# Patent

## Title
Deinococcus N-acylamino acid racemase and use of preparing L-amino acid

## Abstract

The present invention relates to a novel thermostable N-acylamino acid racemase (NAAAR) isolated from Deinococcus radiodurans NCHU1003, the coding sequence and the preparation thereof. The present invention also discloses the process for preparing highly optically pure L-amino acids, such as L-homophenylalanine (L-HPA) and the derivatives thereof, from N-protected amino acid by using the novel NAAAR combined with L-N-carbamoylase.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the novel thermostable N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans NCHU1003, the coding sequence and the preparation thereof. The present invention also relates to an improved process for the preparation of optically active L-amino acid, such as L-homophenylalanine (L-HPA) and the derivatives thereof, from their corresponding N-protected amino acid by using the novel NAAAR combined with L-N-carbamoylase.
2. Background of the Invention
Optically active amino acids, including the L- and D-form amino acids, have been widely used in the industry of food, feedstuff, pharmaceutical synthesis and agricultural chemistry as preparative materials. However, different type of optically active isomers may exhibit totally different biological activities. For instance, some optically active isomer with certain configuration possesses excellent biological activities, but the other isomer does not have such biological activities. When these two isomers are present at the same time, they not only reduce the biological activities but also further inhibit the existing biological activities. For that reason, it is important to find the way for producing enamtiomeric form of amino acid in the pharmaceutical industry.
In the prior art of optically active amino acid preparation, it was performed with chemical synthesis. However, the process of organic synthesis often results in involved environmental pollution and has high cost in isolation and purification. Accordingly, there are lots of limitations in large-scale production. Asymmetric hydrogenation and enzyme-catalyzed reaction were therefore developed for producing high purity optically active amino acids and derivatives thereof. Frank J. Villani Jr et al (1998) isolated the racemic amino acids with tartaric acid and salicylaldehyde, which obtains a product of 32% yield, with e.e. (enantiomeric excess) value of 94%. According to the literatures, there are many bioconversion methods for preparing L-amino acids, but most of such methods still remain nearly 50% of D-formed materials. Based on that the advantages of substrate-specificity, stereochemical selectivity and the like, enzyme-catalyzed process are used in the racemation of amino acid for the production of highly pure optically active amino acids. Non-naturally occurred L-amino acids can be produced by using D,L-5-monosubstituted acetolactams as substrates and Arthrobacter sp. DSM3745 as biocatalyst (Syldatk et al 1992). Similar methods have been applied to other microorganisms, such as Pseudomonas, Achromobacter, Serratia, Aspergillus and so on. Tseng et al (1991) used an enzyme-catalyzed specific stereoselective acetylation of (R)-2-hydroxyl-4-phenylbutyronitrile to produce L-amino acids, but this method required more than 12 days to complete the reaction.
Chen et al (1999) used aspartic acid and 2-oxo-4-phenylbutyric acid as substrates and the enzyme tyrosine aminotransferase to perform an equilibrium shift controlled enzymatic reaction for the production of pure L-HPA. Zhao et al use a commercialized protease—alcalase to produce L-HPA in large-scale in which an e.e. value of 98% can be obtained. This method used N-protected amino acid as substrate, and only L-form N-protected amino acid could be hydrolysed, so that can accomplish the isolation of desired products. However, such enzyme-catalyzed reaction is very complicate, for that the substrate must be protected previously and the protective group should be removed after the enzyme-catalyzed reaction. An alternative enzyme-catalyzed method are preformed by using L-amino acid acylase, which specifically acts on N-protected amino acid, to convert N-protected amino acid racemic mixture to L- or D-formed amino acid (EP99118844 and JP11318442A). Thereafter, the remaining D-formed N-protected amino acid may be converted to L-formed N-protected amino acid by physical or chemical racemation (as described in JP656775A). The difficulty of such process was the reaction must be repeated again and the product and reactants must be isolated after each reaction. Moreover, reaction temperature and pH are increased to racemize the remaining D-formed N-protected amino acid. L-Amino acid may also be produced by using amino acid acyl transferase, which specifically acts on the N-protected amino acid, and further racemation to convert remaining D-formed N-protected amino acid to L-form amino acid (EP99118844.2; and Bommarius A. S. et al, Tetrahedron Asymmetry, 1997, 8:3197-3200).
