WO2003008569A1

WO2003008569A1 - Novel d-stereo specific amino acid amidase, gene thereof, preparation method thereof and production method of d-amino acid by using the same

Info

Publication number: WO2003008569A1
Application number: PCT/KR2001/001235
Authority: WO
Inventors: Moon-Hee Sung; Jae Jun Song; Seung Goo Lee; Dae Heoun Baek; Seung-Pyo Hong; Mi-Sun Kwak; Kwang Kim; Ki-Hong Yoon; Mi-Hwa Lee; Sangchul Lee
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2001-07-14
Filing date: 2001-07-19
Publication date: 2003-01-30
Anticipated expiration: 2004-01-14
Also published as: KR20030006724A; KR100449456B1

Abstract

A D-stereospecific amino acid amidase, its gene,and a method for production of D-stereospecific amino acid using the same are provided. The D-stereospecific amino acid amidase ans its gene originate from thermophilic microorganism Brevibacillus borstelensis, a cloning vector and expression vector containing the same, and a transformant transformed by the expression vector. A method for production of D-stereospecific amino acid uses the D-stereospecific amino acid amidase. A D-stereospecific amino acid amidase and method for large-scale preparation of D-stereospecific amino acid amidase using genetic engineering. The D-stereospecific amino acid amidase can produce various D-stereospecific amino acid useful to industry from a DL-amino acid amid racemic mixture.

Description

NOVEL D-STEREO SPECIFIC AMINO ACID AMIDASE, GENE

THEREOF, PREPARATION METHOD THEREOF AND

PRODUCTION METHOD OF D-AMINO ACID BY USING THE SAME

FIELD OF THE INVENTION

The present invention relates to a novel D- stereospecific amino acid amidase, a method for preparation thereof, and a method for production of d- stereospecific amino acid using the same. Particularly, the present invention relates to a D-stereospecific amino acid amidase derived from thermophilic microorganism Brevibacillus borstelensis, its gene, an expression vector containing the same, transformant transformed by the expression vector, a method of preparing D-stereospecific amino acid, and a method of producing D-stereospecific amino acid using the D- stereospecific amino acid amidase.

BACKGROUND OF THE INVENTION Most natural amino acids have stereospecificity

and a α-carbon exhibiting optical activity and are classified as L-amino acids or D-amino acids by stereospecificity of amino acids. Most natural proteins consist of L-amino acids, except microbial peptidoglycan, peptide antibiotics and biologically active peptides of plants consist of D-amino acids.

So far, D-amino acids have been widely used for foods and drugs as an intermediate in the synthesis of neurotransmitters, vaccines, synthetic sweeteners, antibiotics, and hormones. Accordingly, methods of producing D-stereospecific amino acids are being developed.

For the production of D-amino acids, the conventional production methods include chemical synthesis and enzymatic synthesis. Because the production method of D-amino acids by chemical synthesis produces a racemic mixture containing D- amino acids and L-amino acids, the method needs an additional laborious purification process to obtain pure D-amino acids f om the racemic mixuture . As a production method of D-amino acids by enzymatic synthesis using a biocatalyst, to directly produce pure D-amino acids by one step, the above method is being watched as a clean production technique capable of substituting for a chemical synthesis having environmental pollution problems.

As for the production method by enzyme synthesis using a biocatalyst, methods for purifying D-amino acids from a DL-amino acid mixture have been developed. In such a method, after a DL-amino acid mixture is produced from DL-amino acid amides, D-amino acids are produced by selectively hydrolysis D-amino acid amides from DL-amino acid amide using D-stereospecific amino acid amidase as a biocatalyst. Therefore, the enzymatic synthesis of D-amino acids using D- stereospecific amino acid amidase has the advantage of production of various D-amino acids useful to industry from economical racemic mixtures.

