WO2001070937A1

WO2001070937A1 - A new thermostable d-stereospecific dipeptidase from brevibacillus bostelensis bcs-1 and its use as a biocatalyst for the synthesis of peptides containing d-amino acids

Info

Publication number: WO2001070937A1
Application number: PCT/KR2000/000730
Authority: WO
Inventors: Moon-Hee Sung; Seung-Goo Lee; Seok-Joon Kwon; Dae-Heoun Baek; Seung-Pyo Hong; Sung-Ho Cho; Mi-Hee Jung
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2000-03-24
Filing date: 2000-07-06
Publication date: 2001-09-27
Anticipated expiration: 2002-09-24
Also published as: AU5710500A; KR20010090321A; KR100365838B1

Abstract

The present invention relates to a new D-stereospecific dipeptidase derived from Brevibacillus borstelensis BCS-1 (Accession NO: KCTC 0673BP) and use of the new dipeptidase as a biocatalyst for the synthesis of peptides containing D-amino acid. Particularly, the present invention relates to a new D-stereospecific dipeptidase from Brevibacillus borstelensis BCS-1 selected by screening diverse enzymes hydrolyzing D-peptide derivatives, a gene encoding the dipeptidase, a recombinant vector containing the gene, a microorganism transformed by the vector, the enzyme characteristics (thermostability, pH stability and substrate stability), and a method for synthesis of D-amino acid-containing peptide economically and without environmental pollution.

Description

A new -thermostable D-stereospecific dipeptidase from Brevibacillus bostβlens±s BCS-1 and its use as a biocatalyst for the synthesis of peptides containing D- amino acids

FIELD OF THE INVENTION

The present invention relates to a new D-stereospecific dipeptidase derived from Brevibacillus borstelensis BCS-1 (Accession NO: KCTC 0673BP) and use of the new dipeptidase as a biocatalyst for the synthesis of .peptides containing D- amino acid.

Particularly, the ^" present invention relates to a new D- stereospecific dipeptidase from Brevibacillus borstelensis BCS-1 selected by screening diverse enzymes hydrolyzing D- peptide derivatives, a gene encoding the dipeptidase, a recombinant vector containing the gene, a microorganism transformed by the vector, the enzyme characteristics

(thermostability, pH stability and substrate stability) , and a method for synthesis of D-amino acid-containing peptide economically and without environmental pollution.

BACKGROUND

Most natural amino acids have stereospecificity and an α- carbon that shows optical activity. Although most natural proteins contain L-amino acids, microbial peptidoglycan, antibiotics and biologically active peptides contain D-amino acids .

D-amino acids have been widely used as an intermediate in the synthesis of neurotransmitters, vaccines, synthetic sweeteners, antibiotics and hormones. Especially, industrially applicable antibiotics, neuroactive peptides and sweeteners contain dipeptides composed of dipeptides of L-D- configuration .

For production of D-amino acid-containing peptides, the conventional production methods include chemical synthesis and enzymatic synthesis methods. In particular, industrially useful dipeptide sweeteners have been produced by multi-step chemical synthesis. However, this method has a disadvantage in that the α-amino group and the β-carboxyl group of aspartic acid must be chemically protected (Scheme 1) .

OH

D-AlaOH + Z-Cl ιv Z-D-AlaOH (1)

Z-D-A⁾aOH + X ^§™→ Z-D-AlaOX (2)

Z-D-AlaOX - ^H ' D-AlaOX (3)

L-AspOH ^♦ BzlOH -^M.'.'^ L-Asp(OBzl)OH .(4)

L-Asp⁽OBzl)OH + Z-Cl — OH ^ Z-L-Asp(OBzl)OH (5)

Z-L-Asp⁽OBzl)OH ^♦ D-AlaOX "∞* ^κ » Z-L-Asp(OBzl)-D-AlaOX (6)

UMr

Z-L-Asp⁽OBzl⁾-D-AlaOX ^{W Hi} > L-Asp-D-AlaOX (7)

MeOH

Therefore, a simpler method is required. The enzymatic synthesis method has the following advantages compared with the chemical synthesis method: (i) the enzymatic synthesis of dipeptides containing D-amino acids can use an economical racemic mixture as an acyl group donor and acceptor because of the stereospecificity of the enzyme; (ii) it is possible to omit the protection step and the deprotection step of the protective group .