The N-acylamino acid racemase from actinomycetes was firstly discovered by Takahashi (1991). This enzyme only performs racemation on N-protected amino acids and not on non-protected amino acids. Only few NAAARs were reported to which most of them are from actinomycetes, such as Streptomyces atratus Y-53 (Tokuyama et al, Appl. Microbiol. Biotechnol. 1994, 40: 835-840; and Amycolatopis sp. TS-1-60, Tokuyama et al, Appl. Microbiol. Biotechnol. 1995a, 42: 853-859). The NAAAR from Amycolatopis sp. TS-1-60 can maintains its activity at 55° C. for about 30 min, but the presence of D, L-amino acid acyl transferase in Amycolatopis can interfere the optical purity of methionine, thus limits its application.
L-homophenylalanine (L-HPA) is an optically active unnatured amino acid which can be useful as intermediates for the synthesis of many antihypertension drugs, ACE inhibitors. So far the high purity of L-HPA and derivative thereof is produced by chemical resolution or asymmetric hydrogenation (U.S. Pat. No. 5,981,794 and EP00902011A1). However, the previous methods must be carried out in a condition of high temperature and strong base, which exhibits the problems in expense, safety and environmental conservation and could not achieve best ee value and yield. The process of present invention is an enzyme catalytic method, so its reaction condition may avoid the disadvantages occurred in the chemical methods.
The production of L-HPA by enzymetic processes has been focused on amino transferase using L-aspartic acid or L-glutamic acid as the amino donor, and 2-oxo-4-phenyl butyric acid as the amino acceptor to obtain high purity L-PHA. Syldatk, C et al (1992) used the hydantoinase capable of catalying D, L-5-monosubstituted hydantoins in combination with D-N-carbamoylase or L-N-carbamoylase to produce novel D- or L-formed amino acids. The hydantoinase process had been applied in the production of L-PHA (Lo & Hsu 2003). In this process the hydantoinase is isolated from B. agri, since the substrate specificity of this hydantoinase is favor D-form homophenylalaninyl hydantoin (D-HPAH), a large amount of intermediate (D-NCaHPA) is formed during the process and therefore disadvantaged to the biosynthesis of L-HPA.
The present invention provides an enzymetic process for producing L-HPA with high optical purity by using a novel thermostable N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans NCHU1003 and a carbamoylase from Deinococcus sp.
SUMMARY OF THE INVENTION
In one aspect, the present provides a novel thermostable N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans NCHU1003, and the coding sequence thereof. The present NAAAR possesses excellent thermostability at 55° C. with half-life of about 15 days.
In a preferable embodiment of the invention, the NAAAR coding sequence is cloned in an appropriate expression vector, which is, for example, transformed into E. coli host cell for readily preparing the NAAAR.
In another aspect, the present provides a method for preparing L-HPA from the N-protected amino acid by using the present NAAAR combined with L-carbamoylase.
In an embodiment of the invention, the conversion of N-carbamoyl protected HPA to L-HPA is carried out by the isolated NAAAR and L-carbamoylase. In another embodiment of the invention, the NAAAR and L-carbamoylase are expressed in an E. coli host cell, and L-HPA is produced by the whole cell preparation from the N-protected starting material.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effects of pH on the catalytic activity of the present N-acylamino acid racemase.
FIG. 2 shows the effects of temperatures on the catalytic activity of the present N-acylamino acid racemase.
FIG. 3 shows the conversion efficiency of NCa-D-HPA to L-HPA using whole cell preparation of E. coli tranformants comprising the nucleotide sequences encoding L-carbamoylase and the present N-acylamino acid racemase, with measuring variations of the substrate NCa-D-HPA, the product L-HPA and the possible byproduct NCa-L-HPA during the conversion reaction.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
N-acylamino acid racemase (NAAAR) specifically acts on racemic N-protected amino acids and catalyzes the conversion of racemic N-protected amino acid to optically active N-protected amino acid. The present invention is characterized by first cloning the encoding sequence of NAAAR from Deinococcus radiodurans NCHU1003 genomic DNA. By using genetic engineering technique, the NAAAR gene is expressed in host cells such as E. coli cells and ready for preparing L-HPA in isolated form or in whole cell preparation combined with L-carbamoylase. The amino acid sequence of the present Deinococcus radiodurans NCHU1003 NAAAR and nucleotide encoding sequence are listed in SEQ ID Nos. 1 and 2.
The present invention also provides the recombinant DNA or RNA molecules comprising the NAAAR, which including and not limiting to phages, plasmids, phagemids, cosmids, YACs (yeast artificial fragmentation), BACs (bacteria artificial fragmentation) and the like. The methods for preparing such molecules are well known in the art, such as described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2dn Ed., New York, Cold Spring Harbor Express, 1989.