So far, some proteins such as a D-stereospecific peptidase derived for mesophilic microorganism Bacillus cereus (Asano et al . , J. Biol . Chem . 271:3056-30262, 1996) and D-amino acid amidase derived from Ochrobactrum anthropi (Asano et al . , Biochem. Biophy. Res . Com . 162:470-474, 1989) have been known as biocatalysts that can be used in the production of D-amino acids. However, the production of D-amino acids using these enzymes has not yet been reported. The optimal temperature of the enzymes produced by

these microoganisms is under 37°C. However, there have been no studies about thermostable D-stereospecific amino acid amidases and peptidases derived from thermophilic microorganisms maintaining their enzyme activity over 50°C.

The stability of biocatalysts is the most important factor in the enzymatic synthesis of D-amino acids using biocatalysts. Generally, thermophilic microorganisms produce highly thermostable enzymes that are adaptable to high temperature in habitat. Thermostable enzymes have been reported to have stability for organic solvents, pH, and chemical denaturants as well as high temperature. Therefore, such enzymes are the most suitable biocatalysts in enzymatic synthesis of D-amino acids. Thus, it is important to study thermostable D-stereospecific amino acid amidase and peptidase derived from thermophilic microorganisms maintaining their enzyme activity at high temperature .

Therefore, the present inventors isolated a gene encoding a novel thermostable D-amino acid amidase from a novel thermophilic Brevibacillus borstelensis, and sequenced the gene. In addition, the present inventors have developed a method of producing D-amino acids using the thermostable D-stereospecific amino acid amidase as a biocatalyst. SUMMARY OF THE INVENTION

It is an objective of the present invention to provide thermostable D-stereospecific amino acid amidase derived from thermophilic microorganism having ability of producing D-amino acids by stereospecifically acting on D-amino acid amides and gene thereof.

It is a further objective of the present invention to provide a recombinant expression vector containing the gene of thermostable D-stereospecific amino acid amidase, a transformant thereof, and a method of preparing a D-stereospecific amino acid amidase using the recombinant D-stereospecific amino acid amidase.

It is an additional objective of the present invention to provide a method of producing D-amino acids useful to the industry using the thermostable D- stereospecific amino acid amidase.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a manufacturing process of recombinant plasmid (pDAP) for cloning of thermostable D-amino acid amidase gene derived from microorganism Brevibacillus borstelensis .

Fig. 2 shows a manufacturing process of vector

(pBDA) for large-scale expression of recombinant thermostable D-stereospecific amidase.

Fig. 3 is a graph that represents effect of temperature on activity and thermostability of D- stereospecific amino acid amidase (λ: optimal temperature; O: heat stability) .

Fig. 4 shows the effect of pH on activity and thermostability of D-stereospecific amino acid amidase (D: MES; v: Bis-Tris; 0: Tris; λ: CAPS) .

Fig. 5 is a graph of chromatography producing pure D-phenylalanine from DL-phenylalanine using a recombinant heat stable D-stereospecific amino acid amidase .

Fig. 6 is a graph that represents production of D-phenylalanine from DL-phenylalanine using D- stereospecific aminos acid amidase (λ: 0.674 units; v: 1.348 units; σ: 2.022 units).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the thermostable D-stereospecific amino acid amidase derived from thermophilic microorganism, having the ability of producing D-amino acids by stereospecifically acting D- amino acid amides and gene thereof.

A D-stereospecific amino acid amidase of the present invention comprises the N-terminal amino acid of SEQ ID NO. 1 and preferably has the amino acid sequence of SEQ ID NO. 3. The gene encoding the D-stereospecific amino acid amidase comprises the DNA sequence of SEQ ID NO. 4. However, other proteins having new amino acid sequences by substitutions, deletions, or additions of amino acids are also included in the present invention as said proteins are similar to physical or chemical characteristics of the D-stereospecific amino acid amidase of the present invention. Amino acid sequences having 90% homology with amino acid sequence of SEQ ID NO. 3, amino acid sequences which are translated in SEQ ID NO. 3 or its derivatives, and DNA sequence of SEQ ID NO. 2 are included in the present invention. The present inventors isolated chromosome DNA from thermophilic Brevibacillus borstelensis and produced a recombinant plasmid by inserting the isolated DNA fragments which were partially digested by restriction enzyme into a pUC118 vector and transformed this vector

into E. coli DH5α . Among the transformants, the transformant with D-amino acid amidase activity was selected. The recombinant plasmid in the transformant was named "pDAP" (see Fig. 1), and the transformant

including the said pDAP was named "DH5α /pDAP." The recombinant plasmid pDAP includes a DNA sequence of SEQ ID NO. 2 and this DNA sequence encodes the amino acid sequence of SEQ ID NO. 3. The present inventors deposited the transformant at the Korean Collection for Type Cultures of the Korea Research Institute of Bioscience and Biotechnology on July 11, 2001 (Deposit No. KCTC 10009BP) .