Until now, the kinetically controlled enzymatic synthesis of D-amino acid-containing peptides using α- chymotrypsin (West, J.B., Wong, C.H., J. org. chem . , 51, 2728-2735, 1986) and D-Alanine oligomer synthesis using D- aminopeptidase on an organic solvent have been studied (Kato et al . , Bioca talysis , 3, 207-21, 1989) . However, the synthesis of D-amino acid-containing peptides using D- stereospecific dipeptidase has not yet been reported.

The stability of biocatalysts is the most important factor in the enzymatic synthesis of D-amino acid-containing dipeptides in organic solvents . Generally, thermophilic microorganisms produce highly thermostable enzymes having stability for organic solvents and chemical denaturants. For these reasons, thermostable enzymes can be suitable biocatalysts for enzymatic synthesis .

Many researchers have suggested various screening methods for enzymes hydrolyzing peptides containing D-amino acids from microorganisms. Enrichment culture techniques in media containing D-amino acid derivatives have been used for screening microorganisms producing these enzymes. However, the methods are laborious and time-consuming. Asano et al . used a turbid plate containing hydrophobic D-peptide (D-Phe)₄ in LB media to screen D-stereospecific peptidase (Asano et al . , J. Biol . Chem . , 271, 30256-30262, 1996). However, it is difficult to synthesize a large amount of (D-Phe)₄ and the synthetic peptide is very expensive. Moreover, the above methods cannot be used for screening of various enzymes hydrolyzing D-peptide derivatives (i.e. D-amino acylase and D-amino acid amidase) . Therefore, the present inventors developed a screening method capable of screening various enzymes hydrolyzing D-peptide derivatives . By using this method, we, the inventors of the present invention, isolated a novel thermophilic microorganism living at above 58 ^°C and producing thermostable D-stereospecific dipeptidase which maintains more than 80% enzyme activity after heating for 30 min at 60^°C, and gene encoding the D-stereospecific dipeptidase. In addition, the present inventors have developed a method for the synthesis of D-amino acid peptide by using of thermostable D-stereospecific dipeptidase as a biocatalyst .

SUMMARY OF THE INVENTION

It is an objective of this invention to provide a novel thermophilic microorganism that produces D-stereospecific dipeptidase by developing a method for screening enzymes hydrolyzing D-amino acid-containing D-peptide derivatives. ^■ It is a further objective of this invention to provide D-stereospecific dipeptidase having high ther ostability and unique substrate specificity, and its coding gene.

It is an additional objective of this invention to provide an enzymatic method for the synthesis of D-amino acid-containing D-peptide using the new thermostable dipeptidase purified from the transformed E . coli containing the gene of dipeptidase gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a turbid plate containing N- benzyloxycarbonyl-D-alanyl-D-alanine benzyl ester (herein after, referred to as "Z-D-Ala-D-AlaOBzl") for screening microbial D-stereospecific dipeptidase, D-aminoacylase and D- amino acid amidase, where a: Brevibacillus borstelensis BCS-1; b: 'Negative control strains { E . coli ) ; c: Negative control strain { Bacillus subtilis) ; d: Cell-free extract derived from Brevibacillus borstelensis BCS-1; e: Thermolysin (protease) . FIG. 2 is a graph that represents hydrolysis by us- ing the cell-free extract of Brevibacillus borstelensis BCS-1, where

1: The peak of Z-D-Ala-D-AlaOBzl;

2: The peak of Z-D-AlaOH (it is another product of hydrolysis of the enzyme from Brevibacillus borstelensis BCS- 1. ) ;

3: The peak of BzlOH (it is another product of hydrolysis of the enzyme from Brevibacillus borstelensis BCS-1) . FIG. 3a represents the result of comparing the 16S rDNA sequence of Brevibacillus borstelensis BCS-1 with that of known microorganisms.

FIG. 3b represents the taxonomical position of novel Brevibacillus borstelensis BCS-1 by analysis of the IβS rDNA sequence .

FIG. 3c represents the optimal growth temperature of Brevibacillus borstelensis BCS-1.

FIG. 4a represents construction of the cloning vector containing a gene encoding D-stereospecific dipeptidase from Brevibacill us borstelensis BCS-1.

FIG. 4b represents construction of the expression vector for mass-production from recombinant E . coli, where DPT: D-stereospecific dipeptidase; DAA : D-amino acid aminotransferase .