In a preferable embodiment of the invention, the recombinant DNA technology is provided to produce a fusion protein of L-N-carbamoylase with the present NAAAR from Deinococcus RADIO DURANS NCHU1003. The fusion construct is produced at DNA level by incorporating particular restriction sites for the insertion of desired DNA fragments into a proper expression vector, and is used in a heterologous expression system to express the desired fusion protein.
As used herein, the term “vector” refers to the plasmid which carrying DNA coding the desired protein. The exemplary vectors used in the present invention include, but not limited to, expression vector pQE30.
The gene encoding the protein is operatively linked to a gene expression sequence that directs the expression of the protein within a prokaryotic cell such as E. coli. The “gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the protein to which it is operatively linked. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent, which is known to those of ordinary skill in the art.
The L-N-carbamoylase coding gene is obtained from any appropriate source by polymerase chain reaction (PCR) method. In order to facilitate the subsequent cloning steps, it is preferable to design the primers with unique restriction sites. Similarly, The Deinococcus radiodurans NCHU1003 NAAAR coding sequence may be provided by PCR method using the primers with unique restriction sites for the further cloning into suitable expression vector and/or fusion with the L-N-carbamoylase gene.
The present process for preparing L-HPA by using the NAAAR combined with L-N-carbamoylase may be carried in a condition that the NAAAR and L-N-carbamoylase is added respectively. In such cases, these two enzymes may be added to the reaction mixture containing substrate simultaneously or sequentially. In another way, the enzymes may be immobilized on a support (or supports) and react with the substrate in suitable conditions. The supports useful in the present invention includes, for example, resins, beads, films, surface of microtitration plates and the like.
Alternatively, the method of present invention is preformed in the whole cell preparation of transformed cells which expressing the NAAAR and L-N-carbamoylase genes cloned in the same or individual expression vector. In one embodiment, the whole E. coli cells which have been transformed with a plasmid comprising NAAAR and L-N-carbamoylase encoding sequences is used in the conversion of N-carbamoyl protected phenylalanine to produce highly pure L-HPA.
EXAMPLE
The present invention is further illustrated by the following examples, which however, are not to be construed as limiting the scope of protection.
Example 1
Cloning and Expression of NAAAR Gene
The NAAAR gene was amplified by the polymerase chain reaction using primers DRI-01 (5′-CGCGGATCCATGGCGCATACTGGCCGTATGT-3′, SEQ ID NO: 3), containing BamHI restriction site, and DRI-02 (5′-CCCAAGCTTTCACGCCCGGTGTTCCTCCT-3′, SEQ ID NO: 4), containing HindIII restriction site, and Deinococcus radiodurans NCHU1003 chromosomal DNA as template and cloned into a expression vector pQE30 resultant plasmid, pQE-naaar, was then transformed into host cell E. coli JM109 by CaCl2 method. From the sequencing data of isolated NAAAR gene, SEQ ID NO: 2, the identity of the present NAAAR with known Deinococcus radiodurans strain is 96%.
The E. coli host cells harboring pQE-naaar were cultured with shaking at 37° C. overnight in a 500-mL flask containing 100 ml of LB medium with appropriate concentration of ampicillin. After 4 h incubation at 37° C. (OD600 about 6 to 8), isopropyl-β-D-thiogalactoside was added to a final concentration of 0.1 to 1 mM, and further cultured at 30° C. for 6 h to induce the expression of NAAAR gene. The culture of induced bacteria was centrifuged at 12,000 rpm for 10 min.
The bacteria culture induced as described above was centrifuged at 3,000 rpm for 15 min, and the supernatant was discarded. The collected bacteria were washed with 50 mM Tris-HCl (pH 8.0), and disrupted by ultra-sonicator. The cell lysate was then centrifuged at 13,500 rpm at 4° C. for 20 min. The supernatant was filtrated through 0.45 μm membranes, and the NAAAR proteins were purified by column chromatography using Ni-NTA resin (Qiagene).
The enzymetic activity was determined by measuring the production profile of conversion product at 215 nm via HPLC method as described by Tokuyama et al (1994). The reaction buffer containing 10 mM substrate, 0.5 mM CoCl2 and appropriate amount of the enzyme in 50 mM Tris-HCl buffer (pH 8.0) was incubated at 50° C. for 30 min, and the reaction was terminated by placing in boiling water for 5 min. The enzymetic activity was then measured by a Chiral-HPLC method. The Km values (mM) were determined from the original velocity data and the changes in NAc-D-HPA or NAc-D-HPA concentration. The unit NAAAR activity was defined as the enzyme amount required for catalysing 1 μmole of product formation under used condition. As described in the following Table 1, the specific activity of NAAAR for substrate N-carbamoyl-D-HPA was 1.31 U/mg, see Table 1.