The D-stereospecific amino acid amidase of the

present invention shows optimum activity at about 85^°C and shows a relative stability till about 90^°C (see Fig. 3) . In addition, the D-stereospecific amino acid amidase keeps pH stability from pH 7 to pH 10 and shows optimum enzyme activity at pH 9. EDTA, DTT, or mercaptoethanol shows inhibitory effects of enzyme activity (see Fig. 4) .

Concerning the effect of metal ions, such as Cu²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Hg²⁺, Fe²⁺, Cd²⁺, Li⁺, K⁺, and Na⁺ do not have a large influence on enzyme activity. However, when Co²⁺ and Mn²⁺ are added to reaction solution, enzyme activity highly increases .

The present invention provides a method of preparing the recombinant D-stereospecific amino acid amidase. The method comprises the steps of preparing the expression vector pBDA consists of DNA sequence of SEQ ID NO. 4, preparing the transformant introduced by the expression vector, culturing the transformant, and isolating the D-stereospecific amino acid amidase from the culture solution. The present inventors cloned the coding sequence of the D-stereospecific amino acid amidase from the recombinant plasmid pDAP into the pHCE 19T(II) expression vector. The expression vector including the coding sequence of the D-stereospecific amino acid amidase having the DNA sequence of SEQ ID NO. 4 was named ^MpBDA" (see Fig. 2). The pBDA expression vector was transformed into E. coli XLl-Blue and the transformant was named "XLl-Blue/pBDA. "

The D-stereospecific amino acid amidase of the present invention is obtained by massively culturing the transformant XLl-Blue/pBDA, and after heat treatment, absorbing this culture solution into ion exchange resin, and absorbing this into hydrophobic binding resin and refining this. In addition, the present invention provides a method of producing D-amino acids using the D- stereospecific amino acid amidase.

The D-stereospecific amino acid amidase of the present invention produces D-amino acids and ammonia by stereospecifically activating on the D-amino acid amides. Optically pure D-amino acids can be enzymatically directly produced from amino acid amides of DL-racemic mixture using the enzymatic and stereospecific characteristics of the D-stereospecific amino acid amidase of the present invention. Using the D- stereospecific amino acid amidase of the present invention, the D-amino acids such as D-alanine, D- leucine, D-norleucine, D-norvaline, D-phenylalanine, D- tyrosine, D-valine, D-lysine, D-tryptophan, D-glutamine, D-asparagine, D-methionine, D-proline, and D-aspartic acid can be produced.

The recombinant thermostable D-stereospecific amino acid amidase of the present invention with the substrate-specificity is a useful biocatalyst to produce various D-amino acids from economic DL-amino acid amide mixtures .

EXAMPLE A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention. EXPERIMENTAL EXAMPLE 1: SCREENING AND IDENTIFICATION OF NOVEL THERMOPHILIC MICROORANISM PRODUCING D-AMINO ACID AMINDASE

In order to isolate strains producing thermostable D-amino acid amidase, the present inventors used soil gathered from various environments (a haystack, humus soil, a river, waste water, and a hot spring) .

In detail, the soil inoculated NY medium was composed of 1.5% polypepton, 0.2% glycerol, 0.2% yeast extract, 0.2% meat extract, 0.2% K₂HP0₄, 0.2% KH₂P0₄, and 0.026% NH₄C1 and was incubated at 55°C. Then, the medium inoculated LB medium (1.0% trypton, 0.5% yeast extract, 0.5% common salt, pH 6.8) containing 2% agarose was incubated at 55 ^°C , and pure strain colony was selected. After selected strains inoculated at 55 °C, the strain which was observed growth within 24 hours was selected. Selected strain was inoculated 5ml LB liquid medium and

incubated at 55 ^°C for ten hours. The strain was collected by centrifugation and homogenized by sonication, and then cell-free extract was obtained.

