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WO2003008569A1 - Amidase d'acide amine d-stereospecifique, gene associe, procede de preparation associe et procede de preparation d'acide amine d faisant appel a ladite amidase - Google Patents

Amidase d'acide amine d-stereospecifique, gene associe, procede de preparation associe et procede de preparation d'acide amine d faisant appel a ladite amidase Download PDF

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Publication number
WO2003008569A1
WO2003008569A1 PCT/KR2001/001235 KR0101235W WO03008569A1 WO 2003008569 A1 WO2003008569 A1 WO 2003008569A1 KR 0101235 W KR0101235 W KR 0101235W WO 03008569 A1 WO03008569 A1 WO 03008569A1
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Prior art keywords
amino acid
stereospecific
amidase
acid amidase
gene
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PCT/KR2001/001235
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English (en)
Inventor
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
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Korea Research Institute Of Bioscience And Biotechnology
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Publication of WO2003008569A1 publication Critical patent/WO2003008569A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures

Definitions

  • 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.
  • 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.
  • L-amino acids 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • thermophilic microorganisms maintain their enzyme activity at high temperature .
  • the present inventors isolated a gene encoding a novel thermostable D-amino acid amidase from a novel thermophilic Brevibacillus borstelensis, and sequenced the gene.
  • the present inventors have developed a method of producing D-amino acids using the thermostable D-stereospecific amino acid amidase as a biocatalyst.
  • 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.
  • 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
  • thermostable D-stereospecific amidase (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 ( ⁇ : 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; ⁇ : 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 ( ⁇ : 0.674 units; v: 1.348 units; ⁇ : 2.022 units).
  • 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.
  • 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
  • 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
  • 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) .
  • present invention shows optimum activity at about 85 ° C and shows a relative stability till about 90 ° C (see Fig. 3) .
  • 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) .
  • metal ions such as Cu 2+ , Mg 2+ , Ca 2+ , Zn 2+ , Hg 2+ , Fe 2+ , Cd 2+ , Li + , K + , and Na + do not have a large influence on enzyme activity. However, when Co 2+ and Mn 2+ 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 M pBDA" (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.
  • 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.
  • 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.
  • 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 1 SCREENING AND IDENTIFICATION OF NOVEL THERMOPHILIC MICROORANISM PRODUCING D-AMINO ACID AMINDASE
  • thermostable D-amino acid amidase 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) .
  • the soil inoculated NY medium was composed of 1.5% polypepton, 0.2% glycerol, 0.2% yeast extract, 0.2% meat extract, 0.2% K 2 HP0 4 , 0.2% KH 2 P0 4 , and 0.026% NH 4 C1 and was incubated at 55°C.
  • 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
  • the cell-free extract was added to 0.1M Tris-HCl
  • 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.
  • 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 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.
  • 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.
  • 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)
  • 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.
  • pT7Blue Novagen Inc., USA
  • 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) .
  • 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 pUCll ⁇ 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 DH5 ⁇ 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.
  • the recombinant plasmid including the transformant was named ⁇ pDAP" (Fig. 1) , and the transformant including
  • the pDAP was named "DH5 ⁇ /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) .
  • 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
  • thermostable D-stereospecific amino acid amidase of the present invention is a novel enzyme at the level of DNA, which has been not reported.
  • thermostable 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).
  • PCR polymerase chain reaction
  • 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.
  • ring of yellow color ring by production of para-nitroanilid
  • the recombinant plasmid was confirmed by DNA sequencing.
  • 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 (NH 4 SO 4 ) and was adsorbed into phenyl Sepharose resin and eluted by gradient concentration using 0. IM Tris-HCl (pH 8.0).
  • 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.
  • One "unit” of enzyme activity is defined as the enzyme quantity that was needed to produce lumol amino acids for one minute .
  • Example ⁇ 4-l> In order to the biochemical characteristics of highly purified D-stereospecific amino acid amidase, the method of Example ⁇ 4-l> was used.
  • 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
  • EDTA dithiothreitol
  • 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
  • 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).
  • 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.
  • E. coli XLl-Blue/pBDA transformed by recombinant plasmid pBDA was cultured in LB medium
  • D-stereospecific amino acid amidase derived from Brevibacillus borstelensis BCS-1 had stereospecificity on D-amino acids .
  • D-stereospecific amino acid amidase specifically acted on D-phenylalanine to produce pure D-phenylalanine .
  • 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.
  • the thermostable D-stereospecific amino acid amidase specifically acted on D-amino acids to produce various D-amino acids.
  • 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.
  • 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 .
  • the microorganism identified under I above was accompanied by:
  • 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

