KR100352192B1 - A New thermophilic bacterium Brevibacillus borstelensis BCS-1 and A thermostable D-stereospecific amino acid amidase produced therefrom - Google Patents

Abstract

The present invention relates to a novel high temperature Brevibacillus bosterensis microorganism isolated from Korean soil and a heat-resistant D-stereospecific amino acid amidase produced therefrom, the Brevibacillus bosterensis BCS-1 of the present invention ( D-stereospecific amino acid amidase produced by Brevibacillus borstelensis BCS-1, KCTC 0673BP) is a biocatalyst that hydrolyzes amides containing D-amino acids to D-amino acids and ammonia stably even at high temperatures, and D It can be usefully used as a biocatalyst for the enzymatic synthesis of various new D-amino acids from, L-amino acid containing amides.

Description

A new thermophilic bacterium Brevibacillus borstelensis BCS-1 and A thermostable D-stereospecific amino acid amidase produced therefrom}

The present invention relates to a novel thermophilic microorganism Brevibacillus bosterensis and D-stereospecific amino acid amidase produced therefrom, and more particularly, D, an enzyme that hydrolyzes the amino terminal amino acids of D-amino acid-containing amides. - specific amino acid stereo amidase novel thermophilic microorganism strain to produce (D-Stereospecific amino acid amidase, hereinafter abbreviated to 'DAA') BRAY ratio Bacillus boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) ( KCTC 0673BP) and heat-resistant DAA produced therefrom, and a method for producing a new D-amino acid using an enzyme reaction of DAA.

Most amino acids in nature have alpha carbon which shows optical activity and show stereospecificity. Most proteins in nature are composed of L-stereospecific amino acids, but the components of microorganisms such as peptidoglycans and peptide antibiotics, and those of higher bioactive substances, are D-stereospecific. It consists of amino acids. Accordingly, D-stereospecific amino acids are widely used in food and medicine as intermediates for synthesizing bioactive substances such as neurotransmitters, vaccines, synthetic sweeteners, antibiotics and hormones.

As a method for producing D-amino acids, chemical synthesis and production methods using microbial enzymes as biocatalysts are known. The production of amino acids by chemical synthesis is difficult to undergo a complex optical pure process because the product is obtained in a mixed mixture of D, L-amino acids. On the other hand, D-amino acid production method using biocatalyst can directly produce optically pure D-amino acid in one step, and it is attracting attention as a low pollution clean production technology that can replace the multi-step chemical synthesis method that has the problem of environmental pollution. .

The method for producing D-amino acid using biocatalyst has been studied to produce purely separated D-amino acid from D, L-amino acid mixture, and this method is to convert D, L-amino acid mixture into D, L-amino acid-containing amide mixture. Then, DAA is added thereto to selectively hydrolyze only the D-amino acid amide in the D, L-amino acid-containing amide mixture to produce D-amino acid. Therefore, the use of DAA for the production of D-amino acids has the advantage of producing a variety of industrially useful D-amino acids from relatively inexpensive D, L-amino acid mixtures.

The D- amino acid as the biological catalyst mesophilic foil spectrum not trophy in the microorganism used in the production technology of oak (Ochrobactrum anthropi), Bacillus ssere mouse (Bacillus cereus) specific peptidase derived from the D- solid (Asano et al., J. Biol Chem 271:.. 30256-30262.1996) and no less Arcadia ohrienta (Nocardia orientalis) is D- amino acids derived from the amino dehydratase (Asano et al, Biochem Biophy Res Com 162:...... 470-474 1989) , etc. However, these enzymes exhibit enzymatic activity at 37 ° C. or lower, and no studies have been conducted on heat-resistant DAA enzymes derived from high temperature microorganisms that maintain enzymatic activity at temperatures higher than 50 ° C.

In the bioconversion process using enzymes as biocatalysts, the stability of biocatalysts is the most important factor in determining the productivity of enzyme conversion synthesis technology. In general, thermophilic microorganisms that live in a high temperature growth environment adapt to the high temperature extremes of the habitat and produce heat-resistant enzymes that are very stable against heat. In addition to the heat-stable characteristics, these heat-resistant enzymes have been reported to have stability against organic solvents, stability against pH and chemical denaturation agents, and thus are the most effective biocatalysts used in enzyme conversion synthesis technology.