TABLE 1







Substrate selectivity of present N-acylamino acid racemase












Specific activity
Relative activity



Substrate
(U/mg)
(%)a















N-acetyl-D-HPA
9.17
143.28



N-acetyl-L-HPA
11.97
187.03



N-acetyl-D-Met
6.40
100



N-acetyl-L-Met
8.04
125.63



N-acetyl-D-Phe
1.98
30.94



N-acetyl-L-Phe
1.55
24.22



N-acetyl-L-Gln
0.93
14.53



N-acetyl-L-Leu
0.45
7.03



N-acetyl-L-Trp
0.87
13.13



N-chloroacetyl-L-Phe
1.16
18.13



N-carbamoyl-D-HPA
1.31
20.46



N-carbamoyl-L-HPA
1.91
29.84







aRelative activity is expressed as percentages of the enzyme activity using N-acetyl-D-Met as substrate






To determine the optimal reaction pH of NAAAR, N-carbamoyl-D-HPA was dissolved in buffer with different pH values, including: citrate-Na2HpO4 (pH 2.6 ̃7.6), sodium phosphate buffer (pH 6.0 ̃8.0), Tris-HCl (pH 7.1 ̃8.9) and glycine-NaOH (pH 8.6 ̃10.6). To the substrate solution, 0.5 mM CoCl2 and appropriate amount of the enzyme were added. The reaction mixture was incubated at 50° C. for 30 min, and the reaction was terminated by incubating in boiling water for 5 min. As shown in FIG. 1, preferred reaction buffer for the present NAAAR was Tris-HCl buffer (pH 7.0 ̃9.0).
To determine the optimal reaction temperature, the reaction mixture containing 50 mM Tris-HCl buffer (pH 8.0), 10 mM substrate N-carbamoyl-D-HPA, 0.5 mM CoCl2 and appropriate amount of the enzyme was incubated at different temperature ranged from 20 to 90° C. for 30 min, and the enzyme activity was measured by the Chiral-HPLC method described above. As shown in FIG. 2, the optimal reaction temperature was 40 ̃70° C.
To determine the effect of metal ion on the enzyme activity, a variety of metal ions at same concentration were added to a solution of the purified NAAAR in 50 mM Tris-HCl buffer (pH 8.0). After incubation at 37° C. stood for 15 min, 10 mM N-carbamoyl-D-HPA was added into the reaction mixture and incubated at 50° C. for 30 min. Enzyme activities were then determined by the Chiral-HPLC method. As shown in Table 2, the present NAAAR exhibited an improved enzymetic activity in the presence of bivalent metal ions such as Co++, Mn++, and Ni++.