The cell-free extract was added to 0.1M Tris-HCl

(pH 8.0) containing 2 mM D-alanyl-para-nitroanylid (D-

AlaPNA) at 0.1% (w/w), and incubated at 55^°C, and measured absorbency of yellow color formed by generating para-nitroanylid (PNA) at 405nm. To confirm this, D- amino acid generated from D-amino acid amide as a substrate was analyzed using amino acid analyzer

(Hitachi, Japan) , and a strain producing D-amino acid amidase was selected.

As a result, when the thermophilic microorganism selected above was incubated in LB-agar plate medium, milk-white and saw-toothed edged colony was formed, which grew well in aerobic condition at 55 ^°C. When the microorganism was incubated at liquid or solid medium for long time, the microorganism protruded sporangium outside of filamentous strain and formed elliptic endogenous spore. The microorganism is gram-negative microorganism and identified typical Bacillus genus. In use of carbon sources, the microorganism showed negative in most carbon sources but showed a weak response in fructose and mannose.

The present inventors examined the effect of temperature on growth of the thermophilic microorganism

at various temperature ( 30-58 ^°C) using LB liquid medium

(pH 6.8) . As a result, specific growth rate (μ ) of the strain was 0.006 at 30^°C, 0.016 at 37^°C, 0.017 at 40^°C, and 0.002 at 58 ^°C . It was identified that the strain has optimal growth temperature at 45 ^°C, and maximum growth temperature at 58 ^°C .

Generally, the above-mentioned strain was similar to Bacillus borstelensis in morphological and physiological characteristics, but was different in that Bacillus borstelensis has optimal growth temperature at 30^°C and maximum growth temperature at 50^°C. In addition, Bacillus borstelensis has not been studied in enzymatic characteristics. Therefore, the strain of the present invention was a novel thermophilic microorganism having a new enzymatic activity that was not yet identified. As the present inventor identified physical and biochemical characteristics of the strain, and the strain is gram positive ["gram-negative" above has a hyphen] and novel strain producing tryptopan diaminase (Table 1)

Table 1

In order to identify characteristics of the thermophilic microorganism, gene of 16S rDNA was sequenced. 16S rDNA was amplified using N-terminal primer and C-terminal primer, and the amplified 16S rDNA was inserted into pT7Blue (Novagen Inc., USA) and 16S rDNA was sequenced. As a result of Blast Search of the 16S rDNA sequence with other microorganisms, it has 99.7% homology to Brevibacillus borstelesis and is almost the same as Brevibacillus borstelensis . Thus, the present inventors designated the thermophilic microorganism as "Brevibacillus borstelensis BCS-1," and deposited the microorganism at the Korean Collection for Type Cultures of the Korea Research Institute of Bioscience and Biotechnology on October 21, 1999 (Deposit No. KCTC 0673BP) .

Example 1. Cloning and DNA sequencing of thermostable D- stereospecific amino acid amidase After chromosomal DNA was isolated from thermostable Brevibacillus borstelensis BCS-1, the chromosomal DNA was partially digested by au3Al and separated by electrophoresis on 0.7% agarose gel. From the agarose gel, 3-10kb fragments of DNA were purified using a GENE CLEAN LI kit (Bio-Rad) . The plasmid pUC118 digested by BamH I, 5 terminus phosphate of pUCllδ was removed and mixed with the 3-10kb fragments of the DNA digested by Sau3Al . Recombinant plasmid was constructed by incubating at 16^°C for sixteen hours with T4 DNA ligase and transformed into E. coli DH5α by electroporation. The E. coli transformants transformed by the recombinant plasmid was selected by genetic complementation on LB agar plate containing ampicillin. The method of experimental example 1 was used to measure the D-amino acid amidase activity of the recombinant E. coli transformants.