Therefore, the present inventors grow at a high temperature of 55 ° C. or higher from soils, compost, and high temperature wastewater inhabited by high temperature bacteria in various parts of Korea, and produce heat-resistant DAA capable of synthesizing pure D-amino acid at high temperature. New thermophilic microbes were explored. As a result, among the 1,300 species of high temperature microorganisms isolated from Korean soil, compost, and high temperature wastewater, a novel high temperature microorganism producing D-amino acid synthetic heat resistant enzyme having high thermal stability was isolated and completed the present invention.

It is an object of the present invention to provide a novel thermophilic microbial strain that produces heat resistant DAA which can be usefully used as a biocatalyst for enzymatic synthesis of D-amino acids.

It is also an object of the present invention to provide a novel heat resistant DAA produced from the novel thermophilic microbial strain.

Another object of the present invention is to provide a method for producing enzymatic synthesis of D-amino acid using the novel heat resistant enzyme.

1 is a 16S rDNA nucleotide sequences of various microbes reported the 16S rDNA base sequence of the novel thermophilic microorganism breather ratio Bacillus boss of the invention exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) in the existing by BLAST search Is the result of comparing homology with

FIG. 2 is a graph showing the optimal pH (A) and pH stability (B) of the heat resistant D-stereospecific amino acid amidase purified from the novel high temperature microbial Brevibacillus bosterensis BCS-1 of the present invention,

Figure 3 is a graph showing the optimum temperature (A) and thermal stability (B) of the reaction of the heat-resistant D-stereospecific amino acid amidase purified from the novel high-temperature microbial Brevibacillus bosterensis BCS-1 of the present invention,

Figure 4 is a result of analyzing the D-phenylalanine produced from D, L-phenylalanine amide by the heat-resistant D-stereospecific amino acid amidase produced by the novel high temperature microorganism Brevibacillus bosterensis BCS-1 of the present invention Is a graph.

In order to achieve the above object, the present invention is produced from a separate soil from Korea, a novel thermophilic microorganism Bacillus strain Brescia non boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) and the microbial enzymes of D- amino acids Provided are novel heat-resistant D-stereospecific amino acid amidases that can be usefully used for biosynthesis.

Hereinafter, the present invention will be described in detail.

The present invention relates to Brevi Bacillus BCS-1, a thermophilic microbial strain.

Isolation, identification and enzymatic characteristics of the novel pyrogenic microorganisms of the present invention are as follows.

1) Isolation of New Thermophilic Microorganisms

First, inoculate soil samples collected from haystacks, humus, rivers, hot wastewater, and hot springs across the country into MY medium and enrich the culture. This culture is inoculated in MY flat medium containing 2% agar and cultured to separate pure colonies. The isolated strain is inoculated in LB medium containing 2% agar and cultured to isolate colonies in which microbial growth is observed.

2) microbiological characteristics

As a result of examining the microbiological characteristics of the isolated strains as described above, when cultured in MY agar plate medium, milky white colonies were formed, and the edges of the colonies showed the shape of the fungus having serrated shapes. In addition, filamentous cells were observed in the liquid culture, and when the cells were cultured for a long time in liquid medium, they formed elliptical endospores at the semi-terminus of the cells, showing microbiological characteristics in typical Bacillus . Was characterized by active mobility under the microscope. When the isolates were cultured under anaerobic conditions, the growth of the cells was not observed with the naked eye. However, the microbial growth was weak under the microscope, and the growth of rod-shaped cells was confirmed.

On the other hand, unlike the use of carbon sources represented by most Bacillus strains, the use of the carbon source necessary for the growth of bacteria showed a slight availability when mannose and fructose were incubated at 55 ° C for 48 hours only. In addition, most carbon sources showed negative reactions. And then to a more precise identification determining the 16S rDNA base sequence of the isolated strain, 16S rDNA base sequence of the compared the nucleotide sequence homology with other microorganisms, so that breather ratio Bacillus boss exhilarating sheath (Brevibacillus borstelensis) reported previously Very high homology with

As a result of the analysis of the above physiological and chemical taxonomic characteristics, the optimum growth temperature of the known Bacillus bosterensis (Shida et al., Int. J. Syst. Bacteriol . 45: 93-100 (1995)) is 30 ℃, the microbial strain Brevibacillus bosterenesis of the present invention is the optimum growth temperature of 45 ℃, the maximum temperature is 58 ℃, there is a significant difference in the microbial growth temperature, the microorganism strain of the present invention is identified as Brevibacillus bosterensis and it was named Bacillus non breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1).