FIG. 5 represents the result of comparing the amino acid sequence encoding D-stereospecific dipeptidase of Brevijacillus borstelensis BCS-1 with the amino acid sequence encoding other dipeptidases of known microorganisms (Abbreviation: Bcs-1, D-stereospecific dipeptidase from Brevibacillus borstelensis BCS-1; A.cal, dipeptidase from Acinetobacter calcoaceticus) .

FIG. 6 represents the effect of temperature on D- stereospecific dipeptidase. FIG. 7 represents the thermostability of D-stereospecific dipeptidase .

FIG. 8 represents the effect of pH on D-stereospecific dipeptidase . DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail. The present invention provides a method for screening microorganisms producing D-stereospecific dipeptidase by use of turbid plates containing Z-D-Ala-D-AlaOBzl . The method has the following advantages: (i) the chemical synthesis of Z-D-Ala-D-AlaOBzl is very easy; (ii) it is possible to discover various enzymes hydrolyzing D-peptide derivatives. The present inventors synthesized Z-D-Ala-D-AlaOBzl easily and developed the screening method for microbial D- stereospecific peptidase and esterase by observing halo from turbid plates containing Z-D-Ala-D-AlaOBzl . Particularly, Z-D-Ala-D-AlaOBzl is synthesized by using Z-D-AlaOH and D-AlaOBzl . TosOH as substrates. The Z-D-Ala-D- AlaOBzl dissolved in methanol is added into LB medium, vigorously mixed and poured into Petri dishes . A turbid plate is formed after the medium is solidified. After more than one thousand strains of thermophilic microorganisms isolated from Korean soil were streaked on the turbid plate and incubated overnight at 55 ^°C, a clear zone formed around the colonies producing D-peptide derivatives-hydrolyzing enzymes. This screening method enables various D-peptides derivatives-hydrolyzing enzymes to be screened easily and rapidly.

The present invention also provides a novel microorganism isolated by the above-mentioned screening method.

The novel microorganism screened has the following microbiological characteristics: (i) white colonies and serrated colonies are formed on MY agar; (ii) in liquid culture, filament-shaped bodies are observed; (iii) in stationary phase, endospores are formed, which is a typical characteristic of Bacillus sp . ; (iv) the active mobility of the novel microorganism in stationary phase is observed under microscope; (v) in anaerobic conditions, the microorganism grows a little and rod-like bodies are observed under microscope. As for the carbon source required for their growth, the microorganism uses only mannose and fructose. This is different from the other strains of Bacill us sp.

For exact characterization, the sequence of 16S rDNA derived from the microorganism was compared with those of other known microorganisms . The result showed high homology with the 16S rDNA sequence of Brevibacillus borstelensis (see

FIG. 3b) .

In addition to the above-mentioned physiological and taxonomical characteristics, the novel microorganism provided by this invention has an optimal growth temperature at 45 ^°C and the highest growth temperature at 58 ^°C, whereas the reported Bacillus borstelensis (Shida et al . , Int . J. Syst . Bacteriol . , 45, 93-100, 1995) have optimal growth temperature at 30 ^°C

According to these characteristics, the microorganism provided by the present invention was identified as a novel strain of Breviba cill us borstelensis and designated as Brevibacillus borstelensis BCS-1. The novel thermophilic Brevibacillus borstelensis BCS-1 was deposited in the Korean Collection for Type Cultures (KCTC) on October 21, 1999 (Accession NO: KCTC 0673BP) . This invention also provides a new thermostable D- stereospecific dipeptidase produced by Breviba cill us borstelensis BCS-1.

The cell-free extract from Brevibacill us borstelensis BCS-1 cultured at 55 ^°C was added into the reaction mixture containing Z-D-Ala-D-AlaOBzl . The production of Z-D-AlaOH and benzyl alcohol (BzlOH) by hydrolysis demonstrates that Brevibacillus bors telensis contains thermostable D- stereospecific dipeptidase (see FIG. 2) .

The D-stereospecific dipeptidase of the present invention showed optimal activity at 65 ^°C and ther ostability to 55 °C after heat treatment for 30 min (see FIG. 7) . Also, the range of optimal pH is preferable at pH 7 to pH 10, and most preferable is pH 8.5.