As a result, the transformants having D- stereospecific amino acid amidase activity was selected.

The recombinant plasmid including the transformant was named ^λpDAP" (Fig. 1) , and the transformant including

the pDAP was named "DH5α /pDAP." The present inventors deposited the transformant at the Korean Collection for Type Cultures of the Korea Research Institute of Bioscience and Biotechnology on July 11, 2001 (Deposit No. KCTC 10009BP) .

One transformant producing D-stereospecific amino acid amidase activity was obtained by the above- mentioned selection process, and DNA fragments that were isolated from recombinant plasmid of the transformants were sequenced. As a result, DNA fragment included in the recombinant plasmid has the DNA sequence of 2,166bp including 800bp gene encoding thermostable D- stereospecific amino acid amidase. The gene of thermostable D-stereospecific amino acid amidase was designated "bda . " The total DNA sequence containing gene of D-stereospecific amino acid amidase of the present invention is described SEQ ID NO. 2.

DNA sequence described by SEQ ID NO. 4 of D- stereospecific amino acid amidase compared with the DNA sequence of known other proteins, the DNA sequence has

26% homology with respect to dipeptide transport protein

(DppA) of Bacillus metanorisis and 24% homology with respect to dipeptide transport system (DppA) of Bacillus subtillus . The thermostable D-stereospecific amino acid amidase of the present invention is a novel enzyme at the level of DNA, which has been not reported.

Example 2. Construction of expression vector pBDA containing gene encoding D-stereospecific amino acid amidase

On the basis of the DNA sequence encoding thermostable D-stereospecific amino acid amidase, analyzed as above in Example 1, N-terminus primer described by SEQ ID NO. 5 and C-terminus primer described by SEQ ID NO. 6 was constructed. Using the primers, a polymerase chain reaction (PCR) was performed with recombinant plasmid pDAP as template to amplify DNA fragments. The 0.8kb amplified DNA fragment of SEQ ID NO. 4 was inserted into pHCEl9T(II) vector (TaKaRa Inc. Japan) , and the expression vector containing the gene of thermostable D-stereospecific amino acid amidase, which was designated "pBDA, " was constructed (Fig. 2).

Example 3. Expression of recombinant thermostable D- stereospecific amino acid amidase

In order to produce recombinant termostable D- stereospecific amino acid amidase, expression vector, pBDA which contains the gene of D-stereospecific amino acid amidase was transformed into E. coli XLl-Blue and the transformant was named "XLl-Blue/pBDA. "

The present inventors examined activity of thermostable D-stereospecific amino acid amidase expressed by the transformant, XLl-Blue/pBDA. The XL1- Blue/pBDA containing recombinant plasmid pBDA was streaked in a LB-agar plate medium containing 100 mg/1 ampicillin and ImM D-alanine-para-nitroanilid. As a result, ring of yellow color (ring by production of para-nitroanilid) showed, the recombinant plasmid was confirmed by DNA sequencing.

As a result, XLl-Blue/pBDA containing recombinant plasmid pBDA constantly expressed thermostable D- stereospecific amino acid amidase.

Example 4. Purification and analysis of physiochemical characteristics of thermostable D-stereospecific amino acid amidase <4-l> purification of D-stereospecific amino acid amidase

In order to purify the D-stereospecific amino acid amidase, the recombinant transformant was largely produced by the method of Example 3. The cell-free extract was treated by heating at 50^°C for twenty minutes, and adsorbed into anion exchange resin (Resource A), and then eluted by 0. IM Tris-HCl buffer using gradient concentration. The eluted solution of active fraction was concentrated using a membrane, and this was added to 0. IM Tris-HCl (pH 8.0) containing 1.0M ammonium sulfate (NH₄SO₄) and was adsorbed into phenyl Sepharose resin and eluted by gradient concentration using 0. IM Tris-HCl (pH 8.0). As a result, the D-streospecific amino acid amidase was more purified, by a factor of 5.5, and purification yield was 25%. The purified D- stereospecific amino acid amidase was 29kDa of molecular weight- on SDS-PAGE. The yield and enzyme activity of purified D-stereospecific amino acid amidase at each step is represented at Table 2.

