% of the concentration of the keto acid substrate. If the concentration of the cosubstrate is below 0.1% of the concentration of the keto acid substrate, conversion rate may be low. And, if the concentration of the cosubstrate is above 10% of the concentration of the keto acid substrate, it may be difficult to separate the produced amino acid. Hence, a concentration range of 0.1-10% is preferred.
And, the cascade reaction may be conducted by further adding an organic solvent, specifically hexane, to overcome inhibition of the reactivity of the ω-transaminase by the produced ketone or aldehyde.
Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.
E. coli cells cultured in a complete medium were centrifuged at 4,000 rpm for 10 minutes at 4° C. The supernatant was removed and the remaining cell pellet was lysed in 15 mL of a lysis buffer (15% sucrose, 25 mM EDTA, 25 mM Tris). The resultant was centrifuged at 4,000 rpm at 4° C. After discarding the supernatant and adding lysozyme (5 mg/mL in TSB buffer), the remainder was incubated at 37° C. for 10 minutes. After adding 1.2 mL of 0.5 M EDTA, the resulting solution was allowed to stand at 37° C. for 5 minutes. After adding 1 mL of 10% SDS, the resulting solution was kept at 70° C. and then kept in ice water for 10 minutes. After adding 2.5 mL of 5 M potassium acetate, the resulting solution was kept in ice water for 15 minutes. After adding a phenol-chloroform mixture (50:50) of the same volume as the solution and mixing for 30 minutes, the mixture was centrifuged at 4000 rpm for 10 minutes at 4° C. After adding chloroform corresponding to 0.5 time the volume of the obtained supernatant, the resulting solution was slowly agitated and centrifuged at 4000 rpm at 4° C. The supernatant was recovered and treated with 50 μg/mL RNase at 37° C. for 1 hour. Subsequently, after adding isopropanol of 0.8 volume equivalent and ethanol of 2.5 volume equivalents and agitating slowly, genomic DNA was collected using a heat-sealed Pasteur pipette, completely dried in a tube and then dissolved in TE buffer for further use.
Transaminase was acquired by PCR using the isolated genomic DNA as a template. The following primers were used.
The PCR product was treated with NdeI/BamHI restriction enzyme and the resulting fractions were ligated with pET24ma (acquired from Hiroshi Sakamoto, Paris) and pET23b (Novagen) to prepare a recombinant plasmid.
Paracoccus denitrificans was cultured in an LB broth (10 g/L peptone, 5 g/L yeast extract, 5 g/L sodium chloride, pH 7) at 37° C. for 12 hours and genes expressing ω-transaminase were amplified from a single colony by PCR using synthetic DNA primers. The obtained DNA fragment was inserted into the expression vector DNA pET28a(+) using Nde1 and Xho1 restriction enzymes and ligase.
The following primers were used.
Ochrobactrum anthropi was cultured in an LB broth (10 g/L peptone, 5 g/L yeast extract, 5 g/L sodium chloride, pH 7) at 37° C. for 12 hours and genes expressing ω-transaminase were amplified from a single colony by PCR using synthetic DNA primers. The obtained DNA fragment was inserted into the expression vector DNA pET28a(+) using Nde1 and Xho1 restriction enzymes and ligase.
The following primers were used.
E. coli BL21(DE3) was transformed with the plasmids obtained in Examples 1, 2-(1) and 2-(2). The bacterium was cultured in 300 mL of an LB broth containing kanamycin and IPTG (final concentration=1 mM) was added to OD 0.5. After further culturing for at least 6 hours at 37° C., the bacterial cells were centrifuged at 10000×g for 20 minutes at 4° C. and resuspended in 15 mL of a resuspension buffer (50 mM Tris-HCl, 50 mM calcium chloride, 1 mM β-mercaptoethanol, 0.1 mM PMSF, 20 μM PLP, pH 7). After sonication on ice, followed by centrifugation at 17000×g for 30 minutes at 4° C., the supernatant was obtained as a crude extract.
The desired branched-chain transaminase and ω-transaminase were purified from the crude extract by affinity chromatography.
Branched-chain transaminase having a DNA sequence of SEQ ID NO 7 and an amino acid sequence of SEQ ID NO 8, ω-transaminase derived from Paracoccus denitrificans (PdwTA) having a DNA sequence of SEQ ID NO 9 and an amino acid sequence of SEQ ID NO 10, and ω-transaminase derived from Ochrobactrum anthropi having a DNA sequence of SEQ ID NO 11 and an amino acid sequence of SEQ ID NO 12 were obtained.