In the case of using four diastereomers of alanyl- alanine as substrates, the D-stereospecific dipeptidase shows L-D-stereoselctive hydrolysis of the peptide bond (see Table 3) .

Besides, judging from the result of hydrolyzing the various dipeptides including substrates whose C-terminal and N-terminal are protected chemically, the active site of D- stereospecific dipeptidase of the- present invention has a comparatively flexible structure (see table 4) . The thermostable D-stereospecific dipeptidase could be a useful biocatalyst in the synthesis of D-amino acid-containing peptides and L-D-dipeptides by using the reverse reaction of the hydrolysis reaction.

The present invention also provides the gene encoding the above-mentioned thermostable D-stereospeci ic dipeptidase. The DNA sequence is described by SEQ ID NO: 3. The amino acid sequence (SEQ ID NO: 4) deduced from the DNA sequence shows less than 25% homology with the dipeptidases from A . cal coaceticus, humans, mice, rats, rabbits, pigs and sheep. It demonstrates that the D-stereospecific dipeptidase of the present invention is a novel enzyme at the level of DNA sequence (see FIG. 5) .

Also, the present invention provides a method for producing D-amino acid-containing peptides or peptide sweeteners from the recombinant E. coli overexpressing D- stereospecific dipeptidase.

By using the stereospecificity of D-stereospecific dipeptidase provided by the present invention, dipeptides could be synthesized by one-step reaction by following scheme 2. <Scheme 2>

D-amino acid acyl amide L,D-dipeptide acyl amide or L-amino acid , _or D-amino acid acyl ester L,D-dipeptide acyl ester

H H H f H

H₂ii"- C— COOH + NH₂— C-COX i I NH₂"- C-CONH—C-COX

R R' A R.

In contrast, in the chemical synthesis of peptide sweeteners, the alpha-a ino group and the beta-carboxyl group of aspartic acid used as the acyl donor must be protected chemically. However, in the case of D-stereospecific dipeptidase-catalyzed synthesis of peptide sweeteners, the protection and deprotection of the protecting group could be omitted. Therefore, the synthetic process could be shortened to be a one-step reaction by following Scheme 3.

D-amino acid acvl amide or L,D-peptide acyl amide or ,

L-aspartic acid - D-amino acid acyl ester L,D-ρeptide acyl ester

H H H H ϊ f

H2»'" C-COOH NH2— C-COX -*• NH₂"- C-CONH— C— COX

I I 1 CH₂ R' CH₂ -

I COOH COOH

For the synthesis of D-amino acid-containing peptides based on the above-mentioned principle, the gene (cloned from thermophilic Brevibacill us borstelensis BCS-1) encoding recombinant D-stereospecific dipeptidase was amplified by PCR and was inserted into pTrc99A. The overexpression vector constructed by this process was designated as pDPTl (see FIG. 4b) . pDPTl was introduced into E . coli XLl-Blue. The activity of D-stereospecific dipeptidase of the transformed E . coli increased about 4000 times when compared with original strain. After purification of D-stereospecific dipeptidase overexpressed from the recombinant E. coli, the synthesis of D-amino acid peptides was performed by using it as a biocatalyst. The result of synthesis demonstrated that D- stereospecific dipeptidase provided by the present invention could be industrially applicable . The present invention is further illustrated with reference to the following examples that are not intended to be in anyway limiting to the scope of the invention as claimed.

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples . However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements in the spirit and scope of the present invention.

Example 1: Identification of Brevibacillus bostelensis BCS-1 producing enzymes hydrolyzing D-peptide derivatives .

(Step 1) Chemical synthesis of Z-D-Ala-D-AlaOBzl Z-D-AlaOH (6 mol) and D-AlaOBzl . TosOH (6 mmol) were dissolved in dimethylformamide . 1- [3- (Dimethylamino) propyl -3-ethylcarbodiimide hydrochloride, ^' EDC and 1- Hydroxybenzotriazole hydrate (HOBT) were added into the solution and stirred at room temperature for 16hr. Then, water was added into the solution. The produced Z-D-Ala-D- AlaOBzl was precipitated, filtered off and remaining solvent was evaporated. 1.8 g of the resulting Z-D-Ala-D-AlaOBzl was obtained in white powder form.

(Step 2) Screening of thermophilic microorganisms producing D-stereospecific dipeptidase.