Table 2

One "unit" of enzyme activity is defined as the enzyme quantity that was needed to produce lumol amino acids for one minute .

<4-2> Identification of the Biochemical characteristics of D-stereospecific amino acid amidase

In order to the biochemical characteristics of highly purified D-stereospecific amino acid amidase, the method of Example <4-l> was used.

To measure the effect of temperature, optimal temperature and heat stability was examined at various

temperature (30-100^°C) . D-amino acid of reaction products was quantified at various temperatures, and optimal temperature of purified enzyme was analyzed by measuring relative enzyme activity. After purified enzyme was heated for twenty minutes at various temperatures, heat stability was analyzed by measuring the remaining enzyme activity. After enzyme reaction was performed for thirty minutes at various temperatures, optimal temperature of enzyme activity was analyzed by measuring the production quantity of D-amino acid.

Optimal temperature of enzyme activity was 85 ^°C. After purified enzyme was heated for thirty minutes at various temperatures, heat stability was analyzed by measuring enzyme activity, and the activity of the D- stereospecific amino acid amidase was stable at about

90^°C (Fig. 3) .

In addition, to analyze the effect on the enzyme activity according to pH, after the enzyme was reacted at 50 ^°C for ten minutes at various pH using the buffer of various ranges of pH, such as MES buffer (pH 5.5-6.5), Bis-Tris buffer (pH 6.5-7.5), Tris-HCl buffer (pH 7.0- 9.0), and CAPS buffer (pH 9.0-11), relative activity was analyzed by measuring quantity of D-amino acid. After the enzyme was added to the buffer solution, this solution was placed at 4^°C for four hours. The pH stability was analyzed by measuring relative enzyme activity. As a result, D-stereospecific amino acid amidase keeps stability from pH 7 to pH 10 and shows optimum enzyme activity at pH 9.

Concerning the effect of inhibitor on enzyme activity, 0.003mM of ethylenediaminetetraacetic acid

(EDTA) showed 95% inhibition, 0.004mM of dithiothreitol (DTT) showed 75% inhibition, and 0.007mM mercaptoethanl showed 37% inhibition (Fig. 4).

Concerning the effect of metal ions, Cu²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Hg²⁺, Fe²⁺, Cd²⁺, Li⁺, K⁺, and Na⁺ do not have a large influence on enzyme activity. However, when ImM Co²⁺ and ImM Mn²⁺ are added to the reaction solution, enzyme activity increases by a factor of 62 to 150.

<4-3> analysis of substrate specificity of D- stereospecifc amino acid amidase

In order to identify substrate specificity and stereospecificity of D-stereospecific amino acid amidase derived from Brevibacillus borstelensis BCS-1, enzyme activities of D-stereospecifc amino acid amidase was examined using various D-amino acid amides .

In detail, to measure activity of D-stereospecific amino acid amidase, each 5mM amino acid amide described in Table 3 and the enzyme solution was added to 0. IM Tris-HCl buffer (pH 8.0). The reaction solution placed

at 55^°C for one hour. D-amino acids produced from D- stereospecific amino acid amidase reaction were diluted at 0. ImM with 20mM HC1 solution, and the D-amino acids were analyzed by automatic amino acid analyzer (Hitachi

L-8500A, Japan) , and the D-amino acids were quantified by standard curve of D-amino acid.

In addition, to measure the enzyme activity of each L-amino acid amide, the reaction was performed under the above conditions . L-amino acid amides and aliphatic amides were used as substrates.

As a result, the results of enzyme activity of D- stereospecific amino acid amidase derived from

Brevibacillus borstelensis BCS-1 against various D-amino acid amides and L-amino acid amides are described in Table 3.

Table 3

Example 5. Production of D-amino acids using the recombinant thermostable D-stereospecific amino acid amidase The present inventors produced D-amino acids using D-stereospecific amino acid amidase.