Reaction was conducted using the crude extract containing the branched-chain transaminase obtained in Example 3 and using various
Reaction was conducted using the crude extract containing the branched-chain transaminase obtained in Example 3 and using various trimethylpyruvate (keto acid of
Reaction was conducted using the branched-chain transaminase purified in Example 3 and using various
When trimethylpyruvate and
Reaction was conducted using the branched-chain transaminase purified in Example 3 and using various
Reaction was conducted using the ω-transaminase purified in Example 3 and using various
Reaction was conducted using the ω-transaminase purified in Example 3 and using various keto acids and amine as substrates as described in [Table 6]. After adding 10 μL of the purified enzyme (6 U/mL) to 100 μL of a mixture of 20 mM keto acid, 20 mM (S)-α-methylbenzylamine and 50 mM potassium phosphate (pH 7.0), reaction was conducted at 37° C. for 6 hours and the amount of produced acetophenone (deaminated ketone of (S)-α-methylbenzylamine) was measured. The result was compared with that of the ω-transaminase derived from Ochrobactrum anthropi. The result is shown in [Table 6].
Reaction was conducted using the ω-transaminase purified in Example 3 and using various keto acids and amine as substrates as described in [Table 7]. After adding 10 μL of the purified enzyme (1 U/mL) to 100 μL of a mixture of 20 mM keto acid, 20 mM (S)-α-methylbenzylamine and 50 mM potassium phosphate (pH 7.0), reaction was conducted at 37° C. for 10 minutes and the amount of produced acetophenone (deaminated ketone of (S)-α-methylbenzylamine) was measured. The result was compared with that of the ω-transaminase derived from Ochrobactrum anthropi. The result is shown in [Table 7].
From the comparison of substrate specificity of the two enzymes obtained in Examples 4-10, it was found out that the branched-chain transaminase exhibits higher activity as the number of carbon atoms in the side chain of an amino acid is larger whereas the ω-transaminase exhibits lower activity as the number of carbon atoms in the side chain of an amino acid is larger.
Accordingly,
The change in the production amount of
The rate of conversion from trimethylpyruvate to
The change in conversion rate was monitored while conducting coupled enzyme reactions by varying ω-transaminase concentration with branched-chain transaminase concentration fixed. The rate of conversion to
Up to 5% conversion rate, i.e. when 1 mM cosubstrate was consumed, there was no difference in the conversion rate depending on the ω-transaminase concentration. However, a very significant difference in the conversion rate was observed at higher conversion rate depending on the ω-transaminase concentration. This suggests that the shuttling effect of the cosubstrate by ω-transaminase is very effective in overcoming the low equilibrium constant of the branched-chain transaminase reaction. The result is shown in
The change in conversion rate was monitored while conducting coupled enzyme reactions by varying branched-chain transaminase concentration with ω-transaminase concentration fixed. The rate of conversion to
The change in conversion rate was monitored while conducting coupled enzyme reactions by adding hexane and using racemic cosubstrate in order to overcome inhibition of ω-transaminase reaction by the product.
The addition of hexane resulted in increased conversion rate because of the production of aldehyde which inhibits the reaction was suppressed. Also, the conversion to
The change in conversion rate of
Using the enzyme ratio that showed the best conversion rate in Example 16,
The addition of the organic solvent resulted in the increased conversion to
The change in conversion rate to
When (S)-α-methylbenzylamine was used as the amine substrate of ω-transaminase, the conversion rate was improved by 150% as compared to when benzylamine was used as the amine substrate. The result is shown in
Optically active
Optically active
Various unnatural amino acids and branched-chain amino acids were produced by coupled enzyme reactions. Unnatural amino acids and branched-chain amino acids were produced at 37° C. by conducting reactions using 1 mL of a mixture of 20 mM keto acid, 4 mM
As keto acids for the production of the optically active amino acids, 2-oxopentanoate was used for
Since the ω-transaminase derived from Ochrobactrum anthropi exhibits stronger activity for isopropylamine than that derived from Paracoccus denitrificans,
High-concentration
Various high-concentration unnatural amino acids were produced by coupled enzyme reactions. Unnatural amino acids were produced by conducting reactions at 37° C. using 100 mM keto acid, 5 mM
As keto acids for the production of the optically active amino acids, trimethylpyruvate was used for
Various high-concentration unnatural
As keto acids for the production of the optically active amino acids, 2-oxopentanoate was used for
The present disclosure allows production of various optically active amino acids with high purity and high efficiency by solving the low equilibrium constant problem of transaminase and is applicable to production of various optically active amino acids in industrial scale. Since the present disclosure allows easy production of various unnatural amino acids having high reactivity and stability, which are used as pharmaceutical precursors, it can be usefully employed in preparation of pharmaceuticals, food additives and various animal feeds.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2012-0074035 | Jul 2012 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2013/006028 | 7/8/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/007588 | 1/9/2014 | WO | A |
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| Number | Date | Country | |
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| 20150284750 A1 | Oct 2015 | US |