TABLE 2







Effects of metal ions on N-acylamino acid racemase activities












Concentration
Relative activity



Compounds
(mM)
(%)a















None
2
0



CaCl2
2
0



CoCl2
2
100



CuCl2
2
0



MnCl2
2
79.1



MgCl2
2
20.5



NiCl2
2
40.6



ZnCl2
2
19.7



EDTA
10
0







aRelative activity is expressed as percentages of the enzyme activity with CoCl2.






The present NAAAR was analysed its thermostability at 55° C. in the presence of 0.5 mM CoCl2. The half-life of the NAAAR activity was about 15 days, and remained more than 20% of enzymatic activity after 25 days in the reaction condition. Thus, the NAAAR of present invention is considered to be a thermostable enzyme.
Example 2
Conversion of N-Protected HPA to L-HPA by the NAAAR and L-N-Carbamoylase from Transformed E. coli 
For the co-expression of the NAAAR and L-N-carbamoylase genes in a host cell, we designed two primers at the upstream of T5 promoter and the downstream of λ transcriptional terminator sequence in vector pQE30:
NCAU (5′-ggcgaggatccatgattcaaggggaacgtc-3′, SEQ ID NO: 5), containing BamHI restriction site; and NCAD (5′-aatttaagcttattccccttgggccagttg-3′, SEQ ID NO:6), containing HindIII restriction site. The nucleotide sequences encoding the NAAAR and L-N-carbamoylase were amplified from Deinococcus radiodurans NCHU1003 and Bacillus kaustophilus CCRC11223 chromosome, respectively, by PCR technology. The PCR products contained T5 promoter, lac operator and 6×his-tag sequence at the 5′-end and A transcriptional terminator at the 3′-end. The PCR products were cloned in the pQE30 expression vector. The resultant plasmid, pQEarlnca, was transformed into E. coli JM109 for inductive expression.
The reaction mixture containing 10 mM substrate N-carbamoyl-D-HPA, 0.5 mM CoCl2, 50 mM Tris-HCl buffer (pH 8.0), and the enzymes NAAAR and L-N-carbamoylase isolated from induced transformant was incubated at 50° C. for carrying out the conversion. After analysed by the Chiral-HPLC method, it is shown that the co-expressed enzymes could completely convert the original substrate to the final product L-amino acid in 2 to 3 h in one single reaction. The results are shown in the following Table 3.







TABLE 3







Conversion of NCa-D-HPA with enzymes L-N-carbamoylase/


N-acylamino acid racemase












Time
NCa-D-HPA
NCa-L-HPA
L-HPA



(hour)
(mM)
(mM)
(mM)
















0.5
4.16
0.88
4.96



1.0
1.16
0
8.84



2.0
0
0
10










Example 3
Conversion of N-Protected HPA to L-HPA by Whole Cell Preparation
The E. coli JM109 transfomants harboring co-expression vector, pQEarlnca, were inoculated into 500-mL LB containing 100 ug/ml of ampicillin. The bacteria were cultured at 37° C. until the OD600 reach 1. IPTG was added at final concentration of 1 mM to induce the gene expression.
After the IPTG induction, the bacteria were collected by centrifugation and stored at 4° C. for further usage. In the conversion reaction, 2 g of the wet bacteria preparation was added to 100 ml formulated substrate solution, in which 10 mM N-carbamoyl-D,L-HPA was dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM CoCl2. The reaction was carried out in a 37° C. water bath with magnetic stirrer, and controlled and maintained the pH value at 7.0 to 8.0 during reaction with 1N HCl and 1N NaOH. Aliquots of 0.5 ml were sampled at intervals for the detection of L-HPA production by the Chiral-HPLC method. After 80 h incubation, the L-HPA conversion yield reached 98%, with an ee value of 99% (FIG. 3).
The strain E. coli XLI-Blue containing the recombinant vector pQE-naaar, of the present invention was deposited with the Agricultural Research Service Culture Collection (NRRL), on Nov. 29, 2005, as Deposit No. NRRL-B-30889.