In detail, E. coli XLl-Blue/pBDA transformed by recombinant plasmid pBDA was cultured in LB medium

containing lOOmg/1 ampicillin at 37 ^°C, 180rpm for ten hours. The culture solution was centrifuged at 5,000rpm for twenty minutes, and the cells were collected. Collected cells were resuspended with 0. IM Tris-HCl (pH 8.0), and homogenized by sonication. After the

resuspension were heated at 55 ^°C for thirty minutes, cell extract was obtained by centrifugation. To produce D-phenylalanine by enzyme synthesis, 0.1M Tris-HCl solution (pH 8.0), 0.2M DL-phenylalanine amide, and 0.5mM cobalt was added to 50ml reaction solution. To this reaction solution, each quantity of lOul (0.67 units), 20ul (1.38 units), 30ul (2.02 units) of thermostable D-stereospecific amino acid amidase was

added, and this solution was reacted at 50 ^°C . The reaction conversion rate and optical purity of produced D-phenylalanine was then measured. To measure optical purity of D-phenylalanine, a product of enzyme reaction was induced with o- phthaldialdehyde (Yasuda et al . , Peptides, 1997, 18, 347-354) , and this reaction solution was applied in reverse HPLC column (Rexchrome S5-100-ODS, Regis Chem. Co., 4.6mm by 25cm, 5m) flowed with the solution which was mixed with 50mM sodium acetate (pH 6.8) and methanol in ratio of 55:45. A separate curve was analyzed at 342nm emission and 452nm extinction using a fluorescence detector. As a result, D-phenylalanine produced from the above was produced only by reaction of D-stereospecific amino acid amidase. As unreacted amide compounds remained in uninduced form, only D-phenylalanine produced by D-stereospecific amino acid amidase was analyzed on thin membrane chromatography and amino acid analyzer (Hitach, Japan) (Fig. 5).

As described in Scheme 1 below, D-stereospecific amino acid amidase derived from Brevibacillus borstelensis BCS-1 had stereospecificity on D-amino acids . When DL-phenylalanine amide was used as reaction substrate D-stereospecific amino acid amidase specifically acted on D-phenylalanine, to produce pure D-phenylalanine .

Scheme 1

C

DL-phenylalanine amide D-phenylalanine L-phenylalanine amide

As a result, when conversion rate of D- phenylalanine was identified after reaction of two hours

at 50 ^°C using the D-stereospecific amino acids amidase, conversion rate of D-phenylalanine showed 54% when 0.67 units of enzyme was added, 92% when 1.38 units of enzyme was added, and 100% when 2.02 units of enzyme was added. In addition, D-stereospecific amino acids amidase produced D-phenylalanine at 10g/l/h producibility (Fig 6) . This time, index showing pure purity of D- phenylalanine, enantiomeric excess of the product

(eep = {D-L}/{D+L}) and enantiomeric ratio

(E = {kcat/Km}d/{kcat/Km}L) showed 99.0% and 592, respectively. This result showed that the recombinant thermostable D-stereospecific amino acid amidase is optically separated into DL-phenylalanine amides, and thus the D-stereospecific amino acid amidase was a useful biocatalyst producing D-phenylalanine. As a result, the thermostable D-stereospecific amino acid amidase specifically acted on D-amino acids to produce various D-amino acids.

INDUSTRIAL APPLICABILITY As described hereinbefore, D-stereospecific amino acid amidase of the present invention specifically acted on D-amino acid amides and showed stability for heating, organic solvents, pH, and chemical denaturants. Because optically pure D-amino acids can be enzymatically directly produced from amino acid amides of DL-racemic mixture, the method of producing

D-amino acids is economical and clean. In addition, as aromatic amino acid, D-phenylalanine amide widely used in phamaceutical industry could be produced from economic DL-phenylalaine using the D-stereospecific amino acid amidase. The D-stereospecific amino acid amidase was used as a biocatalyst for producing high value-added amino acids .