As described above, the novel thermophilic microorganism Brevibacillus bosterrensis BCS-1 isolated from soil was deposited with the accession No. KCTC 0673BP dated October 21, 1999 to the Genetic Resource Center in the Biotechnology Research Institute.

The present invention also provides a novel heat-resistant D-stereospecific amino acid amidase produced from the novel high temperature microbial Brevibacillus bosterensis BCS-1.

Heat-resistant DAA produced from the thermophilic microorganism Brevibacillus bosterensis BCS-1 of the present invention is inoculated into the LB liquid medium and incubated, and then the cells are recovered, suspended in Tris buffer and crushed by an ultrasonic crusher. Obtained as a crude enzyme solution.

Using the crude enzyme solution obtained as described above, the amount of amino acid produced by the enzymatic reaction was measured by reacting with the D-amino acid-containing amide, and the amino acid produced by the enzymatic conversion reaction by reacting with the L-amino acid-containing amide under the same conditions. As a result of measuring the amount of, it can be seen that the heat-resistant DAA produced from the thermophilic microorganism Brevibacillus bosterensis BCS-1 of the present invention acts specifically on the amino terminal D-amino acid of the D-amino acid-containing amide. .

DAA acts on D-amino acid-containing amides in the same manner as in the following formula (1) as an enzyme that stereospecifically acts on D-amino acid-containing amides to produce D-amino acids and ammonia.

In addition, the high temperature of the present inventionBrevibacillus bosterensisIn order to investigate the enzymatic properties of heat-resistant DAA produced from BCS-1, the crude enzyme solution obtained by using the above LB medium was purified through pure sedimentation, ion exchange, hydrophobic ion exchange chromatography, and the like. Enzymatic characteristics such as the optimum pH, optimal temperature and thermal stability were investigated. As a result, the molecular weight of the enzyme was confirmed to be 29 kDa, the optimum pH is about 9.5, pH stability range of about 6.0-10.0, The optimum temperature of the reaction was found to be a heat-resistant enzyme having a temperature of 70 ° C. and a thermal stability of about 65 ° C.

The present invention provides a method for producing D-amino acid using the novel heat resistant enzyme.

Amino acid-containing amide mixtures such as D, L-phenylalanine amide, D, L-alanine amide, D, L-leucine amide, D, L-tryptophan amide, etc., using heat-resistant DAA having the above enzymatic properties and stereospecificity From a racemic mixture, pure amino acids such as pure D-phenylalanine, D-tryptophan, D-leucine, and D-alanine can be enzymatically synthesized in one step without direct side impurities.

Hereinafter, the present invention will be described in more detail based on the following examples.

However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Isolation and Identification of Microorganisms

Step 1: Isolation of Microorganisms

Soil samples collected from haystacks, humus, rivers, wastewater, and hot springs around the country were collected from 1.5% polypeptone, 0.2% glycerol, 0.2% yeast extract, 0.2% meat extract, It was inoculated in MY medium having a composition of 0.2% K2HPO4, 0.2% KH2PO4, 0.026% NH4Cl, and concentrated at 55 ° C. The culture was inoculated in LB medium containing 2% agar (1.0% tryptone, 0.5% yeast extract, 0.5% saline, pH 6.8) and incubated at 55 ° C to isolate pure colonies. After culturing the isolated strain at 55 ℃, the bacterial colonies where the growth of microorganisms were observed within 24 hours were separated.

After inoculating the thermophilic microorganism into 5 ml LB liquid medium and incubating at 55 ° C. for 10 hours, the cells were recovered by centrifugation, and the cells were crushed by an ultrasonic crusher to obtain a crude enzyme solution, which was used for measuring enzyme activity.