## Claims

1. A process for preparing an optically active L-amino acid from a racemic mixture of an N-protected amino acid, comprising converting N-protected amino acids with a thermostable N-acylamino acid racemase comprising the amino acid sequence of SEQ ID NO:1 combined with L-N-carbamoylase from Bacillus kaustophilus to obtain the optically active L-amino acid.

2. The process according to claim 1, wherein the optically active L-amino acid is L-homophenylalanine (L-HPA) and the N-protected amino acid is N-carbamoyl-homophenylalanine.

3. The process of claim 2, wherein the converting reaction is carried out in the presence of a bivalent metal ion.

4. The process of claim 2, wherein the bivalent metal ion is Co++, Mn++, or Ni++.

5. The process of claim 2, wherein the N-acylamino acid racemase and the L-N-carbamoylase are co-expressed in a host cell transformed with a vector comprising the N-acylamino acid racemase and L-N-carbamoylase coding sequences.

6. The process of claim 2, wherein the Bacillus kaustophilus is Bacillus kaustophilus CCRC 11223.

7. The process according to claim 1, wherein the N-protected amino acid is selected from the group consisting of N-acetyl-HPA, N-acetyl-Phe, N-acetyl-Met, N-acetyl-Phe, N-acetyl-Gln, N-acetyl-Leu, N-acetyl-Trp, N-carbamoyl-homophenylalanine, and N-chloroacetyl-Phe.

8. A process for preparing L-homophenylalanine (L-HPA), comprising converting N-carbamoyl-HPA with a thermostable N-acylamino acid racemase combined with L-N-carbamoylase from a Bacillus sp. at 20 ̃80° C. in Tris-HCl buffer (pH 5.0 ̃10.0) to obtain optically active L-HPA, and
wherein said thermostable N-acylamino acid racemase is isolated from Deinococcus radiodurans NCHU1003, which converts N-protected amino acids to corresponding L-amino acids when combined with L-N-carbamoylase and comprises the amino acid sequence of SEQ ID NO:1.

9. The process of claim 8, further comprising:
(A) transforming host cells with a recombinant vector comprising a DNA molecule encoding said N-acylamino acid racemase and regulatory elements;
(B) culturing the transformants under a condition sufficient for expression of said N-acylamino acid racemase; and
(C) recovering and/or purify the N-acylamino acid racemase.

10. The process of claim 8, in which the converting reaction is carried out in the presence of a bivalent metal ion.

11. The process of claim 10, in which the bivalent metal ion is Co++, Mn++, or Ni++.

12. The process of claim 8, in which the N-acylamino acid racemase and/or the L-N-carbamoylase are immobilized on a support.

13. The process of claim 8, in which the N-acylamino acid racemase and the L-N-carbamoylase are co-expressed in a host cell transformed with a vector comprising the N-acylamino acid racemase and L-N-carbamoylase coding sequences.

14. The process of claim 13, in which the converting reaction is carried out in a whole cell preparation.

15. A process for preparing L-homophenylalanine (L-HPA), comprising converting N-carbamoyl-HPA with a thermostable N-acylamino acid racemase combined with L-N-carbamoylase from Bacillus kaustophilus at 20 ̃80° C. in Tris-HCl buffer (pH 5.0 ̃10.0) to obtain optically active L-HPA, and
wherein said thermostable N-acylamino acid racemase comprises the amino acid sequence of SEQ ID NO:1.

16. The process of claim 15, wherein the converting reaction is carried out in the presence of a bivalent metal ion.

17. The process of claim 16, wherein the bivalent metal ion is Co++, Mn++, or Ni++.

18. The process of claim 15, wherein the N-acylamino acid racemase and/or the L-N-carbamoylase are immobilized on a support.

19. The process of claim 15, wherein the N-acylamino acid racemase and the L-N-carbamoylase are co-expressed in a host cell transformed with a vector comprising the N-acylamino acid racemase and L-N-carbamoylase coding sequences.

20. The process of claim 19, wherein the converting reaction is carried out in a whole cell preparation.