600 mg of Z-D-Ala-D-AlaOBzl dissolved in methanol (10 mg/ml was added into one liter of the LB medium containing 2.0% agar, vigorously mixed and 20 ml were poured into Petri dishes. Turbid plates formed as the medium solidified. More than one thousand species of thermophilic microorganisms isolated from the Korean soil were streaked on the turbid plates and incubated at 55 ^°C for over 12 hrs . The strain forming halos around colonies was selected and designated as thermophile BCS-1 (FIG. 1) . As shown in Figure 1, only thermophile BCS-1 formed a clear zone around the colony, whereas E. coli and Bacillus subtilis did not. From this result, we, the inventors of the present invention, found that the clear zone was found around the microorganism having

D-stereospecific peptidase activity and that the D- stereospecific peptidase was present in the cytoplasm of thermophile BCS-1. Therefore, the screening method using turbid plates was suitable to isolate microorganisms with D- stereospecific peptidase activity.

(Step 3) Hydrolysis of Z-D-Ala-D-AlaOBzl by using the cell- free extract from thermophile BCS-1.

After the isolated thermophile BCS-1 was incubated in LB medium at 50^°C and centrifuged, the cell pellet was resuspended in 50 mM Tris-HCl containing phenyl methyl sulfonyl fluoride (PMSF ,0.5 M) , disrupted by ultrasonicator and centrifuged at 12,000 rpm. The supernant was used as the cell-free extract.

100 1 of the cell-free extract of thermophile BCS-1 was added into 1 ml of Tris/HCl buffer containing 5 μmol of Z-D- Ala-D-AlaOBzl (pH 8.0), vigorously mixed at 55°C and 100 μl samples of the reaction mixture were assayed by HPLC. From the reaction, Z-D-AlaOH, D-AlaOH and BzlOH were produced. The results demonstrated that thermophile BCS-1 contained D- stereospecific dipeptidase (FIG. 2).

(Step 4) Identification of a novel Brevibacillus borstelensis BCS-1.

Thermophile BCS-1 has the following physiological and biochemical characteristics (Table 1). <Table 1> The physiological and biochemical characteristics of thermophilic Brevibacill us borstelensis BCS-1.

For more exact characterization of the thermophilic microorganism, 16S rDNA was sequenced. 16S rDNA was amplified by N-terminal primer (SEQ ID NO: 1) and C-terminal primer (SEQ ID NO: 2) , inserted into pT7Blue and sequenced. Blast search of the 16S rDNA sequence of the microorganism demonstrated that it is almost the same as the 16S rDNA sequence of Brevibacillus borstelensis . Figure 3 is the result of the comparison of the 16S rDNA sequence of the thermophilic microorganism provided by the present invention with those of several other microorganisms.

In addition to the above physiological and taxonomical characteristics, thermophile BCS-1 has an optimal growth temperature at 45^°C and the highest growth temperature at 58 ^°C (FIG. 3C) , whereas the reported Bacillus borstelensis (Shida et al . , Int . J. Syst . Bacteriol . 45: 93-100, 1995) have an optimal growth temperature at 30 ^°C.

The remarkably difference of growth temperatures demonstrated that the thermophilic microorganism of the present invention is a novel strain of Brevibacillus borstelensis . Thus, the thermophilic microorganism was designated as Brevibacillus borstelensis BCS-1 and deposited in the Korean Collection for Type Cultures (KCTC) on October 21, 1999 (Accession NO: KCTC 0673BP) .

Example 2 : DNA sequence determination of the gene encoding thermostable D-stereospecific dipeptidase.

Chromosomal DNA was isolated from thermophilic Breviba cill us borstelensis BCS-1, partially digested by Sau3AI and separated by electrophoresis on 0.7% agarose gel. From the agarose gel, 3-10 kb fragments of DNA were purified by GENE CLEAN II kit (Bio-Rad) . The plasmid, pUCllδ digested by BamHI, 5' -phosphate of the pUCH8 was removed and mixed with the 3-10 kb fragments of the above DNA digested by Sau3AI . Recombinant plasmid pBCS8 was constructed by incubating at 16^°C for 16 hours in T4 DNA lygase-containing reaction mixture (FIG. 4a) and electrophorated into E . coli WM335 (D-glutamine auxotroph) . The transformed E . coli was selected by genetic complementation on the agar plate containing ampicillin and D-alanyl-D-glutamate (0.2 mM) . The method used in Example 1 was used to measure the D- stereospecific dipeptidase activity of the recombinant E . coli WM335.