Step 2: Characterization and Identification of Microorganisms

The thermophilic microorganism isolated in step 1 formed a milky white colony when the incubated in LB agar plate medium and sawtooth-like colony on the edges, and grew well under aerobic conditions above 55 ° C. in at was identified as a microorganism of a typical Bacillus (Bacillus genus) as gram-positive bacteria to the pojanang (sporangium) projecting from the cell outer shell of filamentary during long incubation forming endospores elliptical, utilization of carbon sources Fu rukto agarose (fructose ) And only mannose, but only a slight reactivity was negative in most carbon sources. In order to investigate the effect of temperature on cell growth, the specific growth rate (μ) of the cells was found to be 30. The growth rate of the cells was confirmed at various temperature ranges (30-55 ℃) using LB liquid medium (pH 6.8). 0.006 at 37 ℃, 0.016 at 37 ℃, 0.017 at 40 ℃, 0.019 at 45 ℃, 0.018 at 50 ℃, 0.007 at 55 ℃, the optimum growth temperature of the isolated bacteria of the present invention was confirmed as 45 ℃ and the maximum growth temperature It confirmed with 58 degreeC. The results reporting that Bacillus boss exhilarating sheath (Bacillus borstelensis) has a maximum cell growth temperature 30 ℃, the maximum growth Temperature 50 ℃ in this similarity in separating bacteria and morphological and physiological characteristics of the present invention (Shida et al ..., Int J. Syst Bacteriol 45:. 93-100 (1995)) showed a significant difference between different from Bacillus boss is not bar the enzymatic characteristics of the system exhilarating research, non-thermophilic Bacillus breather boss exhilarating cis BCS- 1 was identified as a new thermophile with a new enzyme activity that has not been identified to date.

Detailed physiological and biochemical properties of the microorganisms selected according to the present invention are shown in Table 1.

Physiological and Biochemical Properties Results obtained from new microorganisms Growth temperature 30-58 ℃ Cell shape Rod shape Gram Dyeing positivity motility positivity Endogenous spores has exist Voges-Proskauer positivity Nitrate reducing power positivity Acid production from glucose voice Acid production from mannitol voice Acid production from arabinose voice Beta-galactosidase production voice Tryptophan Deamidase Production positivity Catalase production positivity Gelatinase production positivity growth at pH 5.7 weakness Growth in 7% Saline voice Growth in anaerobic conditions positivity

In order to more accurately identify the high temperature bacteria of the present invention having the above microbiological characteristics, gene analysis of the 16S rDNA of the high temperature bacteria was performed.

For gene analysis, 16S rDNA gene was amplified by PCR using N-terminal primer (SEQ ID NO: 1) and C-terminal primer (SEQ ID NO: 2), cloned into plasmid pT7Blue, and the nucleotide sequence was determined. Subsequently, BLAST search of 16S rDNA nucleotide sequence of the high temperature bacterium of the present invention compared the homology with 16S rDNA sequencing of several microorganisms reported, and showed the most similarity to Brevibacillus borstelensis . Confirmed. Figure 1 shows a comparison of the 16S rDNA nucleotide sequence of a variety of microorganisms reported 16S rDNA of the high temperature microorganism screened according to the present invention.

Identification of the isolated strain of the present invention with the above results in non-Bacillus breather boss exhilarating cis and was named Bacillus breather boss ratio exhilarating system BCS-1. This pyrobacterial was deposited on October 21, 1999 with the accession number KCTC 0673BP to the Genetic Resource Center in the Biotechnology Research Institute.

Example 2 Purification of Heat Resistant DAA and Enzymatic and Physicochemical Properties

Isolated bacterium of the present inventionBrevibacillus bosterensisIn order to confirm the enzymatic and physicochemical properties of DAA produced by BCS-1, the crude enzyme solution obtained in step 1 of Example 1 was purified by fluoroprecipitation, ion exchange chromatography, hydrophobic ion exchange chromatography, and the like. DAA was purified. As a result of purification, the final purification was 207 times more, the purification yield was confirmed as 2%, the molecular weight of the purified DAA was confirmed to be 29 kDa on SDS-PAGE. To investigate the effects of pH and temperature on purified enzyme activity, buffers, acetate buffers (acetate, pH 3.0-5.5), bis-tris buffers (BES-Tris, pH 5.5-6.5), phosphate buffers (H2PO4, pH 6.5-7.5), Tris buffer (Tris, pH 8.0-10.5), glycine buffer (Glycine, pH 9.0-10.5), potassium chloride buffer (KCl, pH 11.5-13.0) and the like to determine the optimum pH. , pH stability was measured by adding the enzyme solution to the buffer solution and allowed to stand for 4 hours in a 4 ℃ refrigerator, and then measured relative enzyme activity. For the temperature effect, the optimum reaction temperature and thermal stability of the purified enzyme at various temperature ranges (30 ℃ -100 ℃) were investigated. Optimum temperature of purified enzyme was measured by measuring quantitative reaction of D-amino acid at different temperatures to measure relative enzyme activity.Thermal stability was measured by restoring purified enzyme for 20 minutes at various temperature ranges above. Measured.