BUDAPEST TREATY ON THE INTERNATIONA-. RECOGNITION OF THE DEPOSIT OF MICROORGANISMS POH THE PURPOSE OF PATENT PBOCEDUBE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 O : SUNG, M on-Hee

Korea Research Institute of Biosάence and Hotechnology, #52, Oun-doπg, Yusong-ku, Taejon 305-333, Republic of Korea

I . IDENTIFICATION OF THE MICROORGANISM

Accession number given by the

Identification reference given by the INTERNATIONAL DEPOSITARY DEPOSITOR: AUTHORITY:

Escherichia coli DH5@/pDAP KCTC 10009BP

π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONQMIC DESIGNATION

The microorganism identified under I above was accompanied by:

[ x ] a scientific description

[ ] a proposed taxonomic designation

(Mark with a cross where applicable)

HI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on July 11 2001.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Korean Collection for Type Cultures Signature(s) of person(s) having tϊie power to represent the International Depositary Authority of authorized ofSeial(s):

Address: Korea Research Institute of Biosciert.ee and Biotechnology (KRIBB)

#52, Oun-dong, Yusong-ku,

Taejon 305-333, BAE, Kyung Sook, Director Republic of Korea Date: July 18 2001

Forai BF/d (KCTC Form 17) sole me* 569

JNAllO < . nwm^ 01 TIII

IMT.RN K 1 OHV

RECEIPT IN TEE CASE OE ΛN ORIGINAL DEPOSIT

, _>Uύ p-..- _*. .. — :ivAc 7 1

I SUNG, Vfcon-Hee

Sarrώu Apt. 6-84. ^333-1. Tseoyong 2-ODng, C ung-ku, Taejor, 331-152, Republic of Korea

IDENTIFICATION OF THE MICROORGANISM

Accession number given by the

Identification reference given b the . INTERNATIONAL DEPOSITARY DEPOSITOR: Atrr oRiTY:

BrEvibaallas borstelensis BCS-1 KCTC 0673BP

π. SOENTIHC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The inicroorganism identified under I above was accompanied by: t x 1 a scientific description

[ ] a proposed taxαπomic designation

(Mark -with a cross where applicable)

Dl. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by ft on October 21 1999.

IV. RECEIPT OF REQUEST FOR CONVERSION

The m^jcroorgaπism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

I Name: Korean Collection for Type Cultures Signature(s) of person(s) having the power to represent the International Depositary Authority of authorized ofucial⁽s)^"-

Address- Korea Research Institute of Bϊoscieπce and Biotechnology ⁽ KRIBB) 52. Ouπ-dong. Yusong-ku, Taejon 305-333. BAE, Kyting Sook, Director Republic of Korea Date October 27 1999

Fcrra F!P/< (KCTC form 17⁾

Claims

What is claimed is :

1. A D-stereospecific amino acid amidase comprising an N-terminal amino acid sequence of SEQ ID NO. 1.

2. The D-stereospecific amino acid amidase according to claim 1, wherein the D- stereospecific amino acid amidase comprises an amino acid sequence of SEQ ID NO. 3.

3. A gene encoding the D-stereospecific amino acid amidase of claim 2.

4. The gene according to claim 3, wherein the gene comprises a DNA sequence of SEQ ID NO. 2.

5. A recombinant plasmid pDAP containing the gene of claim 4.

6. A transformant DH5α /pDAP (Deposit No. KCTC 10009BP) transformed by the recombinant plasmid of claim 5.

7. An expression vector pBDA containing a gene of SEQ ID NO. 4 encoding the D-streospecific amino acid amidase.

8. A transformant XLl-pBDA transformed by the expression vector of claim 7.

9. A method of preparing the recombinant D-stereospecific amino acid amidase, wherein the method comprises the step of culturing the transformant of claim 8 and isolating the recombinant D-stereospecific amino acid amidase from the culture solution.

10. A method of producing D-amino acids from DL-amino acid amides using the recombinant D-stereospecific amino acid amidase prepared by the method of claim 9.

11. The method according to claim 10, wherein the D-amino acids are selected from the group consisting of D-alanine, D-leucine,

D-norleucine, D-norvaline, D-phenylalanine,

D-tyrosine, D-valine, D-lysine, D-tryptophane, D-glutamine, D-asparagine, D-methionine, D-proline, and D-aspartic acid.