One strain producing D-stereospecific dipeptidase was isolated by the above-mentioned selection process. The gene encoding D-stereospecific dipeptidase was cloned and the DNA sequence was analyzed. As shown in Figure 5, the deduced amino acid sequence (SEQ ID NO: 4) of the D-stereospecific dipeptidase was compared with that of other reported dipeptidases . The less than 25% homology demonstrated that D-stereospecific dipeptidase of the present invention is a novel enzyme at the level of DNA.

Example 3: Overexpression and measurement of the activity of thermostable D-stereospecific dipeptidase.

On the basis of DNA sequence encoding the D- stereospecific dipeptidase of Example 2, N-terminal primer

(SEQ ID NO: 4) and C-terminal primer (SEQ ID NO: 5) were designed. The gene encoding dipeptidase from Brevibacillus borstelensis BCS-1 was amplified by using the primers by PCR and inserted into pTrc99A. The constructed recombinant expression vector was designated as pDPT-1 (FIG. 4b) and transformed into E . coli XLl-Blue.

The measurement of activity of D-stereospecific dipeptidase was performed by the following method. The cell- free extract containing D-stereospecific dipeptidase was added into 50 mM Tris/HCl containing 5 mM L-alanyl-D-alanine (pH 8.0) and incubated at 55 ^°C for 5 min. 0.1 ml of the reaction mixture was added to 0.75 ml of Cd-Ninhydrin solution (Doi et al . , Anal . Biochem . , 118, 173-184, 1981) and absorbance was measured at 505 nm (Wu Z. et al., Biochemistry, 34, 2455-2463, 1995) . One unit of enzyme activity was defined as the amount of enzyme required for hydrolyzing 1 μmol of L-alanyl-D-alanine for 1 min. The result demonstrated that the activity of cell-free extract from recombinant E . coli XLl-Blue was increased 4000 times when compared with the original strain.

Example 4: The purification of thermostable D-stereospecific dipeptidase and the identi ication of its physicochemical characteristics .

(Step 1) Purification of D-stereospecific dipeptidase.

The method described in Example 3 was used for measuring the activity of D-stereospecific dipeptidase overexpressed in recombinant E . coli . In order to purify the D-stereospecific dipeptidase, the D-stereospecific dipeptidase was overexpressed in recombinant E . coli and cell-free extract from recombinant E . coli was prepared by using the method used in Example 1. The cell-free extract was treated by heating at 50^°C for 20 min, adsorbed on anion exchange resin [Hitrap Q (Pharmacia, Sweden) ] and eluted by 20 mM Tris-HCl buffer containing 1 M NaCl (pH 8.0) . The eluted solution of the active fraction was concentrated using a membrane, added to 50 mM potassium-phosphate buffer (KH₂P0₄/K₂HP0₄, pH 7.2) containing 1 M ammonium sulfate (NH₄S0₄) , adsorbed on phenyl sepharose resin and eluted by 50 mM potassium-phosphate buffer. The eluted solution was reconcentrated, added into 20 mM Tris-HCl (pH 7.5), bound on anion exchange resin [Mono Q (Pharmacia, Sweden)] and eluted by Tris-HCl (pH 7.5) containing 1- M NaCl. By using the above process, D- stereospecific dipeptidase was purified as a single protein. The yield and activity of D-stereospecific dipeptidase purified from each step are shown in Table 2. L-Ala-D-Ala was used as a substrate.

<Table 2> Yield and activity of D-stereospecific dipeptidase from each step.

(Step 2) Identi ication of the biochemical characteristics of D-stereospecific dipeptidase

To identify the biochemical characteristics of D- stereospecific dipeptidase, the method described in Example 3 was used for measuring the activity of D-stereospecific dipeptidase. The result of measuring activity at various temperatures demonstrated that the optimal temperature is 65^°C (FIG. 6) . Also, to identify the thermostability of D- stereospecific dipeptidase, D-stereospecific dipeptidase was heat-treated for 30 min at various temperatures and the activity was measured. The activity of D-stereospecific dipeptidase was stable to about 55^°C (FIG. 7) . In addition, to find the optimal pH, the activity of the enzyme was measured in the range of pH 5.5-10 by bis-Tris, Tris, and CHES buffer. As a result, the pH was favorable in the range of pH 7 to pH 10 and optimal at pH 8.5 (FIG. 8) .