As a result, it was confirmed that the optimum pH was about 9.5 in the effect of pH on the purification enzyme activity (FIG. 2A), and stable in the range of about 6.0-10.0 in pH stability (FIG. 2B). In the temperature effect, the optimum reaction temperature was found to be about 70 ° C. (FIG. 3A), and the thermal stability was found to be stable up to about 65 ° C. (FIG. 3B).

Example 3 Activity Determination of Heat Resistant DAA

In order to investigate the stereospecificity of the heat-resistant DAA purified from the high-temperature Brevibacillus bosterensis BCS-1 of the present invention, various D-amino acid-containing amides and L-amino acid-containing amides shown in Tables 2 and 3 were used to Substrate specificity and stereospecificity were investigated.

In order to measure DAA activity, the reaction solution was prepared in 0.5 ml of 0.1 M Tris buffer solution (Tris-HCl, pH 8.0) to include 5 mM of various amino acid amides shown in Tables 2 and 3 and the enzyme solution. Reacted. The amount of D-amino acid produced by the DAA reaction was analyzed by an automatic amino acid analyzer (Hitachi L-8500A, Japan) by diluting the concentration of D-amino acid to 20 mM in 20 mM hydrochloric acid solution, and according to the D-amino acid standard curve. Quantification Separately, the reaction was carried out under the same conditions as described above to measure the enzyme activity of various L-amino acid-containing amides, and L-amino acid-containing amides and aliphatic amides were used as substrates. The activity of the enzyme (Unit) was defined as the amount of enzyme required to produce 1 μmol of D-amino acid for 1 minute.

The breather non-selective Bacillus in the present invention boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) Enzyme activity measurement results for thermal resistance DAA various amino acids D- and L- amino acid containing an amide-containing amide that is produced from It is as showing in Table 2 and Table 3.

DAA Activity Determination for D-Amino Acid-Containing Amides temperament Enzyme deactivation (Units / mg protein) Relative activity (%) D-methionine amide 131.4 100 D-norvaline amide 103 78 D-norleucine amide 101.8 77 D-lysine amide 79.4 60 D-leucine amide 78 59 D-phenylalanine amide 14.9 11 D-tyrosine amide 13.3 10 D-alanine amide 12.5 10 D-valine amide 11.4 9 D-Tryptophan Amide 9.3 7 D-phenylglycine amide 9.3 7 D-asparagine amide 6.3 5 D-proline amide 3.7 3 D-glutamine amide 2.8 2 D-aspartic acid amide 0.4 0.3 Z-D-alanine amide - 0.0 Z-D-alanine ester - 0.0

Negative reaction

DAA Enzyme Activity on L-Amino Acid-Containing Amides temperament Enzyme deactivation (Units / mg protein) Relative activity (%) L-leucine amide 1.38 1.0 L-phenylalanine amide 0.15 0.1 L-alanine amide - 0.0 L-aspartic acid amide - 0.0 Acetamide - 0.0 n-butyramide - 0.0 Propionamide - 0.0 Benzyl amide - 0.0

Negative reaction

New heat DAA to the new non-thermophilic Bacillus breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) of this invention as the result of production is specific only to the amino terminal amino acid of the D- D- amino acid-containing amide typically action and written to does not represent an L- amino acid-containing polyamide and amide, and aliphatic acyl amides has enzymatic activity with the amino terminal protecting group, the novel thermophilic Bacillus breather ratio of the present invention boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1 DAA produced by KCTC 0673BP was confirmed to be a hydrolytic enzyme that specifically acts on the D-amino acid of the D-amino acid-containing amide.