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Abstract

La présente invention concerne une amidase d'acide aminé D-stéréospécifique, son gène, et un procédé de production d'acide aminé D-stéréospécifique faisant appel à ladite amidase. L'invention concerne l'amidase d'acide aminé D-stéréospécifique et son gène, issus du microorganisme thermophile brevibacillus borstelensis, un vecteur de clonage et un vecteur d'expression le contenant, et un transformant transformé par le vecteur d'expression. L'invention concerne également un procédé de production d'acide aminé D-stéréospécifique faisant appel à l'amidase d'acide aminé D-stéréospécifique. L'invention concerne enfin une amidase d'acide aminé D-stéréospécifique et un procédé permettant la préparation à grande échelle d'amidase d'acide aminé D-stéréospécifique par génie génétique. L'amidase d'acide aminé d-stéréospécifique peut permettre d'obtenir divers acides aminés D-stéréospécifiques utiles à l'industrie à partir d'un mélange racémique d'amide d'acide aminé DL.
PCT/KR2001/001235 2001-07-14 2001-07-19 Amidase d'acide amine d-stereospecifique, gene associe, procede de preparation associe et procede de preparation d'acide amine d faisant appel a ladite amidase WO2003008569A1 (fr)

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KR2001/42606 2001-07-14
KR10-2001-0042606A KR100449456B1 (ko) 2001-07-14 2001-07-14 신규한 ｄ-입체특이적 아미노산 아미다아제, 그의 유전자, 그의 제조방법, 이를 이용한 ｄ-아미노산의 제조방법

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KR101495602B1 (ko) * 2013-10-16 2015-02-26 한국식품연구원 청국장 유래 바실러스속 미생물 및 이를 이용하여 s-아데노실-l-메티오닌을 대량 생산하는 방법
KR101495597B1 (ko) * 2013-10-16 2015-02-26 한국식품연구원 젓갈 유래 바실러스속 미생물 및 이를 이용하여 s-아데노실-l-메티오닌을 대량 생산하는 방법
KR101495609B1 (ko) * 2015-01-23 2015-02-27 한국식품연구원 청국장 유래 바실러스 아밀로리쿼파시엔스 c4-1 및 이를 이용하여 s-아데노실-l-메티오닌을 대량 생산하는 방법
KR102118898B1 (ko) * 2017-10-27 2020-06-05 한국생명공학연구원 신규 d-입체특이적 아미노산 아미다아제와 그 돌연변이체, 및 이를 이용한 d-아미노산 생산 방법

Citations (2)

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Publication number Priority date Publication date Assignee Title
JPH02234678A (ja) * 1989-03-09 1990-09-17 Sagami Chem Res Center アミノ酸アミド加水分解酵素及びその使用
WO1998004733A1 (fr) * 1996-07-29 1998-02-05 Allied Colloids Limited Production d'acides amines et enzymes utilises a cette fin

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Publication number Priority date Publication date Assignee Title
JPH02234678A (ja) * 1989-03-09 1990-09-17 Sagami Chem Res Center アミノ酸アミド加水分解酵素及びその使用
WO1998004733A1 (fr) * 1996-07-29 1998-02-05 Allied Colloids Limited Production d'acides amines et enzymes utilises a cette fin

Non-Patent Citations (1)

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Title
KOMEDA H. ET AL.: "Gene cloning nucleotide sequencing and purification and characterization of the D-stereospecific amino acid amidase from ochrobacterium anthropi SV3", EUR. J. BIOCHEM., vol. 267, no. 7, 2000, pages 2028 - 2035, XP002243586, DOI: doi:10.1046/j.1432-1327.2000.01208.x *

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