(Step 3) Substrate specificity of D-stereospecific dipeptidase .

To identify the stereospecificity of the D-stereospecific dipeptidase for dipeptide substrates, four diastereomers of Ala-Ala dipeptide were used. The inventors of the present invention demonstrated that the enzyme has stereoselectivity recognizing L-amino acid for amino-terminal and D-amino acid for carboxyl-terminal and substrate specificity more selectively hydrolyzing L-D-dipeptides (Table 3) . <Table 3>

' Kinetic parameters of D-stereospecific peptidase,

n*= No reaction

Besides the above-mentioned dipeptide, the enzyme hydrolyzed various dipeptides and Z-L-Asp-D-AlaOMe whose N- terminals and C-terminal are protected chemically (Table 4) . It demonstrated that the active site of the enzyme provided by the present invention has a comparatively flexible structure. From these results, D-stereospecific dipeptidase of the present invention shows the highest stereospecificity for dipeptidases of L-D-configuration, but is weak or inactive toward D-D-dipeptides and D-L-dipeptides .

<Table 4> Substrate specificity of D-stereospecific dipeptidase .

Substrate Relative activity ( % )

L-Ala-D-Ala 100

D-Ala-L-Ala 0

D-Ala-D-Ala 2 . 9

L-Ala-L-Ala 8 . 3

D-Ala-D-Glu 1 . 7

L-Asp-D-Ala 37 . 8

D-Ala-L-Asp 0

L-Asp-L-Asp 1 . 1

D-Ala-D-Ala-D-Ala 0

D-Ala-D-Ala-D-Ala-D-Ala 0

L-Asp-D-Ala-OMe 3 . 2

D-AlaNH, 0

L-AlaNH₂ 0 Example 5 : The enzymatic synthesis of D-amino acid containing peptide by using thermostable D-stereospecific dipeptidase .

(5-1) The enzymatic synthesis of Z-L-Asp-D-AlaOBzl

Chemically protected Z-L-aspartic acid was used as the acyl group donor and D-AlaOBzl was used as an acyl group acceptor in the model reaction system for the production of D-amino acid-containing peptides <Scheme 4>. Synthesis was completed in one-step reaction by using D-stereospecific dipeptidase of the present invention as a biocatalyst.

Z-L-AspOH + D-AlaOBzl >Z-L-Asp-D-AlaOBzl

D-stereospecific dipeptidase

As for the reaction condition, Z-L-AspOH (5 mM) , D- AlaOBzl (10 mM) and D-stereospecific dipeptidase having about 300 units for L-Ala-D-Ala per 1 ml were added, mixed and freeze-dried. The enzyme reaction was performed in several organic solvents such as water-saturated methyl acetate, ethyl acetate, butyl acetate, diethyl ether and hexane. The result demonstrated that Z-L-Asp-D-AlaOBzl was effectively synthesized, yielding 15-30% in methyl acetate, butyl acetate and diethyl ether (Table 5) . <Table 5> The synthesis of Z-D-Asp-D-AlaOBzl by using the D-stereospecific dipeptidase.^'

(5-2) The enzymatic synthesis of L-Asp-D-AlaOMe

To synthesize peptide sweeteners without the chemical protection of the acyl group donor, L-Asp was used as the acyl group donor and D-AlaOMe was used as the acyl group receptor for the synthesis of peptide sweeteners of L-D- configuration as the model reaction system. D-stereospecific dipeptidase of the present invention was used as a biocatalyst in the enzymatic synthesis <Scheme 5>.

L-AspOH + D-AlaOMe >L-Asp-D-AlaOMe

D-stereospecific dipeptidase ,

As for reaction conditions, L-AspOH (5 mM) , D-AlaOMe (10 mM) and D-stereospecific dipeptidase were added, mixed and freeze-dried. Enzymatic synthesis was performed in the organic solvent system containing tetrahydrofuran, dimethyl formamide, dimethyl sulfoxide, ethyl acetate or diethyl ether and in the eutectic system containing small amounts of solvents such as water, glycerol or methanol (Lopez-Fandino et al . , Biotechnol . Bioeng. , 43, 1024-1030, 1994). Synthesis reaction occurred in both the organic solvent system and the eutectic system. By-products except for L-Asp-D-AlaOMe were not synthesized.