Example 4 Synthesis of New Amino Acid Using Enzyme Produced from Microorganism of the Present Invention

The pure D- amino acids from D, L- amino acid mixture by using a selective separation of the novel non-breather boss Bacillus exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) new DAA heat resistance to the produced (KCTC 0673BP) in accordance with the invention The productivity of pure D-phenylalanine was analyzed from D, L-phenylalanine amide, a representative D, L-amino acid containing amide mixture to obtain.

The enzyme reaction was performed at 55 ° C. after adding 0.1 M Tris buffer (pH 8.0), 5 mM D, L-phenylalanine amide and 20 l (0.5 units) of purified enzyme solution to 0.2 ml reaction solution. Heat treatment was carried out in boiling water for 5 minutes to inactivate the enzyme, and then centrifuged to obtain a reaction product.

The prepared reaction product o - au taldi aldehyde (o -phthaldialdehyde) derivatized with (.. Yasudaet al, Peptides 18 : 347-354 1997) after, 50 mM acetate buffer (sodium acetate, pH 6.8) and using a 100% methanol Using a reverse phase HPLC column (Rexchrome S5-100-ODS, Regis Chem. Co., 4.6 mm x 25 cm, 5 m) while increasing the concentration of the organic solvent in proportion to The separation curve of D, L-amino acid in the range (luminescence 342 nm, extinction 452 nm) was confirmed.

In this case, the produced D-phenylalanine can be produced solely by the reaction of DAA, and the unreacted amide compound remains in underivatized form, and only D-phenylalanine produced by DAA is analyzed. .

From these results, it can be seen that the DAA enzyme produced by the thermophilic microorganism Brevibacillus bosterrensis BCS-1 of the present invention acts specifically on D-amino acids and is a biocatalyst that can be used to produce various D-amino acids.

Figure 4 is a non-thermophilic Bacillus breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) performing a matrix of D, L- phenylalanine amide for the kinetic optical resolution by using the purified enzyme obtained from the present invention The resulting D-phenylalanine was analyzed by HPLC. As shown in Figure 4, the DAA to produce a non-thermophilic Bacillus breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) of this invention because they have the D- stereospecific D, L- phenylalanine amide When used as a reactor, it can be seen that the specific action only on D-phenylalanine to produce pure D-phenylalanine. The experimental results showed a breather ratio Bacillus boss exhilarating system BCS-1 produces a conversion rate of 47% after reaction for 24 hours at 30 ℃ using a heat-resistant DAA that, where eep (enantiomeric excess of the product) = 97.1%, E (enantiomeric ratio) = 196, indicating that the heat-resistant DAA produced from the results of the present invention is a very useful biocatalyst capable of producing D-phenylalanine by optically dividing D, L-phenylalanine amide.

As described above, in accordance with the present invention which is produced DAA isolated from soil novel thermophilic microorganism Bacillus non breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP) has D- amino acid-containing amide compound Because it acts only on D-amino acids and is an enzyme that is stable against heat, organic solvents, pH and chemical denaturation agents, the production of D-amino acids using this as a biocatalyst is performed in one step from a relatively inexpensive mixture of D, L-amino acids containing It is an economical and environmentally friendly method that can effectively produce new pure D-amino acids without direct side reactions.

Claims (5)

New non-thermophilic Bacillus breather boss exhilarating system BCS-1 (Brevibacillus borstelensis BCS- 1) (KCTC 0673BP). delete The novel heat-resistant D-stereospecific amino acid amidase according to claim 1, which is obtained by culturing the Brevibacillus bosterensis BCS-1, and characterized by the following (a) to (f). (a) molecular weight 29 kDa; (b) optimum pH 9.5; (c) pH stability range 6.0-10.0; (d) reaction optimum temperature of 70 ° C .; (e) thermal stability temperature up to 65 ° C; (f) Stereospecifically Acts on D-Amino Acid-Containing Amides A method for enzymatically producing new D-amino acids from D, L-amino acid-containing amides using the microorganism strain of claim 1 or the D-amino acid amidase produced by the microorganism. The method according to claim 4, wherein the D-amino acid is D-methionine, D-norvaline, D-norleucine, D-lysine, D-leucine, D-phenylalanine, D-tyrosine, D-alanine, D-aspart At least one selected from acid, D-tryptophan, D-proline, D-glutamic acid and D-aspartic acid.