By using the above-mentioned method, the synthesis of L-D- dipeptides can be synthesized not only in the model mentioned in the Example 5 but also in the synthesis of industrially useful D-amino acid-containing antibiotics, neuroactive peptides and sweetening.

INDUSTRIAL APPLICABILITY

As described above, a new screening method using turbid plates containing Z-D-Ala-D-AlaOBzl enables the isolation of various microorganisms producing enzymes hydrolyzing D- peptide derivatives easily and rapidly. The novel thermophilic Brevibacillus borstelensis BCS-1 screened by the developed method produces a new thermostable D-stereospecific dipeptidase that has a unique substrate specificity and stability for heating, organic solvents, pH and chemical denaturants . Thus, the D-stereospecific dipeptidase is industrially useful for the synthesis of dipeptides of L-D- configuration without by-products by one-step reaction. Also, the reaction uses the racemic mixtures as substrate. Therefore, various peptides containing D-amino acids, such as antibiotics, neuroactive peptide and peptide sweeteners contain dipeptides of L-D-configuration . The dipeptides of L- D-configuration may be synthesized at high yield without protection and deprotection using this new D-stereospecific dipeptidase .

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

BUDAPEST -πtEΛTΪ ON THE !NTEENA.-naNAL RECOGNITION OF THE DEPOSIT OF MKBOOKGANKMS FOR THE FUHPOSE OF PATENT PKOCEDUBE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1

Cc SIM}, ∞n-Hsε

Sarri u Apt 6-84, #383-1, Taepyong 2-ά3ng, Cπung-ku, Taejαn 301-152, Republic of Korea

I . IDENTIFICATION OF THE MICROORGANISM

Accession number given by the

Identification reference given by the INTERNATIONAL DEPOSITARY DEPOSITOR- AUTHORITY:

Breviba illiis borstelensis BCS-1

KCTC 0673BP

π. SCIENTIFIC DESCRIPTION AIND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

[ x ] a scientific description

[ ] a proposed taxonomic designation

(Mark w thr a cross where applicable)

HI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the rπiαoαrganisπi identified under I abαve, which was recdved by it on October 21 1999.

W. RECEIPT OF REQUEST FOR CONVERSION

The πucroorganism identified under I above was recdved by this International Depositary Authority on ^"" . and a request to convert, the original deposit to a deposit under the Budapest Treaty was recdved by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Korean Collection for Type Cultures Sigπatare{s)-of personts) having the power to represent the International Depositary Authority of authorized αffidal(s):

A Address Korea Research Institute of Biosdence and Biotechnology ⁽KRIBB) ^• #52. Oun-dong, Yusong-ku. Taejon 305-333.

Republic of Korea Dale: October 27 1999

fonn BFΛ (KCTC Fonn 17) 26 sole P»ac

Claims

We claim

1. A novel thermophilic Brevibacillus borstelensis BCS-1 (Accession NO: KCTC 0673BP) .

2. A new thermostable D-stereospcific dipeptidase derived from Brevibacillus borstelensis BCS-1 of Claim 1.

3. The dipeptidase according to Claim 2, whose amino acid sequence is described by SEQ ID NO: 4.

4. The dipeptidase according to Claim 2, which has thermostability to 55^°C by heating for 30 min and pH stability in the range of pH 7 to 10.

5. The dipeptidase according to Claim 2, which has substrate specificity for dipeptide having L-amino acid for N-terminal (P-_L site) and D-amino acid for C-terminal (P_x' site) of dipeptides .

6. A gene encoding D-stereospecific dipeptidase of Claim 2 described by SEQ ID NO: 3.

7. An expression vector pDPTl represented in Figure 4b containing the gene encoding D-stereospecific dipeptidase of claim 6.

An E. coli transformant transformed with pDPTl of Claim 7

9. The enzymatic process for synthesis of D-amino acid- containing peptides, peptide-sweeteners or L-D-dipeptides using the D-stereospecific dipeptidase (Claim 2) of Brevibacillus borstelensis (Claim 1).

10. The method for screening the microorganisms producing enzymes hydrolyzing D-peptide derivatives by use of turbid plate containing Z-D-Ala-D-AlaOBzl .